DOI: 10.5061/dryad.q83bk3jsb
title: Molecular mapping of KCNE4-dependent regulation of Kv1.3

description: The voltage-gated potassium channel Kv1.3 plays a crucial role in the
 immune system response. In leukocytes, the channel is coexpressed with the
 dominant negative regulatory subunit KCNE4, which associates with Kv1.3 to
 trigger intracellular retention and accelerating C-type inactivation of
 the channel. Previous research has demonstrated that the main association
 between these proteins occurs through both C-termini. However, these data
 fail to fully elucidate the KCNE4-dependent modulation of channel
 kinetics. In the present study, we analyzed the contribution of each KCNE4
 domain to the modulation of Kv1.3. Our results further confirmed that the
 C-terminus of KCNE4 is the main determinant involved in the
 association-triggered intracellular retention of the channel.
 Additionally, interactions throughout the transmembrane region were also
 observed. Both the C-terminus and, especially, the transmembrane domain of
 KCNE4 accentuated the C-type inactivation of Kv1.3. Our data provide, for
 the first time, the molecular effects that a KCNE peptide, such as KCNE4,
 exerts on a Shaker channel, such as Kv1.3. Our results pave the
 way for understanding the molecular mechanisms underlying potassium
 channel modulation and suggest that KCNE4 participates in the
 conformational rearrangement of the Kv1.3 architecture, altering the
 C-type inactivation of the channel.
description: <strong>Constructs</strong> Rat
 (r)Kv1.3 in pRcCMV was obtained from T. C. Holmes (University of
 California–Irvine, Irvine, CA, USA) and introduced into pCDNA3.1, pEYFPC1
 and pECFP-C1 (Clontech Laboratories). Kv1.3, tagged with hemagglutinin
 (HA) extracellularly between S3 and S4, was obtained from D. B. Arnold
 (University of Southern California, Los Angeles, CA, USA). Murine (m)KCNE4
 in pSGEM was a gift from M. Sanguinetti (University of Utah, Salt Lake
 City, UT, USA). Human (h)KCNE2 in pHA was obtained from S. de la Luna
 (Centre for Genomic Regulation, Barcelona, Spain). mKCNE4 and hKCNE2 were
 subcloned and inserted into pECFP-N1 and pEYFP-N1. The plasma membrane
 marker pDsRed-tagged pleckstrin homology (PH) domain of Akt (Akt-PHpDsRed)
 was obtained from F. Viana (Instituto de Neurociencias de Alicante,
 Universidad Miguel Hernández-CSIC, San Juan de Alicante, Spain).
 MluI sites were introduced into CFP-tagged mKCNE4 and hKCNE2 to
 isolate either their N-terminal (Nt), transmembrane (TMD) or C-terminal
 (Ct) domains. Next, these fragments were obtained by PCR and subcloned and
 inserted into reciprocal KCNE plasmids. Several KCNE2/4 chimeras (X-X-X)
 were generated. KCNE(X-X-X) identifies the Nt, TMD and Ct fragments from
 each peptide: KCNE(2-2-4)-CFP (Nt and TMD of KCNE2 with the Ct of KCNE4),
 KCNE(4-4-2)-CFP (Nt and TMD of KCNE4 with the Ct of KCNE2),
 KCNE(2-4-2)-CFP (Nt and Ct of KCNE2 with the TMD of KCNE4),
 KCNE(4-2-4)-CFP (Nt and Ct of KCNE4 with the TMD of KCNE2),
 KCNE(4-2-2)-CFP (TMD and Ct of KCNE2 with the Nt of KCNE4) and
 KCNE(2-4-4)-CFP (TMD and Ct of KCNE4 with the Nt of KCNE2). The full
 sequence of the KCNE2/4 chimeras can be found in Supplemental Fig.
 S1. Mutations were generated with the QuikChange XL
 SiteDirected Mutagenesis Kit (Agilent Technologies) and transformed into
 XLGold<sup>®</sup> ultracompetent cells. The mutants were
 subsequently sequenced using a BigDye Terminator v3.1 kit (Applied
 Biosystems) at CCiT University of Barcelona. The sequences were analyzed
 using Clone Manager Professional v7 (Sci Ed Software LLC).
 <strong>Cell culture</strong>
 HEK293 cells were seeded on 100-mm culture dishes for protein
 extraction and biochemistry, on 15x15 mm square poly-D-lysine-coated
 coverslips for immunocytochemistry, and on 35-mm dishes for
 electrophysiology experiments. The cells were cultured at 37 °C and 5%
 CO<sub>2</sub> in Dulbecco’s modified Eagle’s medium (DMEM;
 Lonza) supplemented with 10% fetal bovine serum (FBS; Gibco), 10,000 U/mL
 penicillin, 100 µg/mL streptomycin (Thermo Fisher Scientific) and 4 mM
 L-glutamine (Sigma‒Aldrich). Cells were transfected 24
 h before the experiment using Lipotransfectin<sup>®</sup>
 (Attendbio Research). For coimmunoprecipitation, 4 µg of Kv1.3-HA and 4 µg
 of KCNE2/4 chimeras were transfected; for confocal microscopy, 500 ng of
 Kv1.3-YFP and 750 ng of the remaining constructs were transfected; and for
 patch clamp experiments, 150 ng of Kv1.3-pCDNA3.1 and 450 ng of the
 remaining constructs were transfected. No cell size changes were observed
 in any group after transfection being 15.52 ± 1.46, 15.52 ± 1.48, 15.41 ±
 0.74 pF (n=5-6) for Kv1.3, KCNE4 and KCNE2 expressing cells,
 respectively, <strong>Protein extraction, co-IP
 and Western blot </strong> The cells were washed
 twice with cold PBS and lysed for 15 min at 4 °C with gentle mixing in
 lysis buffer (150 mM NaCl, 50 mM HEPES, 10% glycerol, 1% Triton X-100, pH
 7.2) supplemented with protease inhibitors: 1 µg/mL leupeptin, 1 µg/mL
 pepstatin, 1 µg/mL aprotinin and 1 µM phenylmethylsulfonylfluoride (PMSF).
 The homogenates were centrifuged for 10 min at 16,200 × g at 4 °C, after
 which the supernatants were collected. The protein concentration was
 assessed using the Bio-Rad Protein Assay (Bio-Rad Laboratories).
 For coimmunoprecipitation (coIP) experiments, 1 mg of protein per
 condition was precleared with 25 µL of protein
 G-Sepharose<sup>®</sup> beads (GE Healthcare) for 1 h at 4 °C
 with gentle mixing. The beads were removed by centrifugation for 30 s at
 1,000 × g. Next, the supernatants were incubated in a Micro
 Bio-Spin<sup>®</sup> Chromatography column (Bio-Rad
 Laboratories) containing 50 µL of protein
 G-Sepharose<sup>®</sup> beads and 2.5 µg of rabbit anti-GFP
 antibody (Genscript Biotech, Piscataway). The antibody had previously been
 crosslinked to Sepharose beads for 30 min at room temperature (RT, 20-25
 °C) with 5.2 mg/mL dimethyl pimelimidate (DMP). The samples were incubated
 in the column for 2-3 h at RT with gentle mixing. Centrifugation for 30 s
 at 1,000 × g yielded the supernatants. The columns were washed five times
 with 500 µL of wash buffer (150 mM NaCl, 50 mM HEPES, 1% X-Triton, pH 7.4)
 supplemented with protease inhibitors and eluted with 100 µL of 0.2 M
 glycine (pH 2.5). Eluants were added to 20 µL of
 Laemmli buffer with β-mercaptoethanol and 6 µL of Tris-HCl (1 M, pH 10),
 boiled for 10 min and loaded on SDS‒PAGE gels. Next, the proteins were
 transferred to PVDF membranes (Merck Millipore) and blocked for 1 h in
 0.5% Tween<sup>®</sup>-20 in PBS supplemented with 5% skimmed
 powder milk. The membranes were immunoblotted overnight with mouse
 antibodies against Kv1.3 (1/200, UC Davis/NIH Neuromab Facility) or
 against GFP (1/500, Hoffmann-La Roche). The filters were washed with PBS
 containing 0.5% Tween<sup>®</sup>-20 and incubated for 2 h
 with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad
 Laboratories). <strong>Observation of
 colocalization by confocal microscopy</strong>
 The cells were fixed for 10 min with 4% paraformaldehyde (PFA) at
 RT, washed twice with PBS without potassium and mounted on microscope
 slides with Mowiol + DABCO (Sigma Aldrich). The samples were left to dry
 at RT, and images were obtained on a Zeiss LSM 880 Airyscan confocal
 microscope from the Advanced Optical Microscopy Unit (CCiTUB). Images
 analyzed with ImageJ were processed using a previously described method
 (18) with the JACoP plugin
 to calculate colocalization coefficients (19).
 <strong>Electrophysiology</strong>
 Voltage-gated K<sup>+</sup> currents were evoked in
 HEK293 cells using the whole-cell configuration of the patch clamp
 technique on a HEKA EPC10 USB amplifier using the acquisition software
 PATCHMASTER v2x91 (HEKA Elektronik GmbH). Currents were
 recorded at RT at a frequency of 0.1 Hz and filtered and sampled at 2 kHz.
 Pipettes were made from borosilicate capillaries (GC120T-10; Warner
 Instruments) using a programmable puller (P-97; Sutter Instruments) and
 polished on a microforge (MF-830; Narishige). The pipette resistance was
 between 2 and 4 MΩ. The pipette solution contained (in mM) 80 K-aspartate,
 42 KCl, 3 phosphocreatine, 10
 KH<sub>2</sub>PO<sub>4</sub>, 3 MgATP, 5 HEPES-K,
 and 5 EGTA-K (adjusted to pH 7.25 with KOH). The bath solution contained
 (in mM) 145 NaCl, 4 KCl, 1.8 CaCl<sub>2</sub>, 1
 MgCl<sub>2</sub>, 10 HEPES-Na, and 10 D-glucose (adjusted to
 pH 7.4 with NaOH). The data were analyzed using a FITMASTER v2x91 (HEKA
 Elektronik GmbH) and GraphPad Prism 5 (GraphPad Software).
 The cells were held at -80 mV as the holding potential. To study
 the voltage dependency of activation, currents were elicited after a 250
 ms pulse from -80 to +60 mV in 10 mV increments, followed by a 250 ms
 pulse at -40 mV to visualize tails. The tail current was measured,
 normalized, and adjusted to a Boltzmann equation of the form:
 <strong>Equation 1.</strong> where
 V is the independent variable (voltage), V<sub>50</sub> is the
 voltage at which half of the channels are open, and <em>k</em>
 is the slope of the curve. To study the voltage
 dependency of inactivation, the cells were depolarized at +40 mV for 1 s,
 and then a 250-ms pulse was applied from -60 to +40 mV in 10-mV
 increments. The current of the second pulse was normalized and fitted to a
 Boltzmann equation. To study use-dependent
 inactivation, a train of 15 depolarizing pulses of 200 ms at +60 mV was
 applied at a frequency of 2.5 Hz. The peak current of the obtained traces
 was normalized to that of the first pulse and expressed as a function of
 the number of pulses. To obtain the decay constant, we fitted the curve to
 an exponential equation of the form:
 <strong>Equation 2.</strong> where
 t is the independent variable (time), τ is the time constant and A is the
 amplitude. To further characterize the kinetics of inactivation, a long
 depolarizing pulse (5 s at +60 mV) was applied and fitted to an
 exponential decay. To study the molecular
 rearrangements affected by KCNE4, we applied a very long depolarizing
 pulse (1 min at 0 mV) as previously described (20). The elicited traces were
 fitted to a biexponential equation of the form:
 <strong>Equation 3.</strong>  
 where τ<sub>fast</sub> and τ<sub>slow</sub>
 represent the time constants for the fast and slow components,
 respectively. Similarly, A<sub>fast</sub> and
 A<sub>slow</sub> represent the amplitudes of both kinetic
 components. To determine their contributions, the amplitude of the fast
 component was relative to that of
 A<sub>fast</sub>+A<sub>slow</sub> and is expressed
 as a percentage. Recovery from inactivation was studied
 by applying a classic two-pulse protocol separated by a variable time
 interpulse at -90 mV. The first pulse was at +40 mV for 2 s, which allowed
 for full inactivation of the channels. The second test pulse was at +40 mV
 for 250 ms. Fractional recovery (FR) was calculated as:
 <strong>Equation 4.</strong> where
 I<sub>peak</sub> is the peak current at the first pulse,
 I<sub>plateau</sub> is the current at the end of the first
 pulse (steady state) and I<sub>test</sub> is the peak current
 of the test pulse. FR as a function of interpulse duration was fitted to
 an exponential equation. <strong>Free energy
 calculations and cycle analysis</strong> From 5 s
 pulses to +60 mV, free energy calculations were also performed as
 previously described (21).
 Briefly, at long depolarizations, the number of channels in the closed
 state is negligible, so an equilibrium of the form 𝑂 ⇌ 𝐼 can be assumed.
 The free energies of inactivation, recovery, and their difference were
 calculated as: <strong>Equation
 5</strong> where R is the universal gas constant,
 T is the temperature, τ is the time constant of inactivation and r is the
 ratio of the steady-state current at the end of the pulse divided by the
 peak current. The free energy of inactivation
 (∆G<sub>inactivation</sub>) was calculated for all KCNE2/4
 chimeras. Thermodynamic cycle analysis was performed to analyze the
 coupling between the three different domains of KCNE4 as previously
 described (22). Thus, two
 cycles were constructed to assess the coupling
 (∆∆G<sub>C</sub>) of the C-terminus and transmembrane regions
 in the absence (cycle I) or presence (cycle II) of the N-terminus of
 KCNE4. The differences between couplings (∆∆∆G<sub>C</sub>)
 represented the influence of the N-terminus on C-terminus-transmembrane
 coupling. <strong>Equation 6.              
 </strong> Finally, the mean contribution of each
 domain was calculated assuming that their contributions were independent.
 For the contribution of the C-terminus, we averaged the differences in
 free energy in all the transitions that added the C-terminus of KCNE4
 (KCNE2 → KCNE(2-2-4); KCNE(2-4-2) → KCNE(2-4-4); KCNE(4-2-2) →
 KCNE(4-2-4); KCNE(4-4-2) → KCNE4). The same was performed for the
 transmembrane domain (KCNE2 → KCNE(2-4-2); KCNE(2-2-4) → KCNE(2-4-4);
 KCNE(4-2-2) → KCNE(4-4-2); KCNE(4-2-4) → KCNE4) and the N-terminal domains
 (KCNE2 → KCNE(4-2-2); KCNE(2-4-2) → KCNE(4-4-2); KCNE(2-2-4) →
 KCNE(4-2-4); KCNE(2-4-4) → KCNE4).
 <strong>Homology Kv1.3/KCNE4 docking
 model</strong> We used our previously published
 docking model for Kv1.3/KCNE4 (12), which was validated on the
 cryo-EM structure of Kv1.3 (23, 24). Briefly, the largest part of
 Kv1.3 was modeled after Kv1.2 (PDB code 2R9R), except for the first
 N-terminal 49 amino acids (modeled after 1PXE; zinc-binding domain from
 neural zinc finger factor-1) and the C-terminus (3HGF; nucleotide-binding
 domain of the reticulocyte binding protein Py235). KCNE4 was modeled with
 a KCNE1 structure (PDB code 2K21) except for residues 99-170 in the
 C-terminus (HTC; a complex of recombinant hirudin and human α-thrombin).
 Homology modeling was performed on the SwissModel Protein Modeling on the
 Expert Protein Analysis System (ExPASy) Molecular Biology website (http://kr.expasy.org/). Energy minimization was performed with Yet Another Scientific Artificial Reality Application (YASARA) v23.5.19. Docking of Kv1.3 and KCNE4 was performed by first using a Kv7.1-KCNE1 structure (25) as a template and then docking regions of interaction (17, 23, 24). UCSF Chimera (26) was used for visualization. Putatively interacting residues were identified with the Find Clashes/Contacts tool. The overlap between two atoms i and j was calculated as: <strong>Equation 7.</strong> where r<sub>VDW</sub> is the van der Waals radius for each atom and d<sub>ij</sub> is the distance between the two atoms. Only overlap values greater than -0.4 Ǻ were considered to indicate contacts. <strong>Statistical analysis</strong> The results are expressed as the means ± SE from at least three independent experiments. When only two conditions were analyzed, differences were assessed by Student’s t test. For multiple analysis, ANOVA was performed followed by either Tukey’s test (for colocalization analysis) or Dunnett’s test (for electrophysiology experiments). If the residuals were significantly non-Gaussian, as measured with the Kolmogorov‒Smirnov test, differences were assessed with a Kruskal‒Wallis test followed by Dunn’s <em>post hoc</em> test.
description: # Supplemental Materials
 [https://doi.org/10.5061/dryad.q83bk3jsb](https://doi.org/10.5061/dryad.q83bk3jsb) ## Description of the data and file structure Supplemental materials (Supplemental Figures 1 and 2, and supplemental tables) are supporting data to further confirm the main body information ### Files and variables **Supplemental Figure S1. Sequence of the KCNE2/4 chimeras.** **Supplemental Figure S2. KCNE4 does not alter slow rearrangements of Kv1.3 or recovery from inactivation.** \**Supplemental Table S1. **Gibbs free energy calculations for Kv1.3 +YFP and Kv1.3 + KCNE4 **Supplemental Table S2.** Interacting residues between Kv1.3 and the TMD of KCNE4. Per each pair of interacting residues, only the highest overlap and its associated distance is shown. **Supplemental Table S3.** Interacting residues between Kv1.3 and the C-terminus of KCNE4. Per each pair of interacting residues, only the highest overlap and its associated distance is shown.