Application Note

Channel:Cells:
Tools:hHCN2
HEK293
SyncroPatch 384PE

Biophysical modulation of hHCN2 by bPAC
recorded on the SyncroPatch 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells engineered and kindly provided by Axxam S.p.A., Milan.
Summary

Results

Neuronal and cardiac rhythmicity is predominantly

Currents mediated by hHCN2 were activated upon

controlled

cyclicplasma membrane hyperpolarization. Figure 1 shows

nucleotide gated (HCN) channels. The HCN family

current responses to decreasing voltage steps for ancomprises four members (HCN1-4) which are ubiquitously

exemplar HEK cell expressing hHCN2 and the correspond-

expressed in the central and peripheral nervous system1.

ing current-voltage plot for an average of 346 cells. The

Activated by hyperpolarization, HCN channels open

example trace shows typical slow activation upon hyper-

slowly with no inactivation. Cyclic AMP (and other second

polarization. The half maximal activation of HCN2 wasmessenger proteins) affects the activation properties-89.2 mV.

byhyperpolarization

activated

independent of phosphorylation, modulating the voltage
dependence of current activation and accelerating thekinetics of channel opening2. HCN mediates a Na+/K+

AB

conductance (Ih) which contributes to the establishment-0.2

surprising that HCN channels play an important role in-0.4

I (nA)of the resting membrane potential3. It is therefore not
the regulation of neuronal firing and excitability as wellas pacemaking. Disruption of HCN function slows down
100 pA

treatment of neuronal disorders such as epilepsy4 andneuropathic pain5.
384PE showing pharmacology and modulation of hHCN2
expressed in HEK cells. In addition, these cells heterologously express a light-sensitive bacterial phospho-adelynate cyclase (bPAC). We demonstrate two ways oftriggering the cAMP pathway in order to modulate
the HCN2 channel opening kinetics. First, we used theinternal perfusion system of the SyncroPatch 384PE for
direct application of cAMP to the intracellular environment. Second, we triggered the cAMP pathway by
optical stimulation of bPAC. Further, we showed voltage
dependent block of Ih with Cs+ and ZD7288. Ivabradine, adrug used for symptomatic management of stable heart
related chest pain and heart failure blocked the channel
with an IC50 of 0.1 mM in good agreement the literature6&7.
Download more Application Notes from www.nanion.de-0.8-1.0

the heart rate and provides a potential target for theHere we present data collected on the SyncroPatch

Vhalf = -89.2 mV-0.6-1.2
0.5 s-40-60

-80

-100

V (mV)-120

Figure 1: Activation of HCN2 expressed in HEK cells on the SyncroPatch
384PE by hyperpolarizing voltage steps. A. Raw traces from an exemplar cellexpressing hHCN2 recorded on the SyncroPatch 384PE. Shown are current
responses to decreasing voltage steps from -30 to -130 mV. B. Currentvoltage plot for an average of 346 cells. Shown are mean of steady-state
current amplitudes ± S.E.M.

Applicationofinternal

cAMP

did

not

furtherincrease the current amplitude but modulated thecurrent kinetics decreasing the time constant (tau) toreach the peak current by 31% ± 1.1% for an average for204 cells.

Application NoteIn order to study the current kinetics and the voltage
dependent effect of compounds in parallel, voltage steps
of increasing amplitude (-30 mV, -90 mV, -110 mV, -130 mV)
were applied in one sweep. A single concentration ofeither Cs+, ZD7288 or ivabradine was applied to each well

8 ms

250 pAand concentration response curves were calculated by
combining the results across the plate (Figure 4). The IC50 ofCs+ and ZD7288 vary with different voltages (Cs+-90= 124 µM,

0.5 s

Figure 2: Modulation of hHCN2 expressed in HEK cells on the SyncroPatch
384PE by cAMP. Raw current traces from an example cell showing current
activation by stepping the voltage from -30 mV to -130 mV (black trace)and current modulation by additional intracellular application of 2 mM
cAMP (blue trace). The inset displays a higher resolution of current kinetics.
This example cell shows a modulation of tau by 8 ms when cAMP wasapplied.

Intheseintracellularcells

engineered

cAMPbyAxxam

concentration

canS.p.A

thealsobeincreased by light stimulation of bPAC. Figure 3 shows thereversible modulation of Ih current kinetics after blue light

excitation (λ = 470 - 495 nM for 0.5 Hz) decreasing tau of

Cs+-110= 101 µM, Cs+-130= 86 µM (n = 77); ZD7288-90 = 5 µM,ZD7288-110 = 11 µM, ZD7288-130 = 17 µM (n = 105))

demonstrating a voltage dependent blocking effect ofthese compounds on the current. No such an effect wasobserved for ivabradine (IC50 ~ 6 µM). For Cs+ and ZD7288, acumulative concentration response curve was also
performed (data not shown), resulting in comparable IC50s

as calculated from the single-point concentration responsecurves.
AB

activation to a similar extent compared to the response
after intracellular perfusion of cAMP.
AB

Figure 4: Block of hHCN2 by Cs+, ZD7288 and ivabradine. A. Raw current
traces (red traces) from example cells showing activation of Ih by decreasing
voltage steps followed by block with single concentrations of Cs+, ZD7288
or ivabradine (black traces in left panel) and full block with 2 mM Cs+ (bluetraces). B. The concentration response curves were calculated across theplate. The data collected were normalized to the reference current andfitted with a standard Hill-equation. Average concentration response curves
were calculated for the -90 mV, -110 mV and -130 mV voltage steps.

Figure 3: Modulation of Ih by light-induced cAMP production. A. Raw current
traces from 16 example cells showing the effect of blue light stimulation (8cells on the left, black traces) and no light (red traces (left) and red andblack traces (right)). B. Online analysis plot showing tau versus time for thesame cells shown in A.

Nanion Technologies GmbHphone + 49 89 219 095-0Ganghoferstr. 70Afax80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960

Application NoteFigure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384PE. Screenshot depicts raw data traces of hHCN2expressing HEK cells as recorded on one NPC-384 patch clamp chip. A single point concentration response curve for Cs+, ZD7288 and ivabradine wasperformed on individual wells. One control application of physiological solution was made followed by one concentration of compound on each well andincreasing concentrations over the plate. Multi-hole chips were used where 4 holes were present per well. The data of the 384 well plate representation inthe upper left part are color-coded for easy assessment of data. Depending on the seal resistance, pictures are green (Rmemb > 300MΩ), blue (Rmemb 100- 300 MΩ) or light blue (<100 MΩ). One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on the right. Graphs show
raw data traces of hHCN2 channels upon activation by hyperpolarizing voltage steps and application of buffer (red trace), followed by one compound
addition (black trace) and a full block at the end of the experiment (blue trace).

Exp end and chipunload

10 - 40 min

5 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1 buffer application, 1 - 4 compound applications, 1 fullblock application

Analysis

Figure 6: Timeline of an experiment on the SyncroPatch 384PE. The completion of 1 experiment on the SyncroPatch 384 patch clamp chip (384 wells) for a
1 - 4 point concentration response curve on hHCN2-mediated currents took approximately 10-40 min.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70Afax80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960

Application Note
Figure 5 shows a screenshot of the SyncroPatch 384

the SyncroPatch 384PE was used to modulate HCN2 using

software during an experiment. A color-coded over-internal cAMP and alteration in the current kinetics

view (based on seal resistance in this case) of all

could be monitored online. This continuous monitoring of384 wells gives the user a good impression of theanalysis parameters such as tau is important forsuccess rate of the experiment. The user can easily

distinguishing compound effect from increase in leak or

toggle between raw traces and online analysis. In

loss of seal and can be a benefit for assay development.

the example shown, raw traces are chosen and theModulation of HCN2 by cAMP was also triggered bygraphs show control responses to physiological buf-

optical stimulation of bPAC, when using the lightfer (red traces), followed by block with Cs , ZD7288

stimulation tool of the SyncroPatch 384PE. The HCN2or ivabradine (black traces) and full block with 2 mM

mediated current response was inhibited by Cs+, ZD7288

Cs+ (blue traces). The experiment shows that time andand ivabradine in a concentration-dependent mannerstate dependent compound effects can be simultane-

as expected with an IC50 within the range found in the+ously studied. The cells were stable for over 30 minutes

literature6,7.

and compound could be incubated for up to 8 minutes per concentration ensuring that full effect of theThe SyncroPatch 384PE is a high throughput and highly

compound was reached. An individual well can bereliable

automatedhighlighted to monitor progression of the experiment andrecording

hHCN2

mediated

currents.

User-is shown enlarged at the bottom of the screen.

friendly

software,excellent

success

rates,

patch

clamp

deviceforsingle additions or multiple additions of compound
In conclusion, Ih expressed in HEK cells can beto each well, perfusion of internal solution, lightrecorded on the SyncroPatch 384PE with good successstimulation and easy analysis result in reliable highrates (89 % of wells were included in analysis of pharma-quality data at an increased throughput with ancological experiments). The internal perfusion feature of

economical cost per data point.

References

Cell culture
Cells were cultured and harvested according to

1. Ludwig et al., 1999. EMBO J., 18 (9): 2323-9.

Nanion’s standard cell culture protocol.

2. Ludwig et al., 1998. Nature. 393: 587-591
3. Pape. 1996. Annu. Rev. Physiol. 58, 299–327.

Electrophysiology

4. Dyhrfjeld-Johnson et al., 2009. Front. Neurosci. 3:25–33

Whole cell patch clamp recordings were conducted

5. Emery et al., 2011. Science 333 1462–1466.

according to Nanion’s standard procedure for the

6. Stieber et al., 2005. J. Biol. Chem., 280 (41): 34635-43.

SyncroPatch 384PE using multi-hole (4 holes per well)

7. Stieber et al., 2006. Mol. Pharmacol., 69 (4): 1328-37.

chips. Perforated patch recordings were conducted. Theinternal solution was supplemented with 2 or 3 mM ATPMethods

and 2 mM cAMP, either present from the beginning or
washed within the recording using the Internal Exchange

Cells
HEK293 cells co-expressing hHCN2 and the Photo-function of the SyncroPatch 384PE. Ih currents were
evoked by stepping from Vhold of -30 mV in -20 mV

activated Adenylyl Cyclase from Beggiatoa bacterium

increments to -130 mV for 2 s. Blue light excitation (λ=470-(bPAC) were engineered and kindly provided by Axxam495nM) was used to stimulate bPAC and hence to trigger

S.p.A., Milan; (https://axxam.com/).

the cellular cAMP pathway.
hHCN2-mediated currents were calculated as thedifference of the inward current at the beginning andthe end of the voltage step. A mono-exponential decay
fit was used to determine the time constant (tau) of thecurrent activation.

Nanion Technologies GmbHphone +
 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960