Optical control of the cardiac rhythm with photoswitchable Na_V1.5 channel blockers

Domain: biology
Published: 2026-03-10
Editorial outcome: major_revision

Abstract
Voltage-gated sodium channel Na 1.5 is essential for cardiac excitability, mediating the rapid depolarization phase of the cardiac action potential (AP) and ensuring proper electrical conduction in the heart. Dysfunction of Na 1.5 is implicated in life-threatening arrhythmias, making it a critical therapeutic target. Acting as a Na 1.5 open-state blocker, quinidine demonstrates efficacy in arrhythmia treatment, but its low specificity restricts its clinical application. Here, we report an optopharmacological strategy which enables a precise and optical control of Na 1.5 function by means of photoswitchable quinidine derivatives. Through systematic structural optimization, we identify azo-Q2a as a high-performance photoswitchable inhibitor, exhibiting low activity in the dark or under 480 nm light irradiation (trans isomer), while approximately 7-fold higher efficacy is observed under 365 nm light irradiation (cis isomer). Of note, azo-Q2a demonstrates exceptional selectivity for Na 1.5 over cardiac ion channels and other Na 1 subtypes, minimizing potential off-target effects. Furthermore, by solving the cryo-EM structure of the Na 1.5 in complex with the cis-active isomer azo-Q2a (3.0 Å resolution), we reveal the essential binding site that is responsible for the optical control of Na 1.5. Finally, azo-Q2a also attenuates heart rate of living zebrafish larvae with light, showing its potential in cardiac related research and treatment. Our work not only establishes azo-Q2a as a robust photoswitchable inhibitor for Na 1.5 but also provides a structural blueprint for the rational design of next-generation optopharmacological antiarrhythmic agents.

Introduction
The Na 1.5 encoded by the SCN5A gene is the predominant subtype of voltage-gated sodium channels family (Na 1.1-1.9) in the heart, which plays an essential role in the excitability of cardiomyocytes1. The channels mediate the sodium influx into cells resulting in inward sodium current (I ), which induces fast depolarization of rapid response cardiac action potential (AP). Therefore, dysfunction of Na 1.5 channels underlies multiple cardiac channelopathies2-4, including long QT syndrome type 3 (LQT3), Brugada syndrome (BrS), sick sinus syndrome (SSS), dilated cardiomyopathy (DCM), atrial fibrillation (AF), and progressive cardiac conduction disease (PCCD). This underscores its status as a pivotal therapeutic target for rhythm management.

Additionally, given its pivotal role in action potential generation and conduction, Na 1.5 has long been recognized as a canonical pharmacological target for Class I antiarrhythmic drugs (Ⅰ ). Among them, quinidine belongs to the Ⅰ subclass as the first antiarrhythmic drug in the world. Acting as a Na 1.5 channel open-state blocker, quinidine inhibits peak I , thereby reducing the membrane excitability of cardiomyocytes, slowing down the depolarization and conduction velocity of rapid response cardiac AP. It has been previously used for conditions such as atrial premature beats (APB), paroxysmal supraventricular tachycardia (PSVT), AF and LQT35.

However, its therapeutic utility is severely constrained by extensive off-target polypharmacology. Quinidine often accompanies inhibition of hERGN channel, thereby prolonging APD, ERP and QT interval6,7, which increases its off-target activities8. Furthermore, quinidine exhibits promiscuous binding to multiple extracardiac receptors, including nicotinic acetylcholine receptors (nAChRs)9, 5-hydroxytryptamine receptors 3 (5-HT )10 and α adrenergic receptors11. Its nonspecific pharmacodynamic profiles induce intolerable gastrointestinal (nausea, diarrhea) and neurological (tinnitus, cinchonism) adverse effects12 in approximately 20-30 percents5 of patients during clinical application. These limitations underscore the imperative for developing targeted Na 1.5 modulators with improved specificity.

Light has emerged as a powerful biophysical modality for spatiotemporally precise control of biological systems13-15. Through its integration with pharmacology, optopharmacology enables non-invasive and rapidly reversible modulation of target-specific receptors and ion channels. This innovative strategy effectively circumvents the problems of off-target and dose-limiting adverse effects of traditional drugs, and thus achieve a much more precise treatment. Moreover, optopharmacology has been widely applied in ion channels16-18, transporters19, G protein-coupled receptors20,21, receptor-linked enzymes22. Pioneering studies have demonstrated the photopharmacological modulation of cardiac sodium channels23, yet there remains a strong demand for novel compounds that exhibit optimal Na 1.5 photoswitching, guided by a deeper mechanistic understanding.

In this work, we describe a photoactivated quinidine derivative with photoswitchable azobenzene moiety incorporating, which enables reversible optical control over Na 1.5 function and heart rate. We further characterize the binding sites of our lead compound cis-azo-Q2a using single-particle electron cryo-microscopy (cryo-EM). This provides a structural template for achieving selectivity and facilitate rational photoswitchable drug design.

Results
Rational design and synthesis of photoswitchable quinidine derivatives.
As disclosed in the recently cryo-structure of Na 1.5 in complex with quinidine24, quinidine blocks the Na 1.5 channel mainly with the quinoline moiety. While the quinuclidine group directly interrupts the central permeation pathway, the quinolone aromatic ring primarily interacts with the F1760 residue through a - stacking interaction. Much inspired by the above-mentioned crucial interaction, we hypothesized that quinidine derivatives with additional aryl substituents attached to the quinolone core would be beneficial for the binding affinity of Na 1.5.

Initially, we designed three simple quinidine phenyl derivatives with phenyl substituents at the C-2 (Q1), C-3 (Q2), and C-6 (Q3) positions of the quinoline core (Fig. 1a and Supplementary Fig. 1a). Specifically, Q1 was synthesized from quinidine via a Minisci reaction25 while Q2 and Q3 were prepared through Suzuki-Miyaura coupling reactions with phenylboronic acid using 3-I-QD and 6-OTf-QD as starting materials, respectively26,27.

Remarkably, whole-cell patch-clamp recordings in HEK293T cells heterologously expressing Na 1.5 channels revealed that Q1 and Q3 exhibited significantly higher inhibition rates than quinidine at 10 µM concentration (8.8 ± 2.9% for quinidine, 69.5 ± 6.2% for Q1, and 44.7 ± 7.8% for Q3, Fig. 1b and Supplementary Fig. 1b). These results strongly suggest that enhanced π-π stacking interactions may strengthen the binding between quinidine derivatives and the Na 1.5 ion channel.

Next, we explored photoreactive quinidine derivatives, focusing on azobenzene-based chromophores due to their widespread application. In total, nine azo-quinidine derivatives (hereafter referred to as azo-QD) were designed, featuring azobenzene moieties installed at ortho-, meta-, and para-positions with phenyl spacers at the C-2, C-3, and C-6 positions of the quinoline core (Fig. 2a).

Notably, our synthetic approach for azo-QDs utilized Suzuki-Miyaura coupling with three key intermediates: 3-I-QD, 6-OTf-QD, and 2-Br-QD (Fig. 2). The synthesis of 2-Br-QD began with the oxidation of quinidine using m-CPBA, followed by reduction with NaHSO , yielding N-oxide QD in 45% over two steps28. Subsequent bromination via the Yu protocol afforded 2-Br-QD in 46% yield29.

For 3-I-QD, direct derivatization was achieved through Suzuki-Miyaura cross-coupling with azo-BPin1 and azo-BPin2, producing the desired products in 27% and 24% yields, respectively. Meanwhile, 6-OTf-QD and 2-Br-QD were successfully coupled to furnish target compounds in 86%, 48%, 38%, and 33% yields. However, attempts to employ sterically hindered azo-BPin3 in the Suzuki-Miyaura reaction failed to yield any quinidine derivatives.

Photochemical characterization of azo-QDs.
With all 6 azo-QDs in hand, photochemical characterization has been explored (Fig. 3a-b and Supplementary Fig. 1c-g). Using UV-Vis spectroscopy, we characterized the spectral properties of all cis- and trans- azo-QDs. In the dark, they existed as the thermodynamically favored trans isomer. Isomerization to the cis isomer was triggered upon UV-A irradiation (365 nm), and this process was reversed within 5 seconds upon exposure to blue light (480 nm).

Taken azo-Q2a as an example, we observed a rapid and reversable photoswitch upon oscillating between 365 and 480 nm illumination over cycles (Fig. 3c). Using 1H-NMR and 13C-NMR spectroscopy, we assessed the relative concentrations of the two isomers in equilibrium after 365 and 480 nm illumination (Fig. 3d, Supplementary Fig. 2-3). After illumination at 365 nm, trans isomer azo-Q2a was efficiently isomerized to cis isomer (86%); 480 nm illumination resulted in efficient isomerization back to the trans isomer (79%). While we noted that the isosbestic point shifts slightly upon illumination at 480 nm which is small relative to the main spectral features, this might result from minor side- or photoproduct formation or from changes in solvent polarity and ionic strength.

Furthermore, the photoisomerization rates of azo-Q2a were measured. Under 365 nm UV light at 8500 lux, the half-life was 28 min, whereas under 480 nm blue light at 1500 lux, the half-life was 3.5 min. The quantum yields of azo-Q2a were determined to be 0.075 under 365 nm irradiation and 0.32 under 480 nm irradiation. These results demonstrate that azo-Q2a exhibits efficient and controllable photoreactive behavior across different irradiation wavelengths30 (Supplementary Fig. 4 and Supplementary Table 1).

Finally, we examined the chemical stability of azo-Q2a. Under in vitro conditions, azo-Q2a remained remarkably stable, exhibiting a half-life (t ) of 107 h in the absence of reducing agents (Fig. 3e). In contrast, exposure to 1 mM glutathione (GSH) triggered rapid cis-to-trans isomerization with a half-life of 59 min, driven by the reversible reduction of the -N=N- bond. At a higher GSH concentration (10 mM), azo-Q2a underwent predominant reductive degradation with a half-life of 143 min, indicating a shift in the reaction pathway under strongly reducing conditions (Supplementary Fig. 5).

Optical control of Na 1.5 channels with photoswitchable azo-QDs.
To determine whether azo-QDs support optical control of Na 1.5 channels, we initially profiled the concentration-dependent inhibition activities of quinidine and six derivatives to Na 1.5 current in the dark or under 365 nm illumination, using whole-cell patch-clamping recordings in HEK293T cells heterologously expressing Na 1.5 channels. (Fig. 4a-d and Supplementary Fig. 6a-e).

When holding the cellular membrane potential at -120 mV, the expected Na+ currents were observed at the typical voltage steps31 (-120 to -20 mV). All of the examined azo-QDs in the cis isomer conferred stronger inhibition activity than quinidine, and azo-Q1a, azo-Q2a, and azo-Q3b displayed a significant increase in their IC values after 365 nm illumination. (Fig. 4a-c and Supplementary Fig. 6a-e). Among the examined azo-QDs, azo-Q2a showed the most prominent alteration (IC = 11.76 µM in cis; 81.58 µM in trans, Fig. 4c), while quinidine had no obvious difference (IC = 35.87 µM under 365 nm; 41.21 µM in dark, Fig. 4d). IC values are detailed in Supplementary Table 2.

We measured current-voltage (I-V) steps between -120 and +40 mV (5 mV steps) in the presence of 10 µM azo-Q2a (Fig. 4e). In its cis isomer, azo-Q2a strongly suppressed inward currents conducted by Na 1.5 channels, whereas the trans isomer had no discernible effect. In addition, neither isomer altered the activation property of Na 1.5, assessed as conductance-voltage (g-V) relationships and the V value (Fig. 4f, Supplementary Table 3). Note that blockade of Na 1.5 channels was readily reversible as we repeatedly photoswitched azo-Q2a between its trans and cis isomer (Fig. 4g-h).

azo-Q2a is highly selective and significantly reduces hERG toxicity of quinidine.
To further evaluate the selectivity profile of azo-Q2a among other sodium channel subtypes (Na 1.2–Na 1.6) highly expressed in the central nervous system and muscle tissues (Supplementary Fig. 7a). At a concentration of 10 μM, cis-azo-Q2a exhibited potent inhibition of Na 1.5 (61.1 ± 4.8%, use-dependent, Supplementary Fig. 6f) but minimal activity (< 10%) against all other subtypes tested. In contrast, trans-azo-Q2a under dark conditions showed negligible suppression of Na 1.5 (2.5 ± 1.6%) yet significantly inhibited Na 1.3 (36.2 ± 6.1%). Notably, this off-target effect on Na 1.3 was effectively reversed upon optical illumination, which switches the compound to cis-configuration.

These results underscore the light-dependent selectivity of azo-Q2a for Na 1.5. Given that Na 1.5 serves as the predominant sodium channel in cardiac tissue, spatially confined illumination enables the high-affinity cis-azo-Q2a to selectively modulate heart function while minimizing potential off-target effects in neural or other systems.

In addition, recalling that quinidine’s off-target interactions that inhibit hERG channels lead to serious cardiotoxicity7, we investigated the potential inhibition activity of azo-Q2a towards other ion channels known to contribute to cardiac action potential, including K 4.3 (I ), K 2.1 (I ), hERG (I ) and Ca 1.2 (I ) channels (Fig. 5a). The experiments monitored the currents of these channels under both 365 and 480 nm illumination. Currents of these ion channels were recorded using whole-cell patch-clamping in HEK293T and CHO cell lines expressing cardiac ion channels (see Methods section for details).

The inhibition rate of 10 µM azo-Q2a for Na 1.5 channels was significantly different between the cis (61.1 ± 4.8%) and trans (2.5 ± 1.6%) isomers, whereas both azo-Q2a isomers exerted in same inhibition activity against K 4.3, K 2.1, hERG and Ca 1.2 channels (Fig. 5b). Quantification of the inhibition showed that azo-Q2a endows light sensitivity only to Na 1.5 but has negligible effects on K and Ca channels, yielding photoswitching efficacies of 33.1 ± 4.3% for Na 1.5 and far less for the rest channels, of 5.2 ± 1.7% (K 4.3), 3.6 ± 4.2% (K 2.1), 7.9 ± 4.5% (hERG), 7.7 ± 5.4% (Ca 1.2) (Fig. 5h and Supplementary Table 4). To compare the difference in hERG channel inhibition activity between azo-Q2a and quinidine, we repeated these assessments through application of 10 and 30 µM quinidine. APs expected, quinidine showed comparable pharmacological behavior across all tested ion channels without optical control property (Fig. 5b and Supplementary Fig. 7b-d). Notably, at 10 µM, cis-azo-Q2a inhibited Na 1.5 and hERG currents by 61.1% and 45.5%, respectively, yielding a Na 1.5/hERG inhibition ratio of 1.34, whereas quinidine produced 8.8% and 71.7% inhibition (ratio 0.12). This corresponds to an approximately 11-fold increase in the Na 1.5/hERG inhibition ratio, indicating an overall enhancement of functional selectivity relative to quinidine (Fig. 5b and Supplementary Table 5).

In addition, we further evaluated the concentration-response relationship of azo-Q2a and quinidine on hERG channels under 365 and 480 nm illumination, which showed that the IC value of azo-Q2a on hERG was 16-fold lower than that of quinidine (Supplementary Fig. 7e-f).

Together, these results demonstrate that azo-Q2a possesses high selectivity for Na 1.5 channels among cardiac ion channels and other Na 1 subtypes, significantly reducing the hERG channel inhibition activity observed with quinidine. These characteristics make azo-Q2a a promising candidate for the development of photoactivated antiarrhythmic therapies.

azo-Q2a reversibly inhibits Na+ currents in rat primary cardiomyocytes.
We next investigated azo-Q2a as an agent for the selective optical control of Na 1.5 channels in rat primary cardiomyocytes. We recorded voltage-gated Na+ and K+ currents from primary ventricular myocytes under 365 and 480 nm illumination (Fig. 5c).

Consistent with our observations in the transient transfection cell experiments, Na+ current in cardiomyocytes was reduced at 365 nm illumination after the addition of azo-Q2a (10 µM), and this was reversed by blue light (480 nm) (Fig. 5d). The selective optical control of Na 1.5 channels was over multiple on/off cycles (Fig. 5d-e); the photoswitching efficacy was 24.3 ± 3.3%, showing no significant difference from the 33.1 ± 4.3% compared to HEK293T cells expressing Na 1.5 channels (Fig. 5h). No significant inhibition on K+ currents were observed under the same experimental conditions (3.0 ± 2.4%), supporting the selectivity of azo-Q2a towards Na 1.5 channels (Fig. 5f-h). Additionally, quinidine (in 10 and 30 µM) was applied as control which showed insignificant optical control of both Na+ (0.8 ± 0.8%) and K+ currents (0.9 ± 1.9%, in 10 µM), (Supplementary Fig. 7b-d). To further exclude potential effects of UV-induced ROS, we performed illumination-only controls (Supplementary Fig. 8a), which showed no changes in sodium currents, consistent with the unchanged inhibition of quinidine under both wavelengths.

Owing to the significant role of Na 1.5 in the action potential of myocardial cells, we directly evaluate the effect of azo-Q2a on cardiac electrophysiology performing whole-cell current-clamp recordings on primary cardiomyocytes to assess the impact of trans and cis-azo-Q2a on cardiac action potential (Supplementary Fig. 8b-e). The results demonstrated that cis-azo-Q2a significantly reduced the dV/dt by 47.2 ± 8.7%, whereas trans-azo-Q2a showed only a modest inhibition of 16.6 ± 1.1%. Furthermore, we observed that cis-azo-Q2a shortened the action potential duration at 50% repolarization (APD ) by 55.2 ± 4.9% whereas trans-azo-Q2a produced only a modest inhibition of 16.6 ± 3.4%. We attribute this shortening primarily to the inhibition of the late sodium current (I ), which is known to modulate repolarization.

Cryo-EM Structure basis for azo-Q2a optical control of Na 1.5 channels.
To assess azo-Q2a binding we obtained a cryo-EM structure for Na 1.5 in complex with azo-Q2a at the resolution of 3.0 Å (Na 1.5S-azo-Q2a) (Supplementary Table 6). Illuminated by 365 nm light, azo-Q2a was photo-induced as the cis isomer before sample preparation. Note also that, seeking to optimize protein expression, we truncated the carboxyl terminal domain and linker between repeats Ⅰ-Ⅱ and Ⅱ-Ⅲ of Na 1.532 (referred to as Na 1.5S, Supplementary Fig. 9).

We first assessed the inhibition activity of quinidine on Na 1.5S using whole-cell patch-clamping recording, yielding an IC value of 44.66 μM in the dark and of 43.80 μM under 365 nm illumination; these values do not differ from the values for Na 1.5WT (Supplementary Fig. 10a, c and Supplementary Table 7). We then examined the optical control of azo-Q2a towards Na 1.5S following the same recording protocols and conditions used for Na 1.5WT and found that azo-Q2a showed the same inhibition activity and optical control property for Na 1.5S and Na 1.5WT (IC = 10.44 µM for cis; 75.41 µM for trans, Supplementary Fig. 10b, d), suggesting that truncation of Na 1.5 does not interfere with the interaction of quinidine or azo-Q2a in the pore domain.

The unambiguous EM densities for cis-azo-Q2a are shown in the blue mesh support that the cis-azo-Q2a molecule bind to the pore domain (Figure 6a, Supplementary Fig. 11a). Similar to quinidine, cis-azo-Q2a is coordinated by both polar and hydrophobic residues from repeats Ⅰ, Ⅲ, and Ⅳ, but distanced from repeat Ⅱ (Fig. 6a-c). In addition, upon comparing our complex structure of Na 1.5S with the reported Na 1.5-quinidine structure31 (PDB ID: 6LQA), we found that the binding sites of azo-Q2a and quinidine differ in the central cavity of the pore domain (Fig. 6b-d): quinidine is likely hydrogen-bonded to Gln371, Thr1417, and Ser1759 and appears to interact with Phe1760 through π-π stacking; cis-azo-Q2a is surrounded by multiple hydrophobic residues from the S6 segments (Fig. 6b-c), among which Val405 appears to interact with the quinolone aromatic ring through a hydrophobic interaction (Fig. 5b-c), while Phe1760 plays an essential role connecting with the azobenzene group through π-π stacking (Fig. 6b-c). The interactions between Phe1760 and the quinoline moiety of quinidine changed to the azobenzene portion of cis-azo-Q2a, while the S6 of Na 1.5S undergoes an α to π helix transition (Fig. 6e, Supplementary Fig. 11b).

We generated Na 1.5 variants bearing on V405A and F1760A mutations, expressed them in HEK293T cells, and assayed azo-Q2a inhibition activity in the dark or under 365 nm illumination. We assessed the inhibitory effect of 100 μM quinidine on the V405A and F1760A mutants. The results demonstrated that the functional impairment was more pronounced in the F1760A mutant compared to the WT, with inhibition rates of 77.6 ± 3.2 % (WT), 51.2 ± 2.3% (V405A), and 32.6 ± 1.8% (F1760A), (Supplementary Fig. 7g). This finding is consistent with previous cryo-EM structural analyses, which revealed that the quinoline ring of quinidine forms critical interactions with the F1760 residue31.

Assessing the calculated concentration-responses supporting our Na 1.5S-azo-Q2a structural insights: Both mutations V405A and F1760A resulted in a significant rightward shift of the IC curve for cis-azo-Q2a (IC > 100 μ M, Fig. 6f–h and Supplementary Table 8), indicating a substantial reduction in its inhibitor potency. Collectively, these findings confirm that Val405 and Phe1760 are critical residues for the binding and inhibition of Na 1.5 by cis-azo-Q2a, thereby establishing them as essential molecular determinants for the light-dependent control mediated by azo-Q2a.

azo-Q2a reversibly attenuates heart rate and terminates ISO-induced tachycardia in vivo in zebrafish larvae.
To assess the capability of azo-Q2a to confer optical heart rate control in vivo, we used zebrafish larvae (2 days post-fertilization) as the animal model (Fig. 7a), for which light scattering is known to be low33. The Na+ current (I ) generated by Na 1.5 channels is essential for maintaining a normal heart rate. Heart rate was monitored by counting the number of sequential contractions in 10-30 s intervals under a dissecting microscope.

The larvae were exposed to different treatments via soaking (Control; 10 μM azo-Q2a and 10 μM quinidine) for 1-1.5 h in the dark34, and then heart rate was recorded 30 min after 365 or 480 nm light exposure using video microscopy (Fig. 7a). We illuminated control zebrafish larvae that we kept under dissimilar experimental conditions for 5 min (at 365 nm or at 365 nm followed by 480 nm light both continuing for 5 min), aiming to estimate optical effects on the heart rate (Fig. 7a).

We first examined the heart rate of larvae treated with no inhibitor as a control: these showed no significant change after different light exposure, suggesting that the light itself does not affect the heart rate (Fig. 7b). The average heart rate of control experiments measured in the dark was used as baseline to normalize each detection value. After perfusion of trans-azo-Q2a (10 μM) lasting for 1-1.5h in the dark, there was no difference in the calculated heart rate (98.8 ± 1.4%) compared to the control (Supplementary Movie 1) treated with DMSO. However, we did observe a significant slowing of the heart rate for these larvae after 365 nm illumination (56.5 ± 4.3%, Supplementary Movie 2), and this slowing was reversed by the subsequent 480 nm illumination (103.2 ± 2.0%, Fig. 7b, Supplementary Table 9 and Supplementary Movie 3).

We also examined larvae perfused with disposed with quinidine in the dark (90.8 ± 1.4%), under 365 nm illumination (93.9 ± 0.9%) and after subsequent 480 nm illumination (91.6 ± 1.3%), (Fig. 7b and Supplementary Table 9). These results are consistent with previous reports for quinidine’s inhibition activity, demonstrating that azo-Q2a confers optical control and has higher inhibition of the heart rate in vivo compared with quinidine35.

To further evaluate the therapeutic potential of azo-Q2a against arrhythmias, we established a tachycardia model in zebrafish larvae (3–4 dpf, untreated control shown in Supplementary Movie 4) by incubating them in darkness with 10 μM isoproterenol (ISO) for 100 min, which significantly increased heart rate36 (Supplementary Movie 5). Using the same treatment and illumination protocol as described above, we found that both 10 μM cis-azo-Q2a (35.3 ± 2.6%, Supplementary Movie 6) and 100 μM quinidine (Supplementary Movie 7) markedly attenuate heart rate in ISO-pretreated larvae, whereas 10 μM trans-azo-Q2a (Supplementary Movie 8) produced only a mild reduction in heart rate (4.4 ± 1.2%, Supplementary Fig. 12). These results demonstrate that light-induced isomerization of azo-Q2a can effectively terminate ISO-induced tachycardia in vivo, thereby supporting its potential relevance for Na 1.5-related arrhythmias.

Overall, these results indicate that the optical control phenomena of electrophysiological experiments obtained in vitro can be reproduced in living animals and moreover, that azo-Q2a can reversibly control heart rate.

Methods
Ethics statement
All animal procedures were conducted in strict accordance with the “Guide for the Care and Use of Laboratory Animals” and the “Principles for the Utilization and Care of Vertebrate Animals”. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center (DLASB D0572). All zebrafish (AB strain) used in this study were raised and handled according to protocol (IMM-XiongwW-3) approved by the Institutional Animal Care and Use Committee at Peking University, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Every effort was made to minimize animal suffering and to reduce the number of animals used.

Chemical Synthesis and Photochemical Characterizations
azo-QDs were synthesized and characterized by standard chemical methods. The UV-visible absorption spectra of the cis and trans isomers were determined by UV-Vis spectroscopy. 365 nm and 480 nm LED light was used to perform trans ↔ cis isomerization in vitro. Ratio of the trans and cis isomerization and half-life of cis isomerization was determined by NMR spectra. Full design, synthesis, and photochemical characterization of the azo-QDs is detailed in the Supplementary Information.

Cell culture and transfection
The human embryonic kidney cells (HEK293 and HEK-293T) and Chinese hamster ovary cells (CHO) were obtained from ATCC. HEK293 and HEK-293T cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 15 % (volume/volume) Fetal Bovine Serum (FBS, PAN-Biotech) at 37 °C and 5 % CO . In addition, due to poor adherence of HEK293T stable cell lines expressing K 4.3 channels and K 2.1 channels, 0.1 mg/mL Poly-D-lysine (Sigma) was perfused in culture dishes prior to cell culture and the ratio of FBS increased to 20 %. CHO cells were maintained in Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (1:1, DMEM/F12, SinoDetech) supplemented with 10 % (volume/volume) FBS.

Cells with 70 ~ 80 % confluence were transiently transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Then the cells were cultured for 20 ~ 48 h and subjected to experiments.

Voltage-clamp recordings
Voltage-clamp recordings were obtained using a HEKA EPC-10 patch-clamping amplifier (HEKA Electronic) and the data were acquired by the PatchMaster program (HEKA Electronic). For whole-cell patch-clamping recording, patch pipettes were pulled from thin-walled borosilicate glass (Sutter Instrument), polished, and gave resistances of 1.5 to 2.5 MΩ in the experimental solutions. In addition, all recordings were accepted only when series resistance was <6 MΩ and compensated by 70%, and seal resistance exceeded＞1 GΩ. To evaluate the inhibition effect of quinidine and azo-QDs, compounds were delivered by gravity perfusion. The illumination during whole-cell patch-clamping recording was given by LumiCite 9100 LED (OPLENIC). For 480 nm wavelength, 100% light intensity was used (45000 lux), and for 365 nm wavelength, 35% light intensity was used (1200 lux).

As for the voltage-clamp recording analyses, all data were reported as mean ± SEM. Data analyses were performed using Origin 2022 (Origin Lab), Excel 2016 (Microsoft), and GraphPad Prism 10 (GraphPad Software). Inhibition curves were generated using a Hill equation (1)
I = Imax / (1+10(logIC50-[C])×H) (1)
where I is the current at different compounds concentrations, I is the maximal current of ion channels without inhibitor application, [C] is the logarithmic concentration, IC is the half-maximal inhibition concentration and H is the Hill coefficient.

Sodium channel
For sodium currents recording, the pipette solution contained 140 mM CsF, 10 mM HEPES, 1 mM EGTA, 10 mM NaCl (pH = 7.3 with CsOH and an osmolarity of ~295 mOsm/L). The bath solution contained 140 mM NaCl, 3 mM KCl, 10 mM HEPES, 10 mM Glucose, 1 mM CaCl , 1 mM MgCl (pH = 7.3 with NaOH and an osmolarity of ~310 mOsm/L). In whole-cell recordings of Na 1.5 currents, the cells were held at −120 mV and the inward peak sodium currents were elicited by a 50 ms step to −20 mV. Recordings from other sodium channel isoforms (Na 1.2–Na 1.6) were performed under the same holding conditions (−120 mV) but using subtype-specific test pulses: 0 mV for Na 1.2 and Na 1.3 (50 ms), −30 mV for Na 1.4 (20 ms), and −10 mV for Na 1.6 (50 ms). In addition, the current-voltage (I–V) relationships were obtained by a 50 ms step from −120 to +40 mV in 5 mV increment. To calculate the voltage dependence of activation, conductance G was fitting with Boltzmann equation (2)
G / Gmax = 1 / (1+e -(Vm-V1/2)/k) (2)
where G is conductance and G is the maximum conductance between −120 and +40 mV. V is the stimulus potential. V indicates the voltage at half-maximal activation, and k is a slope factor describing voltage sensitivity of the channel. k is the slope factor. G are calculated from the I–V relationships according to equation (3)
I = G * (Vm+ENa) (3)
where I is the peak current, G is conductance, V is the stimulus potential, E is the equilibrium potential which is the calculated reversal potential based on our whole-cell recording of Na 1.5 I-V response curve. Significance of fitted V compared to control was analyzed using extra sum-of-squares F test.

Potassium channel
For potassium currents recording, the pipette solution contained 130 mM KCl, 10 mM HEPES, 5 mM EGTA, 1 mM MgCl , 5 mM Mg-ATP (pH = 7.3 with KOH and an osmolarity of ~295 mOsm/L). The bath solution contained 138 mM NaCl, 4 mM KCl, 10 mM HEPES, 10 mM Glucose, 2 mM CaCl , 1 mM MgCl , 0.33 mM NaH PO ·2H O(pH = 7.3 with NaOH and an osmolarity of ~310 mOsm/L). In whole-cell recordings of K 4.3 currents, the cells were held at −80 mV then currents were elicited by a 600 ms step to +60 mV. For hERG currents, the cells were held at −80 mV then currents were elicited by a step to +20 mV following a step to -40 mV, each stimulation last for 2 s40,41. For K 2.1 currents, the cells were held at −80 mV then currents were elicited by a 100 ms step to -120 mV.

Calcium channel
For calcium currents recording, the pipette solution contained 135 mM K-gluconate, 10 mM HEPES, 5 mM EGTA, 2 mM MgCl , 5 mM NaCl, 4 mM Mg-ATP (pH = 7.3 with KOH and an osmolarity of ~295 mOsm/L). The bath solution contained 105 mM NaCl, 30 mM TEA-Cl ,10 mM BaCl ·2H O, 10 mM HEPES, 10 mM Glucose, 5 mM CsCl, 4 mM KCl, 1 mM MgCl (pH = 7.3 with NaOH and an osmolarity of ~310 mOsm/L). In whole-cell recordings of Ca 1.2 currents, the cells were held at −80 mV then currents were elicited by a 200 ms step to -10 mV.

Isolate and culture rat ventricular primary cardiomyocytes
Ventricular primary cardiomyocytes were isolated from 1- to 2-day-old Sprague-Dawley rats42 provided by Peking University Health Science Center Department of Laboratory Animal Science. All animals were handled in strict accordance with the “Guide for the Care and Use of Laboratory Animals” and the “Principles for the Utilization and Care of Vertebrate Animals,” and all animal work was approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center (DLASBD0572). Each effort was made to minimize animal suffering and the number of animals used. The experiments were blind to viral or drug treatment conditions during behavioral testing. The gender of the fetal rats was not screened during the experiment.

Briefly, primary cardiomyocytes were isolated with 0.05% trypsin (Gibco) and 0.1% collagenase II (Sigma). Cardiomyocytes were separated from the fibroblasts by pre-plating the digested cell suspension for 2 h. Cells were maintained in DMEM (Gibco) supplemented with 10 % (volume/volume) FBS (PAN-Biotech) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) (Gibco) at 37 °C and 5% CO .

The voltage-gated whole-cell patch-clamping recording of sodium and potassium currents in primary cardiomyocytes follow the protocol in HEK293T cells. For cardiac action potential recordings, data were collected using a Multiclamp 700B amplifier (Molecular Devices), filtered at 10 kHz and sampled at 50 kHz. Patch pipettes (3–6 MΩ) were filled with an internal solution containing 130 mM KCl, 10 mM HEPES, 5 mM EGTA, 1 mM MgCl₂, and 5 mM Mg-ATP (pH 7.3 adjusted with KOH; ~295 mOsm/L). The external bath solution contained 138 mM NaCl, 4 mM KCl, 10 mM HEPES, 10 mM glucose, 2 mM CaCl₂, 1 mM MgCl₂, and 0.33 mM NaH₂PO₄·2H₂O (pH 7.3 adjusted with NaOH; ~310 mOsm/L). Series resistance (10–30 MΩ) was compensated by ~60–80%, and recordings were discarded if series resistance varied by more than 20% during the experiment. Action potentials were evoked by 4-ms depolarizing current injections (500 pA) applied at 1 Hz. Data were acquired and analyzed using pClamp 10.4 (Molecular Devices).The maximal upstroke velocity (V ) was determined as the peak of the first derivative of the membrane voltage (dV/dt) during the depolarization phase. APD₅₀ was measured as the time from the overshoot to 50% repolarization.

Heart rate recording in zebrafish
To determine heart rate of zebrafish, 2 dpf embryos were loaded in a recording chamber filled with E3 solution at the desired stage. Heart rate was calculated by counting the number of sequential contractions in 30 s intervals under a dissecting microscope (S8APO; Leica)43. The gender of the embryos was not screened during the experiment. All zebrafish in this study were raised and handled according to a zebrafish protocol (lMM-XionguW-3) approved by the Institutional Animal Care and Use Committee at Peking University, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care international.

In order to detect the effects of compounds on zebrafish heart rate under different illumination, the control group was without treatment, and the quinidine and azo-Q2a were prepared with E3 buffer at the applicated concentrations. Then, zebrafish larvae were exposed to the compounds by soaking and incubated in a 28.5 ℃ incubator in the dark for 1.5 h under perfusion. Next the heart rate labeled darkness was recorded as described above. After that, giving to each group respectively 365 nm light (4500 Lux) or 365 nm after a subsequent illumination with 480nm light (180000 Lux), the duration of each illumination is 5 minutes. Finally, we recorded the heart rate of groups labeled 365 nm or 365 nm + 480 nm after placing the larvae in the dark for 30min after light exposure.

To further examine the therapeutic potential of azo-Q2a against Na 1.5-related arrhythmia, a tachycardia model was established in 3–4 dpf zebrafish larvae by incubation with 10 μM isoproterenol (ISO) in Hanks’ solution for 100 min in darkness. Following ISO treatment, larvae were exposed to 10 μM cis-azo-Q2a, 10 μ M trans-azo-Q2a or 100 μM quinidine under 365 nm illumination for 5 min or kept in darkness. Heart rate was measured after an additional 30 min of darkness.

Transient co-expression of human Na 1.5S and β1
The methods for transient co-expression, protein purification, cryo-EM analysis were conducted following a standard protocol44. The optimized coding DNAs for human Na 1.5 (Uniprot: Q14524) was cloned into the pEG BacMam vector with twin Strep-tag and FLAG tag in tandem at the amino terminus, while β1 (Uniprot: Q07699) was cloned into the pCAG vector without affinity tag45,46. To optimize the protein behavior, carboxyl terminal domain (1895-2016) and linker between repeat Ⅰ-Ⅱ (461-657) and Ⅱ-Ⅲ (1066-1187) of Na 1.5, named Na 15S, were deleted based on the truncated construct rNa 1.532. HEK293F cells (Invitrogen) were cultured in SMM 293T-I medium (Sino Biological Inc.) under 5 % CO in a Multitron-Pro shaker (Infors, 130 r.p.m.) at 37 ℃. When cell density reached 1.8 ~ 2.2 ×106 cells/ml, 2.0 mg plasmids (1.5 mg Na 1.5S and 0.5 mg β1) and 4 mg of 40-kDa linear polyethyleneimines (PEI, Yeasen) were mixed up in 15 mL fresh medium and pre-incubated for 15- 30 min before adding into 1 liter cell culture. In addition, 10 mM sodium butyrate was added to cell culture. Transfected cells were cultured for 48 h before harvesting.

Protein purification of Na 1.5S and β1
14 liter transfected cells were harvested by centrifugation at 1431 g and resuspended in the lysis buffer containing 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl. The suspension was supplemented with 1 % (w/v) n-dodecyl-β-D-maltopyranoside (DDM, Anatrace), 0.1 % (w/v) cholesteryl hemisuccinate Tris salt (CHS, Anatrace), and protease inhibitor cocktail containing 2 mM phenylmethylsulfonyl fluoride (PMSF), 6.5 μg/ml aprotinin, 3.5 μg/ml pepstatin, and 25 μg/ml leupeptin. After incubation at 4 ℃ for 2 h, the cell lysate was centrifuged at 16751 g for 1 h, and the supernatant was applied to anti-Flag M2 affinity gel (Sigma) at 4 °C. After flow through by gravity, the resin was rinsed four times with the Wash buffer (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.02% (w/v) glycol-diosgenin (GDN, Anatrace), and the protease inhibitor cocktail). Protein was eluted with the wash buffer plus 200 μg/mL FLAG peptide (GL.biochem). The eluent was then applied to Strep-Tactin Sepharose (IBA Lifesciences). The purification protocol was similar to the previous steps except for the elution buffer, which was Wash buffer plus 2.5 mM D-Desthiobiotin (IBA Lifesciences). The eluent was then concentrated via 100-kDa cutoff Centricon (Millipore) and further purified with size exclusion chromatography (Superose-6 Increase 10/300 column, GE Healthcare) in Wash buffer. Peak fractions were pooled and concentrated to ∼30 μL. Then, 1 mM azo-QD (effectiveness 82 %) was added to the concentrated sample 30 min before cryo sample preparation.

Cryo-EM data acquisition
4 μl aliquots of concentrated Na 1.5S-azo-Q2a complex were applied to Quantifoil 300 mesh R1.2/1.3 Au grids which were glow-discharged for 35 s at medium RF level of Plasma Cleaner PDC-32G (Harrick). Then grids were blotted from both sides for 4 s at 8 °C and 100 % humidity and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Prepared grids were subsequently transferred to a Titan Krios electron microscope (Thermo Fisher) operating at 300 kV and equipped with Cs corrector, Gatan K3 Summit detector and GIF Quantum energy filter. A total of 9,406 movie stacks were automatically collected, using AutoEMation47 with a slit width of 20 eV on the energy filter and a preset defocus range from -1.8 µm to -1.5 µm in super-resolution mode at a nominal magnification of 64,000×. Each stack was exposed for 2.56 s with the exposing time of 0.08 s per frame, resulting in total of 32 frames per stack. The total dose rate was 50 e-/Å2 for each stack. The stacks were motion corrected with MotionCor248 and binned 2 fold, resulting in a pixel size of 1.0979 Å/pixel. In addition, dose weighting was performed49. The defocus values were estimated with Gctf50.

Image processing
The data processing procedure was nearly identical to the one we previously reported51. In brief, a total of 6,525,104 particles were automatically picked in cryoSPARC52. 2D classification identified 614,955 good particles that were subsequently applied to Hetero refinement and non-Uniform refinement. Finally, a 3D EM map with an overall resolution of 3.03 Å was generated using 287,376 particles. The resolution was estimated with the gold-standard Fourier shell correlation 0.143 criterion53 with high resolution noise substitution54.

Model building and structure refinement
The initial model of Na 1.5S was based on the coordinate of human Na 1.5-quinidine (PDB:6LQA)31, and the model building and structure refinement processes were the same as we used before51. In brief, the coordinate of Na 15-quinidine was fitted into the EM map by CHIMERA (Detailed workflow were shown in Supplementary Fig. 9b)55. And every residue was manually checked in COOT56 carefully. The chemical properties of amino acids were considered during model building. The cis-azo-Q2a was fitted into the EM map. Structure refinement was performed using phenix.real_space_refine application in PHENIX57 real space with secondary structure and geometry restraints. Over-fitting of the overall model was monitored by refining the model in one of the two independent maps from the gold-standard refinement approach and testing the refined model against the other map58. Statistics of the map reconstruction and model refinement can be found in Supplementary Table 6.

Molecular docking simulation
The Cryo-EM structure of the Na 1.5–cis-azo-Q2a complex was refined in Schrödinger Maestro v13.5, followed by restrained minimization using the Impref module of Impact with the OPLS4 force field, in which hydrogen atoms were optimized while allowing limited heavy-atom relaxation59-61. The trans-azo-Q2a ligand was prepared in LigPrep to generate low-energy, all-atom conformers, with ionization states enumerated at pH 6.5 ± 1.0 and minimized using OPLS461. A receptor grid was defined from the cis-azo-Q2a binding pocket, and trans-azo-Q2a was docked into Na 1.5 in both unconstrained and pose-constrained modes, the latter referencing the cis-azo-Q2a orientation. Docking was performed using Glide in extra-precision (XP) mode, and poses were ranked by GlideScore and evaluated by MM/GBSA to estimate relative binding free energies62.

Data analysis and Statistics
For in vitro experiments, the cells were evenly suspended and then randomly distributed in each well tested. For in vivo experiments, the zebrafish were distributed into various treatment groups randomly. Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software). Before statistical analysis, variation within each group of data and the assumptions of the tests were checked. Comparisons between two independent groups were made using unpaired Student’s two-tailed t test. Comparisons among nonlinear fitted values were made using extra sum-of-squares F test. Comparisons among three or more groups were made using ordinary one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. Values of IC are presented as mean (95 % CI (Profile likelihood)). Other data are presented as mean ± SEM.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this interesting manuscript, Shiqi Liu and co-workers reported a photoactive inhibitor of the cardiac sodium channel Nav1.5 based on the antiarrhythmic drug quinidine. They presented electrophysiology, cryo-EM structure and living zebrafish in vivo results, demonstrating that the inhibitory activity of azo-Q2a is photoswitchable. The manuscript is clearly written and the experiments are well-performed. This reviewer has several suggestions concerning their figure presentation and the optical control mechanism, which, hopefully, could help the authors to clarify their manuscript. After revision, this reviewer supports its publication in Nature Communications.\nMajor concerns:\n1. Quinidine is an open state blocker of Nav1.5, whether azo-Q2a is also an open state blocker or not?\n2. Selectivity. I suggest the author to tune down the selectivity of azo-Q2a. First, it still inhibits hERG as shown in Fig. 4b (45% for hERG and 60% for Nav1.5); second, it is not known the efficiency of azo-Q2a for other sodium channel subtypes.\n3. The EM complex structure supports the binding of an optical control blocker in Nav1.5. However, the structure figure is not clearly presented. It’s difficult to interpret the precise interactions between cis-azo-Q2a and the channel, and the detailed differences between cis-azo-Q2a and quinidine. For example, how confident to claim a carbon-hydrogen---π interaction between Val405 and the quinolone ring?\n4. EM map. The EM density in Fig. 5b appears to support the binding of cis-azo-Q2a. For clarity, this reviewer suggests that the authors should present the density map of cis-azo-Q2a from three different views, as well as density maps for the S6 helices, because of conformational changes (Fig. 5e).\n5. The author used a loop truncated version human Nav1.5S instead of full-length Nav1.5 for improved protein behavior, based on the construct of rNav1.5. Please provide reference.\n6. It is not clear how Val405 contributes to the inhibitory activity of azo-Q2a but not optical control, while F1760 determines the optical control as interpreted in Lines 246-255. From the results in Fig. 5f-h, the response of azo-Q2a to V405A and F1760A looks similar under darkness or 365 nm light.\n7. The optical control mechanism is unclear. Is it because the cis form of azo-Q2a prefers binding in the identified pocket in Nav1.5 while the trans form could cause steric clashes with the channel, or because of different accessibility for the two forms? The authors should dock the trans form in the pocket and discuss possible molecular mechanism for optical control.\nMinor points:\n1. In Line 187, Supplementary Fig. 3d should be Supplementary Fig. 3e?\n2. In Line 148, what is azo-2P, azo-Q2a, and azo-6M? These names are not consistent in Fig. 3 and Supplementary Fig. 2.\n3. In Lines 133, 142, 275 and others, 365nm should be 365 nm.\n4. In Line 305, Inhibitors should be inhibitors.\n5. In Line 458, what is “NaV1.5Short-azo-Q2a”? Double-check other typos.", "reviewer_2": "The authors present a photoswitchable quinidine derivative, azo-Q2a, targeting the cardiac sodium channel NaV1.5. While NaV1.5 is essential for cardiac depolarization and quinidine has long-standing utility as an antiarrhythmic agent, its poor specificity and off-target effects—particularly hERG inhibition—limit its clinical use. Through the synthesis of azobenzene–quinidine derivatives, the authors identify azo-Q2a as a lead compound with reversible, light-dependent activity: it is highly potent in its cis form (activated by 365 nm light) and largely inactive in its trans form (480 nm light or dark), demonstrating approximately a 7-fold difference in efficacy. Azo-Q2a exhibits strong selectivity for NaV1.5 over other cardiac ion channels and significantly reduces hERG-related cardiotoxicity relative to quinidine. Cryo-EM structural analysis reveals key binding interactions with NaV1.5, notably involving residues Phe1760 and Val405. In vivo, azo-Q2a enables reversible, light-mediated modulation of heart rate in zebrafish larvae, underscoring its potential as a research tool and therapeutic lead for arrhythmia.\nDespite these strengths, the manuscript notably fails to acknowledge significant prior work in this area. In particular, the study omits reference to “Optical Control of Cardiac Electrophysiology by the Photochromic Ligand AzoBupivacaine 2” (Fehrentz et al., Br. J. Pharm., 2025, 82, 1125–1142), which introduced azobupivacaine (AB2) as a reversible photoswitchable NaV1.5 blocker. That earlier work demonstrated optical control of cardiac function in more advanced mammalian models—including mouse left ventricular slices and Langendorff-perfused hearts—and achieved optical termination of ventricular tachycardia. Conceptually, the current study does not extend meaningfully beyond these findings. While the zebrafish data are elegant, they do not surpass the physiological relevance achieved with AB2. The cryo-EM structural data are novel and represent a valuable contribution to mechanistic understanding and future drug design. However, in isolation, they may not justify publication in a high-profile venue, particularly given that azo-Q2a’s action spectrum provides no clear advantage over that of AB2.\nIn summary, without a thorough acknowledgment of prior work and a demonstrable advance in biological scope or translational relevance, this manuscript is not suitable for publication in its current form in Nature Communications.", "reviewer_3": "In their manuscript, Liu et al. describe the azologization of quinidine and assess the optical control of their most promising analogue, azo-Q2a, both in vitro and in vivo in zebrafish larvae. The approach is scientifically compelling. However, previous studies have largely reported similar strategies and outcomes on other channels or receptors, which reduces the novelty of the current work.\nTo be considered for publication in Nature Communications, several critical issues must be addressed.\nFirst, in the experimental section, essential information is missing regarding the light intensity used for photoisomerization. Since the efficiency of azobenzene switching is intensity-dependent, the authors must report the irradiance used and the quantum yields for both trans-to-cis and cis-to-trans conversions. These details are crucial for the reproducibility of the study.\nSecond, the UV-Vis spectra presented in Figure 2 were obtained in MeOH:H₂O, while those in Supplementary Figure 1 were acquired in DMSO. The rationale behind the use of different solvents is not provided. This inconsistency complicates the comparison of isomerization efficiency. The authors should use the same solvent system across experiments and clearly state the solvent and ratio employed in the in vitro and in vivo assays.\nMoreover, the kinetics of photoisomerization for azo-Q2a are not reported. Providing the time course for both trans-to-cis and cis-to-trans conversions would significantly strengthen the photochemical characterization.\nRegarding chemical synthesis, it would be beneficial to present ^1H-NMR data confirming the structure of azo-Q2a in both trans and cis conformations.\nIn the main text (line 102), the authors state that Q1 and Q3 exhibit significantly higher inhibition than quinidine at 10 µM, as shown in Figure 1a. However, the data supporting this claim are not clearly presented. This is a potentially important observation, independent of optical control, and should be explicitly detailed.\nIn Figure 2, the isosbestic point shifts following illumination at 480 nm. The authors should comment on the origin of this shift, which is also observed in Supplementary Figure 1a, as it may reflect photoproduct formation or solvent effects.\nSupplementary Figure 2 provides a compelling analysis of sodium current inhibition following trans-to-cis conversion. However, to demonstrate reversibility, it is essential to show that subsequent cis-to-trans conversion restores the initial binding affinity. Furthermore, there is inconsistent nomenclature in the figure panels. For instance, panel a refers to azo-Q1a, while current traces are labeled \"azo-2p.\" It is unclear what \"azo-2p\" denotes, and consistent compound labeling must be maintained throughout the manuscript and supplemental figures, including line 148.\nIn Figure 3, the authors should clarify whether the IV and conductance curves were obtained after sequential illumination at 365 nm followed by 480 nm, or only after 480 nm exposure.\nThe authors appear to use the same electrophysiological conditions for sodium current recordings in HEK cells and isolated cardiomyocytes. However, the sodium current amplitude in cardiomyocytes often precludes proper voltage clamping under high extracellular Na⁺ conditions. The use of reduced Na⁺ concentrations (e.g., 20 mM) is standard to achieve adequate space clamp and reliable measurements. The authors should report the seal, access and series resistance criteria used in their recordings.\nIn Figure 4b, the authors show that azo-Q2a is less potent than quinidine on the hERG channel, a positive outcome. However, since the two isomers of azo-Q2a differ structurally, it is surprising that both exhibit nearly identical inhibition profiles. Supplementary Figure 3e shows superimposable concentration–response curves and IC₅₀ values for the cis and trans forms.\nThe legends should be corrected to refer to \"concentration\" (µM), not \"dose\" (mg/kg), for in vitro experiments.\nThe authors should also clarify whether illumination-only controls were performed on isolated cardiomyocytes. These cells are highly sensitive to UV-induced reactive oxygen species (ROS), which could confound interpretation of the data.\nAs sodium channel inhibition should decrease conduction velocity and shorten ventricular action potential duration, direct measurement of these parameters—preferably in neonatal cardiomyocytes or even in ex vivo hearts—would considerably strengthen the physiological relevance of the study.\nThe structural data presented in Figure 5 are promising, showing cryo-EM of Nav1.5 in complex with cis-azo-Q2a. However, the figure does not clearly depict the molecular interactions between azo-Q2a and the channel. It is also unclear which moieties of azo-Q2a engage in binding. Did the authors test whether quinidine-mediated inhibition is altered in V405A or F1760A mutants? Given that the two isomers of azo-Q2a exhibit different affinities, the authors should indicate whether cryo-EM structures were also obtained for the trans isomer. Such data would be highly informative regarding the structural basis of photoswitchable control. The authors suggest that V405 plays a minor role in optical control based on the small effect observed in the mutant, but this conclusion seems overstated given the limited evidence.\nThe zebrafish methods lack clarity. It is not clear whether the compounds were bath-applied or microinjected into the larvae. While the authors convincingly show that azo-Q2a can modulate heart rate in vivo, they do not include results for 100 µM quinidine, a concentration known to inhibit multiple ion channels. The lack of effect observed with quinidine is mentioned but not supported by citations. Moreover, the hypothesis that increasing selectivity toward sodium channels would enhance bradycardia is questionable and requires a more nuanced explanation.\nHave the authors tested whether repeated cycles of 365/480 nm illumination can reproducibly modulate heart rate in larvae? Demonstrating reversibility and repeatability is essential for potential translational applications.\nIn terms of pharmacokinetics, the in vivo stability of azo-Q2a must be addressed. Azobenzenes can undergo reductive degradation in biological environments; therefore, azo-Q2a sensitivity to GSH, a physiologically relevant reducing agent, should be evaluated.\nThe authors claim that azo-Q2a could be useful for treating Nav1.5-related arrhythmias. However, none of the current data support this application. Experiments demonstrating that light-induced isomerization can terminate arrhythmias—either in cell-based models or ex vivo heart preparations—would significantly increase the study's impact.\nFinally, the authors state (line 318) that red-shifted azobenzene analogs failed to retain optical control of Nav1.5. This is a key point and should be backed by experimental data. If these experiments were performed, the corresponding results must be provided, at least in the Supplementary Information.\nMinor comments:\n•There are multiple typographical and grammatical errors throughout the text and figure legends that need to be corrected.\n•It is not clear whether the authors used calculated or experimentally determined reversal potentials to derive ENa.\n•The rationale for choosing +20 mV to activate hERG channels is unclear. Higher test voltages typically provide more robust activation.\n•Line 241 incorrectly states that the IC₅₀ of the cis isomer decreased. In fact, the IC₅₀ increased, indicating reduced affinity.\n•Lines 303–304: capitalization errors need correction."}, "author_response": "We sincerely appreciate the reviewers for their time and thoughtful comments on our manuscript.\nIn response to their valuable feedback, we have conducted additional data analyses and performed\nfurther experiments to address all concerns raised. The manuscript has been thoroughly revised,\nwith all changes clearly highlighted. Below, we have provided a detailed point-by-point response to\nthe reviewers' comments. We believe these revisions have substantially strengthened our manuscript.\nPoint-by-point response to reviewers’ comments\nReviewer #1 (Remarks to the Author):\nIn this interesting manuscript, Shiqi Liu and co-workers reported a photoactive inhibitor of the\ncardiac sodium channel Na 1.5 based on the antiarrhythmic drug quinidine. They presented\nV\nelectrophysiology, cryo-EM structure and living zebrafish in vivo results, demonstrating that the\ninhibitory activity of azo-Q2a is photoswitchable. The manuscript is clearly written and the\nexperiments are well-performed. This reviewer has several suggestions concerning their figure\npresentation and the optical control mechanism, which, hopefully, could help the authors to clarify\ntheir manuscript. After revision, this reviewer supports its publication in Nature Communications.\nResponse: We sincerely thank the reviewer for the positive assessment of our work and the\nconstructive suggestions. We have carefully addressed each point raised, and we believe the\nrevisions have significantly improved the clarity and rigor of the manuscript. Our point-by-point\nresponses are detailed below.\nMajor concerns:\nQuestion:1. Quinidine is an open state blocker of Na 1.5, whether azo-Q2a is also an open state\nV\nblocker or not?\nResponse: We thank the reviewer for this insightful question regarding the mechanism of action of\nazo-Q2a. To determine whether it acts as an open-state blocker similar to quinidine, we evaluated\nthe inhibitory effects of cis-azo-Q2a on Na 1.5 at varying stimulation frequencies (0.625 Hz and 5\nV\nHz). The results revealed a substantial enhancement in inhibition at the higher frequency (5 Hz),\nindicative of a pronounced use-dependent effect (revised Supplementary Figure 6f). This frequency-\ndependent potentiation is a characteristic of open-state blockers, as it indicates the compound\npreferentially binds to the channel's open conformation, which is accessed more frequently during\nelevated stimulation rates. Therefore, we conclude that azo-Q2a functions as an open-state blocker\nof Na 1.5. We have revised our wording in the revised manuscript as following:\nV\n“At a concentration of 10 μM, cis-azo-Q2a exhibited potent inhibition of Na 1.5 (61.1 ±\nV\n4.8%, use-dependent, Supplementary Fig. 6f) but minimal activity (< 10%) against all other\nsubtypes tested.” (Page 7, Lines 190-192)\nSupplementary Figure 6f | Use-dependent inhibition of Na 1.5 by cis-azo-Q2a.\nV\nInhibitory effect of 10 μM cis-azo-Q2a on Na 1.5 currents at stimulation frequencies of 0.625 Hz\nV\nand 5 Hz (n = 6). Data are shown as mean ± SEM.\nQuestion: 2. Selectivity. I suggest the author to tune down the selectivity of azo-Q2a. First, it still\ninhibits hERG as shown in Fig. 4b (45% for hERG and 60% for Na 1.5); second, it is not known\nV\nthe efficiency of azo-Q2a for other sodium channel subtypes.\nResponse: We thank the reviewer for this insightful comment on the selectivity profile of azo-Q2a.\nWe have now emphasized the comparative improvement in functional selectivity relative to\nquinidine (approximately 11-fold increase). To eliminate ambiguity and prevent any misleading\ninterpretations, we have incorporated these quantitative comparisons and revised our wording in the\nrevised manuscript as following:\n“Notably, at 10 µM, cis-azo-Q2a inhibited Na 1.5 and hERG currents by 61.1% and 45.5%,\nV\nrespectively, yielding a Na 1.5/hERG inhibition ratio of 1.34, whereas quinidine produced 8.8%\nV\nand 71.7% inhibition (ratio 0.12). This corresponds to an approximately 11-fold increase in the\nNa 1.5/hERG inhibition ratio, indicating an overall enhancement in functional selectivity relative\nV\nto quinidine (Fig. 4b and Supplementary Table 5).” (Page 8-9, Lines 221-226)\nIn terms of the efficiency of azo-Q2a for other sodium channel subtypes, we performed a systematic\nevaluation using whole-cell patch-clamp electrophysiology (Data showed in Supplementary Figure\n7a). We assessed the inhibitory effects of both trans/cis-azo-Q2a, as well as quinidine, at 10 μM on\nsodium channel subtypes highly expressed in the central nervous system and muscle tissues\n(Na 1.2-1.6). Our results demonstrated that cis-azo-Q2a exhibited potent inhibitory activity against\nV\nNa 1.5 (61.1 ± 4.8%) while showing minimal inhibition (< 10%) against all other sodium channel\nV\nsubtypes tested. Furthermore, although trans-azo-Q2a exhibited negligible inhibition of Na 1.5 (2.5\nV\n± 1.6%) under dark conditions, it showed an increase inhibitory effect on Na 1.3 (36.2 ± 6.1%).\nV\nNotably, this effect could be effectively reversed by optical illumination, which switches the\ncompound to its cis-configuration (See figure below). These findings indicate a favorable light-\ninduced selectivity profile for azo-Q2a on Na 1.5. We have revised our wording in the revised\nV\nmanuscript as following:\n“To further evaluate the selectivity profile of azo-Q2a among other sodium channel subtypes\n(Na 1.2–Na 1.6) highly expressed in the central nervous system and muscle tissues (Supplementary\nV V\nFig. 7a). At a concentration of 10 μM, cis-azo-Q2a exhibited potent inhibition of Na 1.5 (61.1 ±\nV\n4.8%, use-dependent, Supplementary Fig. 6f) but minimal activity (< 10%) against all other\nsubtypes tested. In contrast, trans-azo-Q2a under dark conditions showed negligible suppression of\nNa 1.5 (2.5 ± 1.6%) yet significantly inhibited Na 1.3 (36.2 ± 6.1%). Notably, this off-target effect\nV V\non Na 1.3 was effectively reversed upon optical illumination, which switches the compound to cis-\nV\nconfiguration.\nThese results underscore the light-dependent selectivity of azo-Q2a for Na 1.5. Given that Na 1.5\nV V\nserves as the predominant sodium channel in cardiac tissue, spatially confined illumination enables\nthe high-affinity cis-azo-Q2a to selectively modulate heart function while minimizing potential off-\ntarget effects in neural or other systems.” (Page 7-8, Lines 188-201)\nMost importantly, given that Na 1.5 is the predominant sodium channel in the heart, targeted\nV\nillumination of cardiac tissue allows the high-affinity, light-generated cis-azo-Q2a to exert its\ninhibitory effect selectively in the heart, thereby minimizing potential off-target effects in the\nnervous system or other tissues.\nSupplementary Figure 7a | Selectivity profile of azo-Q2a on other voltage-gated sodium\nchannel subtypes.\nInhibition of Na 1.2-1.6 by 10 μM azo-Q2a (480 nm or 365 nm illumination, n = 6-11) and\nV\nquinidine (n = 6-7). Data are shown as mean ± SEM.\nQuestion:3. The EM complex structure supports the binding of an optical control blocker in Na 1.5.\nV\nHowever, the structure figure is not clearly presented. It’s difficult to interpret the precise\ninteractions between cis-azo-Q2a and the channel, and the detailed differences between cis-azo-\nQ2a and quinidine. For example, how confident to claim a carbon-hydrogen---π interaction between\nVal405 and the quinolone ring?\nResponse: We thank the reviewer for this insightful observation. To improve the visual clarity and\ninterpretive accuracy of the structural data, we have comprehensively revised Figure 5 by\nimplementing a ball-and-stick representation for both the azo-Q2a ligand and key surrounding\nresidues (e.g., F1760, V405), which more effectively illustrates atomic distances and coordination\ngeometry. We also sincerely apologize for misinterpreting the C–H–π interaction between Val405\nand the quinolone ring, which has now been corrected in the revised manuscript as follows:\n“cis-azo-Q2a is surrounded by multiple hydrophobic residues from the S6 segments (Fig. 5b-c),\namong which Val405 appears to interact with the quinolone aromatic ring through a hydrophobic\ninteraction (Fig. 5b-c), while Phe1760 plays an essential role connecting with the azobenzene group\nthrough π-π stacking (Fig. 5b-c).” (Page 11, Lines 288-292)\nQuestion: 4. EM map. The EM density in Fig. 5b appears to support the binding of cis-azo-Q2a.\nFor clarity, this reviewer suggests that the authors should present the density map of cis-azo-Q2a\nfrom three different views, as well as density maps for the S6 helices, because of conformational\nchanges (Fig. 5e).\nResponse: We thank the reviewer’s question. To clarity the binding of cis-azo-Q2a and Na 1.5, we\nV\nhave presented the local density for azo-Q2a and S6 in supplementary Figure 11.\nⅣ\nSupplementary Figure 11a-b | EM densities for the representative segments of Na 1.5S-azo-\nV\nQ2a.\na, The local densities for cis-azo-Q2a and sodium ions are presented from three different views. All\nEM densities presented here are contoured at 3 σ. b, Binding of azo-Q2a induces an α to π helix\ntransition in S6 . Shown here are the local densities for the helical turn of S6 which undergoes\nⅣ Ⅳ\nconformational change.\nQuestion:5. The author used a loop truncated version human Nav1.5S instead of full-length Nav1.5\nfor improved protein behavior, based on the construct of rNav1.5. Please provide reference.\nResponse: Thanks for the reviewer’s kind advice. The loop-truncated version of human Na 1.5S\nV\nwas designed based on the Na 1.5 structure first reported by Jiang D, et al (Cell. 2020;180(1):122-\nV\n134) and the Na 1.5-quinidine complex structure (Angew Chem Int Ed Engl. 2021;60(20):11474-\nV\n11480). Reference added into the revised manuscript:\n“30. Li, Z. et al. Structural Basis for Pore Blockade of the Human Cardiac Sodium Channel\nNa(v) 1.5 by the Antiarrhythmic Drug Quinidine*. Angew Chem Int Ed Engl 60, 11474-\n11480 (2021).\n31. Jiang, D. et al. Structure of the Cardiac Sodium Channel. Cell 180, 122-134.e10 (2020).”\n(Page 25, Line 714-717)\nQuestion: 6. It is not clear how Val405 contributes to the inhibitory activity of azo-Q2a but not\noptical control, while F1760 determines the optical control as interpreted in Lines 246-255. From\nthe results in Fig. 5f-h, the response of azo-Q2a to V405A and F1760A looks similar under darkness\nor 365 nm light.\nResponse: We thank the reviewer for this constructive comment. Our integrated analysis, combining\ncryo-EM structural data and electrophysiological results, reveals that both residues are essential for\nhigh-affinity binding and light-dependent inhibition. Specifically, the quinoline aromatic ring of cis-\nazo-Q2a engages in hydrophobic interactions with Val405, while its azobenzene group participates\nin π–π stacking with Phe1760 (Fig. 5b–c). Consistent with these structural observations, whole-cell\npatch-clamp recordings showed that mutations V405A and F1760A resulted in a significant\nrightward shift of the IC curve for cis-azo-Q2a (Fig. 5g–h), indicating a substantial reduction in\n50\nits inhibitory potency. Collectively, these findings confirm that Val405 and Phe1760 are critical\nresidues for the binding and inhibition of Na 1.5 by cis-azo-Q2a, thereby establishing them as\nV\nessential molecular determinants for the light-dependent control mediated by azo-Q2a. We have\nrevised our wording in the revised manuscript as following:\n“Both mutations V405A and F1760A resulted in a significant rightward shift of the IC curve for\n50\ncis-azo-Q2a (IC > 100 μM, Fig. 5f–h and Supplementary Table 8), indicating a substantial\n50\nreduction in its inhibitory potency. Collectively, these findings confirm that Val405 and Phe1760 are\ncritical residues for the binding and inhibition of Na 1.5 by cis-azo-Q2a, thereby establishing them\nV\nas essential molecular determinants for the light-dependent control mediated by azo-Q2a.” (Page\n11, Line 305-310)\nQuestion: 7. The optical control mechanism is unclear. Is it because the cis form of azo-Q2a prefers\nbinding in the identified pocket in Nav1.5 while the trans form could cause steric clashes with the\nchannel, or because of different accessibility for the two forms? The authors should dock the\ntrans form in the pocket and discuss possible molecular mechanism for optical control.\nResponse: We thank the reviewer for this insightful comment. To gain insight into the potential\noptical control mechanism of azo-Q2a, we performed both unconstrained and constrained docking\nfor trans‑azo‑Q2a. In the absence of spatial constraints, the predicted binding poses of the trans\nisomer were displaced from the well-resolved cis-azo-Q2a binding site, revealing diverse\nconformations (trans1–3 in Supplementary Fig. 11c). These results indicate that trans-Q2a is unable\nto adopt a stable, low-energy conformation within the Na 1.5 binding pocket, thereby preventing\nV\neffective interaction with the channel. When docking was performed with the pose constrained to\nmatch that of cis-azo-Q2a, the principal divergence between the two binding modes was localized\nto the azobenzene moiety (trans4 in Supplementary Fig. 11c). In addition, the cryo-EM map shows\nthat the local density for the azobenzene group in the docked trans isomer is poor and that it lacks\nstable interactions with the surrounding residues (Supplementary Fig. 11d). Such structural\nplasticity account for the different binding affinities between cis and trans isomers of azo-Q2a for\nNa 1.5. We have revised our wording in the revised manuscript as following:\nV\n“To gain insight into the potential optical control mechanism of azo-Q2a, we docked trans-azo-Q2a\ninto the Na 1.5 structure. In the absence of spatial constraints, the predicted binding poses of the\nV\ntrans isomer were displaced from the well-resolved cis-azo-Q2a binding site (Supplementary Fig.\n11b). When docking was performed with the pose constrained to match that of cis-azo-Q2a, the\nprincipal divergence between the two binding modes was localized to the azobenzene moiety\n(Supplementary Fig. 11c). In addition, the cryo-EM map shows that the local density for the\nazobenzene group in the docked trans isomer is poor and that it lacks stable interactions with the\nsurrounding residues (Supplementary Fig. 11d). Such structural plasticity may account for the\ndifferent binding affinities between cis and trans isomers of azo-Q2a for Na 1.5.“(Page 14, Line\nV\n372-381)\nSupplementary Figure 11c-d | EM densities for the representative segments of Na 1.5S-azo-\nV\nQ2a.\nc, The docking results of trans-azo-Q2a are shown. Trans1–3 represent unconstrained docking\nposes of trans-azo-Q2a in the central cavity of Na 1.5, while trans4 represents the pose-constrained\nV\ndocking in which the ligand orientation was aligned to that of cis-azo-Q2a. The docking scores and\npredicted ΔΔG binding energies are shown in the table. The trans-azo-Q2a molecules are colored\naccording to their docked binding poses. d, The corresponding EM density for the modeled trans-\nazo-Q2a. The density for the azobenzene group in the trans isomer is poor, as indicated by the red\ncircle.\nMinor points:\nQuestion: In Line 187, Supplementary Fig. 3d should be Supplementary Fig. 3e?\nResponse: Thank you for pointing this out. Supplementary Fig. 3d has been corrected to\nSupplementary Fig. 7f.\nQuestion: In Line 148, what is azo-2P, azo-Q2a, and azo-6M? These names are not consistent in\nFig. 3 and Supplementary Fig. 2.\nResponse: Thank you for pointing this out. The inconsistency was due to a name change of the\ncompounds. We have now updated the text to be consistent with the nomenclature used in the main\nfigures.\nQuestion: In Lines 133, 142, 275 and others, 365nm should be 365 nm.\nResponse: Thanks for pointing out this issue. We have revised “365nm” to “365 nm” throughout\nthe manuscript.\nQuestion: In Line 305, Inhibitors should be inhibitors.\nResponse:Thank you for pointing this out. “Inhibitors” has been corrected to “inhibitors”.\nQuestion: In Line 458, what is “Na 1.5Short-azo-Q2a”? Double-check other typos.\nV\nResponse: Thank you for noticing this. The term should refer to Na 1.5S as mentioned earlier. We\nV\nhave corrected it and unified the nomenclature throughout the manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors present a photoswitchable quinidine derivative, azo-Q2a, targeting the cardiac sodium\nchannel NaV1.5. While NaV1.5 is essential for cardiac depolarization and quinidine has long-\nstanding utility as an antiarrhythmic agent, its poor specificity and off-target effects—particularly\nhERG inhibition—limit its clinical use. Through the synthesis of azobenzene–quinidine derivatives,\nthe authors identify azo-Q2a as a lead compound with reversible, light-dependent activity: it is\nhighly potent in its cis form (activated by 365 nm light) and largely inactive in its trans form (480 nm\nlight or dark), demonstrating approximately a 7-fold difference in efficacy. Azo-Q2a exhibits strong\nselectivity for NaV1.5 over other cardiac ion channels and significantly reduces hERG-related\ncardiotoxicity relative to quinidine. Cryo-EM structural analysis reveals key binding interactions\nwith NaV1.5, notably involving residues Phe1760 and Val405. In vivo, azo-Q2a enables reversible,\nlight-mediated modulation of heart rate in zebrafish larvae, underscoring its potential as a research\ntool and therapeutic lead for arrhythmia.\nResponse: We sincerely appreciate the reviewer's positive feedback on the achievements presented\nin this manuscript. We would like to further highlight the key novel aspects of our work, which\ninclude:\n1. The first synthesis of a series of new photoswitchable Na 1.5 channel blockers derived\nV\nfrom quinidine;\n2. A unique photoswitchable inhibition behavior between the cis-active and trans-inactive\nforms of azo-Q2a againstNa 1.5 but no other channels, by exhibiting a 7-fold difference\nV\nin activity;\n3. The first Cryo-EM structure of Na 1.5 in complex with the cis-active isomer azo-Q2a,\nV\nwhich reveals key binding interactions and paves the way for designing next-generation\nphotoswitchable Na 1.5 channel blockers.\nV\nDespite these strengths, the manuscript notably fails to acknowledge significant prior work in this\narea. In particular, the study omits reference to “Optical Control of Cardiac Electrophysiology by\nthe Photochromic Ligand AzoBupivacaine 2” (Fehrentz et al., Br. J. Pharm., 2025, 82, 1125–1142),\nwhich introduced azobupivacaine (AB2) as a reversible photoswitchable Na 1.5 blocker. That\nV\nearlier work demonstrated optical control of cardiac function in more advanced mammalian\nmodels—including mouse left ventricular slices and Langendorff-perfused hearts—and achieved\noptical termination of ventricular tachycardia. Conceptually, the current study does not extend\nmeaningfully beyond these findings. While the zebrafish data are elegant, they do not surpass the\nphysiological relevance achieved with AB2. The cryo-EM structural data are novel and represent a\nvaluable contribution to mechanistic understanding and future drug design. However, in isolation,\nthey may not justify publication in a high-profile venue, particularly given that azo-Q2a’s action\nspectrum provides no clear advantage over that of AB2.\nResponse: We thank the reviewer for bringing this reference to our attention and appreciate their\nperspective on its relevance. However, we respectfully suggest that the interpretation of the study\n“Optical Control of Cardiac Electrophysiology by the Photochromic Ligand AzoBupivacaine 2” (Br\nJ Pharmacol. 2025;182(5):1125-1142) may mislead certain aspects, as outlined below:\n1. Lack of structural novelty: The compound azobupivacaine (AB2) featured in the\nreferenced study is not a novel entity; it was previously introduced by the Trauner group\n(ACS Chem Neurosci. 2018;9(12):2886-2891).\n2. Lack of channel selectivity: As demonstrated in both Leippe et al. (ACS Chem Neurosci.\n2018;9(12):2886-2891) and Fehrentz et al., AB2 exhibits concurrent photoswitchable\nactivity across various channels (e.g., K 2.1, TREK1, Na 1.5), indicating limited\nV V\nselectivity under light irradiation. Specifically, Fehrentz et al. reported only a 1.4-fold\nphotoswitching effect on Na 1.5, which prompts questions about the compound's practical\nV\neffectiveness as a selective photoswitchable modulator for this channel.\nIn summary, while the work by Fehrentz et al. represents a valuable attempt to explore\nphotoswitchable Na 1.5 blockers using AB2, the compound’s limited photoswitching performance\nV\nacross different channels (K 2.1, TREK1, and Na 1.5) and between its cis and trans isomers\nV V\nunderscores the challenges in developing highly effective photoswitchable Na 1.5 blockers. This\nV\nhighlights the need for novel compound designs and a deeper mechanistic understanding of\nphotoswitching behavior. We are pleased that our current work addresses these challenges and\ncontributes to the rational design of photoswitchable Na 1.5 channel blockers.\nV\nIn summary, without a thorough acknowledgment of prior work and a demonstrable advance in\nbiological scope or translational relevance, this manuscript is not suitable for publication in its\ncurrent form in Nature Communications.\nReviewer #3 (Remarks to the Author):\nIn their manuscript, Liu et al. describe the azologization of quinidine and assess the optical control\nof their most promising analogue, azo-Q2a, both in vitro and in vivo in zebrafish larvae. The\napproach is scientifically compelling. However, previous studies have largely reported similar\nstrategies and outcomes on other channels or receptors, which reduces the novelty of the current\nwork. To be considered for publication in Nature Communications, several critical issues must be\naddressed.\nResponse: We sincerely appreciate your recognition of our work and your thorough reviews. We\nbelieve the primary contribution of this study lies in the first synthesis of a series of new\nphotoswitchable Na 1.5 channel blockers derived from quinidine. Unlike previous studies on other\nV\nchannels, our approach is guided by the resolved cryo-EM structure of quinidine bound to Na 1.5,\nV\nthe predominant cardiac sodium channel, which facilitated a structure-based design aimed at\nachieving optical control of heart rate.\nOur optimized compound, azo-Q2a, exhibits promising photoswitching behavior and reversibility\n(~ 7 fold), as validated consistently in both primary cells and zebrafish models. Furthermore, we\nresolved the cryo-EM complex of azo-Q2a with Na 1.5, revealing key interacting residues and\nV\nproviding mechanistic insights that may inform the development of next-generation\nphotoswitchable Na 1.5 inhibitors.\nV\nThese integrated efforts in rational design, functional validation, and structural elucidation highlight\nthe potential novelty and translational potential of our study. Below we provide a point-by-point\nresponse to your constructive suggestions and comments.\nQuestion: First, in the experimental section, essential information is missing regarding the light\nintensity used for photoisomerization. Since the efficiency of azobenzene switching is intensity-\ndependent, the authors must report the irradiance used and the quantum yields for both trans-to-cis\nand cis-to-trans conversions. These details are crucial for the reproducibility of the study.\nResponse: We thank the reviewer for this important comment. To determine the quantum yields of\nazo-Q2a, we used azobenzene (365 nm) and 1,2-bis (2-methyl-1-benzothiophen-3-yl)-\nperfluorocyclopentene (480 nm) as reference compounds (Nat Chem. 2023;15(9):1285-1295) The\ncorresponding results are presented in Supplementary Figure 12 and Supplementary Table 9. In\naddition, the light intensities were measured using a digital lux meter. The detailed values have been\nincorporated into our revised manuscript to ensure reproducibility:\n“Furthermore, the photoisomerization rates of azo-Q2a were measured. Under 365 nm UV light at\n8500 lux, the half-life was 28 min, whereas under 480 nm blue light at 1500 lux, the half-life was\n3.5 min. The quantum yields of azo-Q2a were determined to be 0.075 under 365 nm irradiation and\n0.32 under 480 nm irradiation. These results demonstrate that azo-Q2a exhibits efficient and\ncontrollable photoreactive behavior across different irradiation wavelengths29 (Supplementary Fig.\n4 and Supplementary Table 1).” (Page 6, Line 148-154)\n“In order to determine the photoisomerization isomerization kinetics in CDCl at 100 mM solution\n3\nof azo-Q2a was irradiated using 3W LED lamps (365 and 480 nm peak emission) until no further\nchange was observable by 1H NMR. The irradiance of LED is 8500 lux for 365 nm and 1500 lux for\n480 nm.” (Page 39, Line 934-938)\nSupplementary Figure 4 | Kinetics of photoisomerization for azo-Q2a.\na-b, Shown are the ln (cis% or trans% isomer) to time (min). In order to determine the\nphotoisomerization isomerization kinetics in CDCl at 100 mM solution of azo-Q2a was irradiated\n3\nusing 3W LED lamps (365 and 480 nm peak emission) until no further change was observable by\n1H NMR. The irradiance of LED is 8500 lux for 365 nm and 1500 lux for 480 nm.\nSupplementary Table 1 | Quantum yields for photoisomerization of ionized azo-Q2a in CDCl .\nstandard azo-Q2a standard azo-Q2a\n365 0.04864 0.02475 0.15 0.075\n480 1.834 0.1980 0.30 0.32\nQuestion: Second, the UV-Vis spectra presented in Figure 2 were obtained in MeOH:H₂O, while\nthose in Supplementary Figure 1 were acquired in DMSO. The rationale behind the use of different\nsolvents is not provided. This inconsistency complicates the comparison of isomerization efficiency.\nThe authors should use the same solvent system across experiments and clearly state the solvent\nand ratio employed in the in vitro and in vivo assays.\nResponse: We thank the reviewer for highlighting this important issue regarding solvent consistency.\nTo address this, we have remeasured the UV-Vis spectra of the six compounds (azo-Q1a to Q3b) in\na uniform solvent system of 100 µM DMSO/H O (1:100, v/v). All compounds exhibited behavior\n2\ncharacteristic of regular azobenzenes, isomerizing from their thermally stable trans-configuration\nto the cis-configuration upon irradiation with 365 nm light, with the process being reversible under\n480 nm light. These results are now presented in Supplementary Figure 1.\nSupplementary Figure 1c-g | Inhibition rate of Na 1.5 by Q1-3 and UV-Vis absorption spectra\nV\nof azo-QDs.\nc-g, Shown are the UV-Vis spectrum of azo-QDs (100 μM in DMSO/H2O = 1/100, rt), in darkness\n(black, trans), 365 nm light (violet, cis) and 365 nm then 480 nm light (blue, trans) illuminations.\nc, azo-Q1a. d, azo-Q3a. e, azo-Q1b. f, azo-Q2b. g, azo-Q3b.\nQuestion: Moreover, the kinetics of photoisomerization for azo-Q2a are not reported. Providing the\ntime course for both trans-to-cis and cis-to-trans conversions would significantly strengthen the\nphotochemical characterization.\nResponse: We appreciate this insightful comment. During the measuring of quantum yields,\nwe\nhave complemented the t of the photochemical characterization. Data is reported in revised\n1/2\nSupplementary Figure 4. We have revised our wording in the revised manuscript as following:\n“Furthermore, the photoisomerization rates of azo-Q2a were measured. Under 365 nm UV light at\n8500 lux, the half-life was 28 min, whereas under 480 nm blue light at 1500 lux, the half-life was\n3.5 min.” (Page 6, Line 148-150)\nSupplementary Figure 4 | Kinetics of photoisomerization for azo-Q2a.\na-b, Shown are the ln (cis% or trans% isomer) to time (min). In order to determine the\nphotoisomerization isomerization kinetics in CDCl at 100 mM solution of azo-Q2a was irradiated\n3\nusing 3W LED lamps (365 and 480 nm peak emission) until no further change was observable by\n1H NMR. The irradiance of LED is 8500 lux for 365 nm and 1500 lux for 480 nm.\nQuestion: Regarding chemical synthesis, it would be beneficial to present ^1H-NMR data\nconfirming the structure of azo-Q2a in both trans and cis conformations.\nResponse: We apologize for omitting this in the initial submission, and the NMR data has now been\nattached as a new supplementary document (Page 65-87, line 1358-1381). All new compounds are\nnow reported. Particularly, both the 1H and 13C NMR spectra of trans and cis-azo-Q2a have been\nincluded (Supplementary Figure 2-3).\nSupplementary Figure 2 | Full 1H-NMR spectra of azo-Q2a irradiated with different\nwavelengths.\na, Shown are the 1H-NMR spectrum of azo-Q2a (CDCl ), in the dark-adapted (black), UV-A-\n3\nadapted (360 nm, gray) and blue-adapted (480 nm, blue) states. All compounds behaved as regular\nazobenzenes, and could be isomerized from their thermally stable trans-configuration to the cis-\nconfiguration with 365 nm light. 480 nm irradiation reversed this effect. Meanwhile, both the trans\nand cis isomers were not pure system. From synthesis and column chromatography, 18% cis isomer\nwas residual in trans. By UV irradiation, only 81% of trans isomer could be translated to cis, while\n24% cis isomer was left by irradiation of 480 nm.\nSupplementary Figure 3 | Full 13C-NMR spectra of azo-Q2a irradiated with different\nwavelengths.\na, b, Shown are the 13C-NMR spectrum of azo-Q2a (CDCl ), between cis (blue) and trans (red)\n3\nisomer. Among these peaks, 131.9, 129.4 and 123.2 are peaks belong to trans isomer, while 129.0,\n127.7 and 120.8 belong to cis.\nQuestion: In the main text (line 102), the authors state that Q1 and Q3 exhibit significantly higher\ninhibition than quinidine at 10 µM, as shown in Figure 1a. However, the data supporting this claim\nare not clearly presented. This is a potentially important observation, independent of optical control,\nand should be explicitly detailed.\nResponse: We thank the reviewer for this valuable suggestion. In the revised manuscript, we have\nincorporated the explicit inhibition values (quinidine: 8.8 ± 2.9%, Q1: 70.0 ± 2.3%, Q2: 17.6 ± 2.8%,\nQ3: 46.0 ± 6.7%) in the main text and added statistical diagram in Supplementary Figure 1 to more\nclearly quantify this observation. We have revised our wording in the revised manuscript as\nfollowing:\n“Remarkably, whole-cell patch-clamp recordings in HEK293T cells heterologously expressing\nNa 1.5 channels revealed that Q1 and Q3 exhibited significantly higher inhibition rates than\nV\nquinidine at 10 µM concentration (8.8 ± 2.9% for quinidine, 70.0 ± 2.3% for Q1, and 46.0 ± 6.7%\nfor Q3, Figure 1b and Supplementary Fig. 1b).” (Page 5, Line 112-115)\nSupplementary Figure 1a-b | The inhibition rates of quinidine and Q1-3 on Na 1.5 channels.\nV\na, Chemical structure of quinidine and Q1-3. b, The Na 1.5 inhibition (10 μM) of quinidine (gray)\nV\nand quinidine derivatives Q1 (black), Q2 (red) and Q3 (green) through whole-cell patch clamp\nrecording, n = 3-7.\nQuestion: In Figure 2, the isosbestic point shifts following illumination at 480 nm. The authors\nshould comment on the origin of this shift, which is also observed in Supplementary Figure 1a, as\nit may reflect photoproduct formation or solvent effects.\nResponse: We sincerely appreciate the reviewer’s thoughtful comment and suggestion. We note that\nthe isosbestic point in Figure 2 shifts slightly upon illumination at 480 nm. Such shifts have been\nreported in the literature when (i) there are minor side‐ or photoproducts being formed (Anal\nBiochem.2000;287(2):329-333) or (ii) changes in solvent polarity, ionic strength, or local\nenvironment affect the absorption spectra (Journal of Molecular Structure.2020;1200:127125). We\nhave confirmed that the spectral changes are reproducible and that the shift in the isosbestic point\nis small relative to the main spectral features. Therefore, we conclude that while such effects exist,\nthey do not materially affect our interpretation of the primary photo-conversion pathway.\nQuestion: Supplementary Figure 2 provides a compelling analysis of sodium current inhibition\nfollowing trans-to-cis conversion. However, to demonstrate reversibility, it is essential to show that\nsubsequent cis-to-trans conversion restores the initial binding affinity. Furthermore, there is\ninconsistent nomenclature in the figure panels. For instance, panel a refers to azo-Q1a, while\ncurrent traces are labeled \"azo-2p.\" It is unclear what \"azo-2p\" denotes, and consistent compound\nlabeling must be maintained throughout the manuscript and supplemental figures, including line\n148.\nResponse: We thank the reviewer for this valuable comment. We agree with the reviewer's points\nand have conducted detailed measurements of the reversibility of Na 1.5 inhibition for the optimal\nV\ncompound azo-Q2a, under both trans-to-cis and cis-to-trans photoisomerization conditions.\nSpecifically, Fig. 3g shows representative Na 1.5 current traces under alternating 365 nm and 480\nV\nnm light, while Fig. 3h quantifies the peak currents across multiple cycles, confirming the\nreversibility of inhibition. Then, in Fig. 4d-e we demonstrated the reversibility of azo-Q2a in\ncardiomyocytes. However, for the other compounds screened in Supplementary Figure 6, we\ndetermined that further detailed testing was not warranted due to their poor photocontrol activity\nduring trans-to-cis conversion. Furthermore, we have modified the description. (From\n“Azobenzene-quinidines reversibly inhibit Na 1.5 channels with light in HEK293T Cells.” to\nV\n“Inhibition rate of Na 1.5 by azo-QDs in the two photoisomeric states”.)\nV\nRegarding the second point, we apologize for the inconsistency in nomenclature. This arose from\ncompound renaming during revisions, and we have carefully cross-checked the entire manuscript\nand supplemental figures to ensure that all compound names are now consistent.\nQuestion: In Figure 3, the authors should clarify whether the IV and conductance curves were\nobtained after sequential illumination at 365 nm followed by 480 nm, or only after 480 nm exposure.\nResponse: We thank the reviewer for pointing this out. To clarify, the I-V and conductance curves\nfor Na 1.5 channels were obtained under illumination at either 365 nm or 480 nm separately,\nV\nwithout sequential exposure. We have incorporated this detail into the revised manuscript:\n“I–V (e) and g–V (f) relationship in 10 µM azo-Q2a under only 365 nm (n = 15) or 480 nm (n = 12)\nillumination and without azo-Q2a (control, n = 49).” (Page 31, Line 842-844)\nQuestion: The authors appear to use the same electrophysiological conditions for sodium current\nrecordings in HEK cells and isolated cardiomyocytes. However, the sodium current amplitude in\ncardiomyocytes often precludes proper voltage clamping under high extracellular Na⁺ conditions.\nThe use of reduced Na⁺ concentrations (e.g., 20 mM) is standard to achieve adequate space clamp\nand reliable measurements. The authors should report the seal, access and series resistance criteria\nused in their recordings.\nResponse: We thank the reviewer for this constructive opinions. For the electrophysiological\nconditions in primary cardiomyocytes, we referred to some publications, that employed high\nextracellular Na⁺ concentrations for recording sodium currents in primary cardiomyocytes, similar\nto those used in HEK cells. (a, Nat Commun. 2016;7:12035; b, J Clin Invest. 2010;120(1):369-378).\nMoreover, to explore the influence of Na+ concentration on the inhibitory efficacy of azo-Q2a in\nthe primary cardiomyocytes, we reduced the sodium concentration in the bath solution to 20 mM\nand reassessed the inhibition by cis-azo-Q2a on Na 1.5 (as shown in the figure below). The results\nV\ndemonstrated that the inhibitory rate of cis-azo-Q2a under the 20 mM sodium ion condition (41.4\n± 56.0%) was consistent with that observed previously under high sodium solution condition (42.9\n± 6.9 %). This result indicating that Na⁺ concentration did not affect the observed pharmacological\neffects.\nIn addition, all recordings were accepted only when series resistance was <6 MΩ and compensated\nby 70%, and seal resistance exceeded＞1 GΩ. To further ensure data reliability, we maintained a\nrelatively high number of replicates (n) in all experiments, minimizing the impact of any occasional\nvariations in seal quality. These criteria and details have been added to the Methods section:\n“In addition, all recordings were accepted only when series resistance was <6 MΩ and\ncompensated by 70%, and seal resistance exceeded＞1 GΩ.” (Page 16, Line 435-437)\nEffect of extracellular Na⁺ concentration on cis-azo-Q2a inhibition in cardiomyocytes.\nInhibition of Na 1.5 by cis-azo-Q2a(10 μM) with 140 mM or 20 mM extracellular Na⁺, showing\nV\nno significant difference (n = 6-7). Data are shown as mean ± SEM.\nQuestion: In Figure 4b, the authors show that azo-Q2a is less potent than quinidine on the hERG\nchannel, a positive outcome. However, since the two isomers of azo-Q2a differ structurally, it is\nsurprising that both exhibit nearly identical inhibition profiles. Supplementary Figure 3e shows\nsuperimposable concentration–response curves and IC₅₀ values for the cis and trans forms.\nResponse: We thank the reviewer for this insightful comment. The similar hERG inhibition profiles\nobserved for the cis and trans isomers of azo-Q2a can be attributed to fundamental structural\ndifferences between the Na 1.5 and hERG channels. As our cryo-EM data reveal (Fig. 5), the light-\nV\ndependent activity of azo-Q2a on Na 1.5 relies on specific interactions with key pore residues\nV\nF1760 and V405. Since the isomers differ only in the configuration of this azobenzene group, while\nsharing an identical quinidine-derived pharmacophore core, the hERG channel which lacks an\nanalogous structural motif thus cannot distinguish between them. Consequently, we assume that\nhERG inhibition is driven primarily by the conserved core, resulting in superimposable dose-\nresponse curves. This outcome highlights a key advantage of azo-Q2a: it delivers highly targeted\nphotocontrol by activating only on channels with the requisite molecular determinants, thereby\nminimizing off-target effects.\nQuestion: The legends should be corrected to refer to \"concentration\" (µM), not \"dose\" (mg/kg),\nfor in vitro experiments.\nResponse: We thank the reviewer for this correction. We have carefully revised the manuscript and\ncorrected all instances to use “concentration” instead of “dose” for in vitro experiments.\nQuestion: The authors should also clarify whether illumination-only controls were performed on\nisolated cardiomyocytes. These cells are highly sensitive to UV-induced reactive oxygen species\n(ROS), which could confound interpretation of the data.\nResponse: We thank the reviewer for this important comment. To address the potential effects of\nillumination, particularly UV light, on myocardial cells during the experiment, we utilized quinidine\nas a photoswitchable negative control. Under identical UV illumination conditions, the UV exposure\ngroup did not significantly alter quinidine's inhibitory effects (Fig. 4f-g). In addition, we included\nan illumination-only control group with 365 nm exposure for 90 s (longer than the drug application\ntime) and found that illumination alone did not alter the sodium current, indicating that light\nexposure itself does not interfere with the drug’s inhibitory effect (Supplementary Figure 8a). We\nhave revised our wording in the revised manuscript as following:\n“To further exclude potential effects of UV-induced ROS, we performed illumination-only controls\n(Supplementary Fig. 8a), which showed no changes in sodium currents, consistent with the\nunchanged inhibition of quinidine under both wavelengths.” (Page 9-10, Line 251-254)\nSupplementary Figure 8a | Illumination-only control in cardiomyocytes.\nInhibition of sodium current in primary cardiomyocytes by 10 μM cis-azo-Q2a and by 365 nm\nillumination-only for 90 s. (n = 6-7). Data are shown as mean ± SEM.\nQuestion: As sodium channel inhibition should decrease conduction velocity and shorten\nventricular action potential duration, direct measurement of these parameters—preferably in\nneonatal cardiomyocytes or even in ex vivo hearts—would considerably strengthen the\nphysiological relevance of the study.\nResponse: We thank the reviewer for this important suggestion. To directly evaluate the effect on\ncardiac electrophysiology, we performed whole-cell current-clamp recordings on primary\ncardiomyocytes to assess the impact of trans and cis-azo-Q2a on cardiac action potential. Our\nresults demonstrated that cis-azo-Q2a significantly reduced the depolarization rate by 15.8 ± 2.0%,\nwhereas trans-azo-Q2a showed no significant effect. Furthermore, we observed that cis-azo-Q2a\nshortened the action potential duration at 50% repolarization (APD ) by 26.1 ± 6.2%. We attribute\n50\nthis shortening primarily to the inhibition of the late sodium current (I ), which is known to\nNa, L\nmodulate repolarization (Supplementary Figure 8b-d). We have revised our wording in the revised\nmanuscript as following:\n“Owing to the significant role of Na 1.5 in the action potential of myocardial cells, we directly\nV\nevaluate the effect of azo-Q2a on cardiac electrophysiology performing whole-cell current-clamp\nrecordings on primary cardiomyocytes to assess the impact of trans and cis-azo-Q2a on cardiac\naction potential. (Supplementary Fig. 8b-d). The results demonstrated that cis-azo-Q2a significantly\nreduced the depolarization rate by 15.8 ± 2.0%, whereas trans-azo-Q2a showed no significant effect.\nFurthermore, we observed that cis-azo-Q2a shortened the action potential duration at 50%\nrepolarization (APD ) by 26.1 ± 6.2%. We attribute this shortening primarily to the inhibition of\n50\nthe late sodium current (I ), which is known to modulate repolarization.” (Page 10, Line 255-\nNa, L\n263)\nSupplementary Figure 8b-d | Effect of cis-azo-Q2a on action potential duration in primary\ncardiomyocytes.\nThe influence of cardiac action potential in primary cardiomyocytes by trans and cis-azo-Q2a. b,\nRepresentative action potential traces under control (black), cis-azo-Q2a (violet), and trans-azo-\nQ2a (blue) conditions. c, Quantification of the inhibition rate of the depolarization rate (n = 3). d,\nPercentage inhibition of APD by cis-azo-Q2a and trans-azo-Q2a (n = 3–9). Data are shown as\n50\nmean ± SEM.\nQuestion: The structural data presented in Figure 5 are promising, showing cryo-EM of Na 1.5 in\nV\ncomplex with cis-azo-Q2a. However, the figure does not clearly depict the molecular interactions\nbetween azo-Q2a and the channel. It is also unclear which moieties of azo-Q2a engage in binding.\nDid the authors test whether quinidine-mediated inhibition is altered in V405A or F1760A mutants?\nGiven that the two isomers of azo-Q2a exhibit different affinities, the authors should indicate\nwhether cryo-EM structures were also obtained for the trans isomer. Such data would be highly\ninformative regarding the structural basis of photoswitchable control. The authors suggest that\nV405 plays a minor role in optical control based on the small effect observed in the mutant, but this\nconclusion seems overstated given the limited evidence.\nResponse: We thank the reviewer for acknowledging our structural data and for this insightful\ncomment. To enhance the clarity of molecular interactions, we have revised the figure by adopting\na ball-and-stick model for azo-Q2a and key Na 1.5 channel residues to better illustrate their binding\nV\npose. Furthermore, we assessed the inhibitory effect of 100 μM quinidine on the V405A and F1760A\nmutants using whole-cell patch-clamp recordings. The results demonstrated that the functional\nimpairment was more pronounced in the F1760A mutant compared to the WT, with inhibition rates\nof 77.6±3.2 % (WT), 51.2±2.3% (V405A), and 32.6±1.8% (F1760A), (Supplementary Figure 7g).\nThis finding is consistent with previous cryo-EM structural analyses, which revealed that the\nquinoline ring of quinidine forms critical interactions with the F1760 residue (Angew Chem Int Ed\nEngl. 2021;60(20):11474-11480). We have revised our wording in the revised manuscript as\nfollowing:\n“The results demonstrated that the functional impairment was more pronounced in the F1760A\nmutant compared to the WT, with inhibition rates of 77.6 ± 3.2 % (WT), 51.2 ± 2.3% (V405A), and\n32.6 ± 1.8% (F1760A), (Supplementary Fig. 7g). This finding is consistent with previous cryo-EM\nstructural analyses, which revealed that the quinoline ring of quinidine forms critical interactions\nwith the F1760 residue30.” (Page 11, Line 298-303)\nFurther, based on the cryo‑EM maps, we could not ascertain the presence of trans‑azo‑Q2a.\nNevertheless, we performed both unconstrained and constrained docking for trans‑azo‑Q2ainto our\nstructure suggested poor coordination of the azobenzene group. The unconstrained docking revealed\ndiverse binding poses, whereas the constrained docking yielded a binding pose similar to that of\ncis‑azo‑Q2a. Consistent with the EM density, we infer that the lower binding affinity of\ntrans‑azo‑Q2a may arise from reduced interactions with the azobenzene group, as shown in\nSupplementary Figure 11c-d. We have revised our wording in the revised manuscript as following:\n“To gain insight into the potential optical control mechanism of azo-Q2a, we docked trans-azo-Q2a\ninto the Na 1.5 structure. In the absence of spatial constraints, the predicted binding poses of the\nV\ntrans isomer were displaced from the well-resolved cis-azo-Q2a binding site (Supplementary Fig.\n11b). When docking was performed with the pose constrained to match that of cis-azo-Q2a, the\nprincipal divergence between the two binding modes was localized to the azobenzene moiety\n(Supplementary Fig. 11c). In addition, the cryo-EM map shows that the local density for the\nazobenzene group in the docked trans isomer is poor and that it lacks stable interactions with the\nsurrounding residues (Supplementary Fig. 11d). Such structural plasticity may account for the\ndifferent binding affinities between cis and trans isomers of azo-Q2a for Na 1.5.” (Page 14, Line\nV\n372-381)\nMoreover, combining cryo-EM structural data and electrophysiological results, we clarify that while\nboth Phe1760 and Phe405 residues are essential for high-affinity binding and light-dependent\ninhibition. The quinoline aromatic ring of cis-azo-Q2a engages in hydrophobic interactions with\nV405, while its azobenzene group participates in π–π stacking with F1760 (Fig. 5b–c). Consistent\nwith these structural observations, whole-cell patch-clamp recordings showed that mutations V405A\nand F1760A resulted in a significant rightward shift of the IC curve for cis-azo-Q2a (Fig. 5g–h),\n50\nindicating a substantial reduction in its inhibitory potency. Collectively, these findings confirm that\nV405 and F1760 are critical residues for cis-azo-Q2a binding and inhibition of Na 1.5, thereby\nV\ndefining them as essential molecular determinants for the light-dependent control mediated by azo-\nQ2a. We have revised our wording in the revised manuscript as following:\n“Both mutations V405A and F1760A resulted in a significant rightward shift of the IC curve for\n50\ncis-azo-Q2a (IC > 100 μM, Fig. 5f–h and Supplementary Table 8), indicating a substantial\n50\nreduction in its inhibitory potency. Collectively, these findings confirm that Val405 and Phe1760 are\ncritical residues for the binding and inhibition of Na 1.5 by cis-azo-Q2a, thereby establishing them\nV\nas essential molecular determinants for the light-dependent control mediated by azo-Q2a.” (Page\n11, Line 305-310)\nSupplementary Figure 7g | Inhibition rate of Na 1.5 mutations by quinidine.\nV\nInhibition of sodium current of Na 1.5 WT, V405A and F1760 by 100 μM quinidine in darkness.\nV\n(n = 6-7). Data are shown as mean ± SEM.\nQuestion: The zebrafish methods lack clarity. It is not clear whether the compounds were bath-\napplied or microinjected into the larvae.\nResponse: We thank the reviewer for highlighting this lack of clarity in the zebrafish methods. The\ncompounds were delivered via bath application (soaking), rather than micro-injection. We have\nclarified this point in the revised manuscript to ensure methodological transparency and reliability:\n“Then, zebrafish larvae were exposed to the compounds by soaking and incubated in a 28.5 ℃\nincubator in the dark for 1.5 h under perfusion.” (Page 19, Line 515-517)\nQuestion: While the authors convincingly show that azo-Q2a can modulate heart rate in vivo, they\ndo not include results for 100 µM quinidine, a concentration known to inhibit multiple ion channels.\nThe lack of effect observed with quinidine is mentioned but not supported by citations. Moreover,\nthe hypothesis that increasing selectivity toward sodium channels would enhance bradycardia is\nquestionable and requires a more nuanced explanation.\nResponse: We appreciate the reviewer for raising this point. In our optical control assays evaluating\nazo-Q2a’s effect on zebrafish heart rate, the cis-azo-Q2a isomer exhibited significant retard activity\n(57 ± 4.3% inhibition, Fig. 6b). Based on this pronounced effect, we selected 10 μM quinidine as a\nnegative control to benchmark the baseline inhibitory activity of the parent compound under\nequivalent experimental conditions. This concentration demonstrated weak heart rate suppression\n(94 ± 0.9% under 365 nm, 92 ± 1.3% under 480 nm), consistent with previously reported data\nshowing low efficacy (88 ± 5%) at 10 μM in zebrafish models. (J Appl Toxicol. 2012;32(10):834-\n842)\nIn addition, we apologize for any lack of clarity in our original manuscript. Our hypothesis posits\nthat the enhanced bradycardic effect of cis-azo-Q2a lies primarily in its increased efficacy against\nthe Na 1.5 current, rather than an improvement in channel selectivity. The superior selectivity\nV\nprofile of azo-Q2a over quinidine, however, confers a significant advantage by minimizing off-\ntarget interactions, thereby reducing potential side effects.\nQuestion: Have the authors tested whether repeated cycles of 365/480 nm illumination can\nreproducibly modulate heart rate in larvae? Demonstrating reversibility and repeatability is\nessential for potential translational applications.\nResponse: We thank the reviewer for this thoughtful suggestion. In our zebrafish assay system, we\nhave successfully demonstrated the optical reversibility of azo-Q2a effect. Specifically, the heart\nrate of larvae inhibited by cis-azo-Q2a under 365 nm illumination was rapidly and completely\nreversed upon subsequent 480 nm exposure (Fig. 6). While repeated cycles of 365/480 nm\nillumination could not be performed because the larvae require a 30 min of recovery after each\nillumination to stabilize heart rate, given their sensitivity to light stimulation. This inherent\nlimitation of the experimental system prevents us from directly demonstrating repeatability across\nmultiple cycles of lighting of heart rate modulation in vivo.\nQuestion: In terms of pharmacokinetics, the in vivo stability of azo-Q2a must be addressed.\nAzobenzenes can undergo reductive degradation in biological environments; therefore, azo-Q2a\nsensitivity to GSH, a physiologically relevant reducing agent, should be evaluated.\nResponse: We thank the reviewer for this constructive comment. GSH can influence azo compounds\nin two ways: (i) by mediating the reductive cleavage of the nitrogen–nitrogen double bond, and (ii)\nby catalyzing cis-to-trans isomerization. We have experimentally examined both effects, The results\nindicated that the half-life of trans-azo-Q2a in 10 mM GSH reduction was 143 minutes, whereas\nthe half-life for the isomerization of cis-azo-Q2a in 1 mM GSH was 59 minutes (Supplementary\nFigure 5). Notably, the isomerization catalyzed by 10 mM GSH was too rapid to be reliably\nquantified. As an alternative, we measured the catalytic rate in a 1 mM GSH buffer solution. We\nhave revised our wording in the revised manuscript as following:\n“Finally, we examined the chemical stability of azo-Q2a. Under in vitro conditions, azo-Q2a\nremained remarkably stable, exhibiting a half-life (t ) of 107 h in the absence of reducing agents\n1/2\n(Fig. 2e). In contrast, exposure to 1 mM glutathione (GSH) triggered rapid cis-to-trans\nisomerization with a half-life of 59 min, driven by the reversible reduction of the -N=N- bond. At a\nhigher GSH concentration (10 mM), azo-Q2a underwent predominant reductive degradation with\na half-life of 143 min, indicating a shift in the reaction pathway under strongly reducing conditions\n(Supplementary Fig. 5).” (Page 6, Line 155-162)\nSupplementary Figure 5 | GSH catalysted Kinetics of isomerization and reduction for azo-Q2a.\na, Kinetics of reduction azo-Q2a in the PBS buffer solution of 10 mM GSH. Shown are the ln (trans%\nisomer) to time (min). b, Kinetics of isomerization of azo-Q2a in the PBS buffer solution of 1 mM\nGSH. Shown are the ln (cis% isomer) to time (min).\nQuestion: The authors claim that azo-Q2a could be useful for treating Na 1.5-related arrhythmias.\nV\nHowever, none of the current data support this application. Experiments demonstrating that light-\ninduced isomerization can terminate arrhythmias—either in cell-based models or ex vivo heart\npreparations—would significantly increase the study's impact.\nResponse: We thank the reviewer for this constructive suggestion. To address this concern, we\nperformed additional experiments in zebrafish larvae (3-4 dpf). Larvae were incubated in darkness\nwith 10 μM isoproterenol (ISO) in Hanks’ solution for 100 min, which significantly increased heart\nrate, indicating successful establishment of a tachycardia model. Subsequently, treatment with 10\nμM cis-azo-Q2a and 100 μM quinidine significantly reduced heart rate in the ISO-pretreated larvae\n(Supplementary Figure 12). These results directly support the reviewer’s suggestion by showing\nthat light-induced isomerization of azo-Q2a can effectively terminate ISO-induced tachycardia in\nvivo, thereby strengthening the translational relevance of our study. We have revised our wording in\nthe revised manuscript as following:\n“To further evaluate the therapeutic potential of azo-Q2a against arrhythmias, we established a\ntachycardia model in zebrafish larvae (3–4 dpf, Supplementary Movie 4) by incubating them in\ndarkness with 10 μM isoproterenol (ISO) for 100 min, which significantly increased heart rate35\n(Supplementary Movie 5). Using the same treatment and illumination protocol as described above,\nwe found that both 10 μM cis-azo-Q2a (Supplementary Movie 6) and 100 μM quinidine\n(Supplementary Movie 7) markedly attenuate heart rate in ISO-pretreated larvae (Supplementary\nFig. 12). These results demonstrate that light-induced isomerization of azo-Q2a can effectively\nterminate ISO-induced tachycardia in vivo, thereby supporting its potential relevance for Na 1.5-\nV\nrelated arrhythmias.” (Page 13, Line 342-351)\nSupplementary Figure 12 | Light-induced termination of ISO-induced tachycardia in\nzebrafish larvae.\na, Experimental protocol for tachycardia modeling and treatment in zebrafish larvae (3–4 dpf).\nLarvae were incubated in 10 μM ISO in Hanks’ solution for 100 min in darkness to induce\ntachycardia, followed by drug treatment (10 μM cis-azo-Q2a or 100 μM quinidine) and\nillumination with 365 nm light (5 min) plus 30 min darkness before final recordings. b, Heart rate\nmeasurements showing significant ISO-induced tachycardia, its reduction by cis-azo-Q2a and\nquinidine, and the additional light-induced effect of cis-azo-Q2a. Data are shown as mean ± SEM.\nQuestion:Finally, the authors state (line 318) that red-shifted azobenzene analogs failed to retain\noptical control of Na 1.5. This is a key point and should be backed by experimental data. If these\nV\nexperiments were performed, the corresponding results must be provided, at least in the\nSupplementary Information.\nResponse: Thanks for your constructive suggestions. To get the red-shifted azobenzene analogs, we\ninserted an ortho-fluorine modification onto the azobenzene group of azo-Q2a(azo-4F), based on\npreviously reports, ortho-fluoroazobenzene derivatives are known to offer two-way isomerization\nunder visible light irradiation. (J Am Chem Soc. 2012;134(51):20597-20600) However, the\nphotoisomerization wavelengths of azo-4F did not exhibit the expected red-shift as anticipated.\nOnly the cis-to-trans isomerization wavelength shifted from blue (480 nm) to green (550 nm) light,\nwhile the trans-to-cis photoactivation wavelength remained in the UV range. Furthermore,\nelectrophysiological tests revealed that the inhibitory efficacy of cis-azo-4F (8.3 ± 4.6% in 10 μM)\non Na 1.5 currents was unfortunately reduced compared to cis-azo-Q2a (61.1 ± 4.8% in 10 μM).\nV\nConsequently, we initially chose not to present these data in the original version of the manuscript.\nIn the newly revised manuscript, we have included detailed results on the structural modification of\nazo-4F, photochemical properties, and electrophysiological recordings in the Supplementary Figure\n13. We have revised our wording in the revised manuscript as following:\n“Only the cis-to-trans isomerization wavelength shifted from blue (480 nm) to green (550 nm) light,\nwhile the trans-to-cis photoactivation wavelength remained in the UV range. While\nelectrophysiological tests revealed that the inhibitory efficacy of cis-azo-4F (8.3 ± 4.6% in 10 μM)\non Na 1.5 currents was unfortunately reduced compared to cis-azo-Q2a (61.1 ± 4.8% in 10 μM).”\nV\n(Page 15, Line 398-403)\nSupplementary Figure 13 | Structure, photochemical characterization and inhibitory on\nNa 1.5 currents of red-shifted azobenzene analogs (azo-4F).\nV\na, Design and synthesis of quinidine red-shifted azobenzene analogs (azo-4F). b, The UV-Vis\nabsorption spectra of azo-4F (100 μM in DMSO/H O = 1/100, rt) in darkness (black, trans) or at\n2\n365 nm (violet, cis) and 550 nm (green, trans) illuminations. C, Whole-cell patch clamp recordings\nof Na 1.5 currents through application of trans- or cis-azo-4F (10 µM and 30 µM).\nV\nMinor comments:\nQuestion: There are multiple typographical and grammatical errors throughout the text and figure\nlegends that need to be corrected.\nResponse: We thank the reviewer for this comment. We have carefully checked and corrected\ntypographical and grammatical errors throughout the text and figure legends.\nQuestion: It is not clear whether the authors used calculated or experimentally determined reversal\npotentials to derive E .\nNa\nResponse: We appreciate the careful check. E was the calculated reversal potential based on our\nNa\nwhole-cell recording of Na 1.5 I-V response curve, which assuming that the I-V relationship from\nV\n0 mV to +40 mV was nearly linear and calculated the value of the intersection point of the line and\nthe abscissa (E ). In the revised manuscript, we have added this to method.\nNa\n“where I is the peak current, G is conductance, V is the stimulus potential, E is the equilibrium\nm Na\npotential which is the calculated reversal potential based on our whole-cell recording of Na 1.5 I-\nV\nV response curve.” (Page 17, Line 469-471)\nQuestion: The rationale for choosing +20 mV to activate hERG channels is unclear. Higher test\nvoltages typically provide more robust activation.\nResponse: When quantifying the inhibition rate of the hERG channel current, we measured the peak\ntail currents by 2 s repolarizing pulses to −40 mV, while depolarizing pulses at +20 mV, the tail\ncurrent of the hERG channel approaches its maximum value. (a, Circ Res. 2001;89(12):1168-1176.;\nb, J Biol Chem. 2011;286(40):34664-34674.)\nBesides, we further assessed the inhibition of hERG channel currents by azo-Q2a and quinidine (10\nμM) at a more depolarized potential of -40 mV. The results showed that the inhibition rates under\nboth 480 nm and 365 nm illumination were comparable to those observed at the standard activation\nvoltage of +20 mV.\nThe inhibition of hERG channel currents by azo-Q2a and quinidine at -20 mV and -40 mV.\nThe inhibition rate (%) of hERG channel currents by azo-Q2a and quinidine in 10 μM at -20 mV\n(n=8-15) and -40 mV (n=4-6) under 480 nm or 365 nm illumination. Data are shown as mean ±\nSEM.\nQuestion: Line 241 incorrectly states that the IC₅₀ of the cis isomer decreased. In fact, the IC\n50\nincreased, indicating reduced affinity.\nResponse: Thank you for pointing this out. The text has been corrected.\nQuestion: Lines 303–304: capitalization errors need correction.\nResponse: We appreciate the careful check. The capitalization errors have been corrected in the\nrevised manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done a superb job in addressing all the concerns from me and other referees. In my opinion, the revised manuscript has been substantially strengthened. I strongly support publication of this interesting work.", "reviewer_2": "This paper cannot be published without citing thee previous, pioneering work by Fehrtentz, Trauner, and Klöcker and putting it into context:\n\"Optical Control of Cardiac Electrophysiology by the Photochromic Ligand AzoBupivacaine 2\" Fehrentz, T.; Amin, E.; Görldt, N.; Strasdeit, T.; Moussavi-Torshizi, S. E.; Leippe, P.;Trauner, D.; Meyer, C.; Frey, N.; Sasse, P.; Klöcker, N. British J. Pharm., 2025, 82, 1125-1142.\nIt is unethical to completely ignore this work. I find it hard to believe that the authors ignored this aspect of my review and chose not to cite and discuss prior art.", "reviewer_3": "The authors have replied to almost all of my previous comments and have added substantial and valuable data to the revised manuscript. I appreciate the quality of their additional experiments and the effort made to strengthen the study. Nevertheless, I have a few remaining remarks that could further improve the clarity and completeness of the manuscript.\n1. Isosbestic point (Figure 2):\nI agree with the authors that the slight shift of the isosbestic point could result from minor side- or photoproduct formation or from changes in solvent polarity and ionic strength. It would be helpful to include one brief sentence in the text acknowledging this possible explanation.\n2. Recording solutions for HEK cells and cardiomyocytes:\nI remain somewhat surprised that cardiomyocytes can be well clamped under the conditions used, given the large ionic gradient applied. A short justification or discussion of how good voltage control was ensured under these conditions would improve the transparency of the electrophysiological methodology.\n3. Depolarization rate (Supplementary Figure 8c):\nIt would be appreciated if the authors could present dV/dtmax values instead of (or in addition to) the “Inhibition of depolarization rate” currently shown. This parameter would provide a more direct and quantitative measure of the effect on excitability.\n4. Repeatability in zebrafish experiments:\nThe authors could mention as a limitation the lack of repeatability in zebrafish assays, which may arise from the high sensitivity of larvae to light stimulation. Including this point would clarify the practical challenges associated with the in vivo experiments.\n5. Supplementary Figure 12 – Heart rate quantification:\nI acknowledge the authors for adding these new and informative data. It would be of interest to include quantitative comparison of heart rate in the “ISO-trans-azoQ2a” (darkness) condition versus “ISO-cis-azoQ2a.” Such data would strengthen the physiological interpretation of the observed effects and confirm whether the two illumination states indeed differ functionally."}, "author_response": "We sincerely thank the reviewers for allowing us to submit a revised version of our manuscript. We are also deeply grateful for the reviewers' continued engagement and their additional insightful comments. In accordance with their feedback, we have thoroughly addressed all remaining points and polished the language to enhance the clarity and precision of our manuscript. All modifications have been clearly highlighted. We believe this second round of revisions has significantly strengthened the manuscript and hope it now meets the high standards of Nature communications for publication. Our point-by-point responses follow.\nPoint-by-point response to reviewers’ comments\nReviewer #1 (Remarks to the Author):\nThe authors have done a superb job in addressing all the concerns from me and other referees. In my opinion, the revised manuscript has been substantially strengthened. I strongly support publication of this interesting work.\nResponse:\nWe sincerely thank reviewer for their strong support and positive assessment of our revisions. We are pleased that the reviewer finds the manuscript substantially strengthened and suitable for publication. A final proofread has been conducted to ensure clarity and consistency throughout the manuscript.\nReviewer #2 (Remarks to the Author):\nThis paper cannot be published without citing thee previous, pioneering work by Fehrtentz, Trauner, and Klöcker and putting it into context:\n\"Optical Control of Cardiac Electrophysiology by the Photochromic Ligand AzoBupivacaine 2\" Fehrentz, T.; Amin, E.; Görldt, N.; Strasdeit, T.; Moussavi-Torshizi, S. E.; Leippe, P.;Trauner, D.; Meyer, C.; Frey, N.; Sasse, P.; Klöcker, N. British J. Pharm., 2025, 82, 1125-1142.\nIt is unethical to completely ignore this work. I find it hard to believe that the authors ignored this aspect of my review and chose not to cite and discuss prior art.\nResponse:\nWe sincerely thank the reviewer for their feedback and for highlighting this important prior work. We fully agree on the ethical importance of properly citing and contextualizing relevant literature. We have incorporated the pioneering study by Fehrentz et al. (British Journal of Pharmacology, 2025, 182, 1125-1142) as reference 23 in the current manuscript. This citation appears in the introduction (lines 90-94), where we discuss it in detail as follows:\n“Preliminary attempts at photoswitchable modulation of cardiac sodium channels have been reported, while it shows low photoswitching (1.4-fold) at Na 1.5 and poor selectivity over other channels23. This underscores the need for development of novel photoswitchable modulators with higher Na 1.5 selectivity, stronger photoswitching efficacy and clearer mechanistic definition.”\nReference:\n23.Fehrentz, T. et al. Optical control of cardiac electrophysiology by the photochromic ligand azobupivacaine 2. Br J Pharmacol 182, 1125-1142 (2025).\nReviewer #3 (Remarks to the Author):\nThe authors have replied to almost all of my previous comments and have added substantial and valuable data to the revised manuscript. I appreciate the quality of their additional experiments and the effort made to strengthen the study. Nevertheless, I have a few remaining remarks that could further improve the clarity and completeness of the manuscript.\nResponse:\nWe sincerely appreciate the reviewer's positive feedback on the additional data and our overall revisions. We are encouraged that the reviewer finds the newly added experiments substantial and valuable. In accordance with the remaining suggestions, we have carefully implemented further revisions to enhance the clarity and rigor of the manuscript. Our point-by-point responses are detailed below.\nQuestion: Isosbestic point (Figure 2):\nI agree with the authors that the slight shift of the isosbestic point could result from minor side- or photoproduct formation or from changes in solvent polarity and ionic strength. It would be helpful to include one brief sentence in the text acknowledging this possible explanation.\nResponse:\nWe thank the reviewer for their agreement with our explanation regarding the slight shift in the isosbestic point. As suggested, we have incorporated a brief sentence acknowledging this possibility into the manuscript as following: (lines 151-154 in the revised text)\n“While we noted that the isosbestic point shifts slightly upon illumination at 480 nm which is small relative to the main spectral features, this might result from minor side- or photoproduct formation or from changes in solvent polarity and ionic strength.”\nQuestion: Recording solutions for HEK cells and cardiomyocytes:\nI remain somewhat surprised that cardiomyocytes can be well clamped under the conditions used, given the large ionic gradient applied. A short justification or discussion of how good voltage control was ensured under these conditions would improve the transparency of the electrophysiological methodology.\nResponse:\nWe thank the reviewer for this insightful comment. We agree that ensuring robust voltage-clamp quality is essential for reliable electrophysiological data interpretation, particularly given the large ionic gradients involved in sodium channel recordings. In our experiments with rat primary ventricular cardiomyocytes, cells were held at a hyperpolarized potential of –120 mV, where Na 1.5 channels (with an activation threshold around –70 mV) remain predominantly in the closed resting state. This minimizes voltage-dependent sodium currents during seal formation, promotes a stable baseline, and facilitates effective clamp control prior to channel activation during test pulses. To enhance methodological transparency, we have added a brief justification to the Methods section in the revised manuscript (lines 469-474).\n“In whole-cell recordings of Na 1.5 currents, the cells were held at −120 mV and the inward peak sodium currents were elicited by a 50 ms step to −20 mV. Recordings from other sodium channel isoforms (Na 1.2–Na 1.6) were performed under the same holding conditions (−120 mV) but using subtype-specific test pulses: 0 mV for Na 1.2 and Na 1.3 (50 ms), −30 mV for Na 1.4 (20 ms), and −10 mV for Na 1.6 (50 ms). ”\nQuestion: Depolarization rate (Supplementary Figure 8c):\nIt would be appreciated if the authors could present dV/dtmax values instead of (or in addition to) the “Inhibition of depolarization rate” currently shown. This parameter would provide a more direct and quantitative measure of the effect on excitability.\nResponse:\nWe thank the reviewer for this insightful suggestion. In response, we have incorporated the maximal upstroke velocity (dV/dtmax) values into Supplementary Figure 8c. The original percentage inhibition data for dV/dtmax have been retained in panel 8d to offer complementary insights. This revision provides a more direct and quantitative assessment of the compound's impact on cardiomyocyte excitability, as recommended.\nSupplementary Figure 8 b-d | Effect of cis-azo-Q2a on action potential in primary cardiomyocytes.\nb, Representative action potential traces under control (black), 10 μM cis-azo-Q2a (violet) and trans-azo-Q2a (blue) conditions. c, The maximal upstroke velocity (dV/dtmax) of action potentials under control, 10 μM cis-azo-Q2a and trans-azo-Q2a conditions (n = 3–5). Each line represents paired measurements from the same cell. d, Percentage inhibition rate of the dV/dtmax by 10 μM cis-azo-Q2a and trans-azo-Q2a (n = 3–5). Data are presented as mean ± SEM.\nQuestion: Repeatability in zebrafish experiments:\nThe authors could mention as a limitation the lack of repeatability in zebrafish assays, which may arise from the high sensitivity of larvae to light stimulation. Including this point would clarify the practical challenges associated with the in vivo experiments.\nResponse:\nWe thank the reviewer for this valuable suggestion. We have added a statement in the Discussion section explicitly acknowledge this methodological constraint. This addition clarifies the practical challenges of repeated photostimulation in zebrafish larvae, thereby enhancing the transparency of our work. The additional text in the revised manuscript (lines 400-404) as follows:\n“It should be noted that zebrafish larvae are highly sensitive to repeated light exposure. Consequently, extended recovery periods (30 minutes) must be incorporated between light cycles to minimize disturbance34. This requirement poses an inherent limitation on the repeatability of optical manipulations for controlling heart rhythm in this model.”\nQuestion: Supplementary Figure 12 - Heart rate quantification:\nI acknowledge the authors for adding these new and informative data. It would be of interest to include quantitative comparison of heart rate in the “ISO-trans-azoQ2a” (darkness) condition versus “ISO-cis-azoQ2a.” Such data would strengthen the physiological interpretation of the observed effects and confirm whether the two illumination states indeed differ functionally.\nResponse:\nWe thank the reviewer for this constructive suggestion. We have performed additional zebrafish experiments and quantitative analyses comparing the heart rates under the ISO-trans-azo-Q2a and ISO-cis-azo-Q2a conditions, as now detailed in the revised Supplementary Figure 12. The new analysis reveals that under isoproterenol (ISO)-induced tachycardia, heart rate inhibition by trans-azo-Q2a was 4.4 ± 1.2%, whereas cis-azo-Q2a produced a significantly greater inhibition of 35.3 ± 2.6%. This result intuitively provides direct physiological evidence that the functional efficacy of azo-Q2a is markedly dependent on its photoisomeric state, with the cis-configuration being essential for potent heart rate inhibition.\nSupplementary Figure 12 | Light-induced termination of ISO-induced tachycardia in zebrafish larvae.\na, Experimental protocol for tachycardia modeling and treatment in zebrafish larvae (3–4 dpf). Larvae were incubated in 10 μM ISO in Hanks’ solution for 100 min in darkness to induce tachycardia, then treated with 10 μM trans-azo-Q2a, 10 μM cis-azo-Q2a or 100 μM quinidine, and subsequently exposed to 365 nm illumination or kept in darkness (5 min), followed by 30 min darkness before final recordings. b, Heart rate measurements showing significant ISO-induced tachycardia (green, n = 27), its reduction by cis-azo-Q2a (violet, n = 23) and quinidine (grey, n = 21), and the mild decrease observed with trans-azo-Q2a (blue, n = 18). Data are shown as mean ± SEM."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have responded to my critique as follows:\n“Preliminary attempts at photoswitchable modulation of cardiac sodium channels have been reported, while it shows low photoswitching (1.4-fold) at NaV1.5 and poor selectivity over other channels'.\nThis sentence needs to be corrected. The study by Fehrentz et al. was in no way “preliminary,” as it clearly demonstrated photopharmacological modulation of cardiac NaV1.5 in vitro and ex vivo. In ex vivo mouse hearts, cardiac excitability could be controlled including the effective refractory period and the conduction velocity. Furthermore, the effect allowed to defibrillate ventricular arrhythmia by photopharmacology. The present work rather extends the former study by Fehrentz et al. making use of another Nav-photoswitch and applying it to zebrafish.", "reviewer_3": "The authors have addressed all my concerns. In my opinion, the revised manuscript is acceptable for publication"}, "author_response": "Response: We sincerely thank the reviewer for this important correction and for underscoring the foundational role of the study by Fehrentz et al. We agree that our original characterization of this work as “preliminary” was inaccurate, and we have revised the manuscript accordingly. The relevant sentence has been amended to:“Pioneering studies have demonstrated the photopharmacological modulation of cardiac sodium channels[23], yet there remains a strong demand for novel compounds that exhibit optimal Na 1.5 photoswitching, guided by a deeper mechanistic understanding.” This revision appropriately acknowledges the prior seminal contribution while framing the objective of our current work. We are grateful to the reviewer for their guidance in ensuring the accuracy and respectfulness of our scholarly references.\nResponse: It is highly rewarding to receive such a positive endorsement from the reviewer. We extend our sincere gratitude for their valuable time and for acknowledging the improvements made. Prior to resubmission, we have conducted a final comprehensive check of the manuscript to ensure the highest quality."}]

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