Cardiac and neuronal rhythmicity is mainly controlled by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. The HCN family includes four members (HCN1-4) which are universally expressed in the peripheral and central nervous systems1.
Initiated by hyperpolarization, HCN channels open slowly with no inactivation. Cyclic AMP (and other second messenger proteins) affects the activation properties independent of phosphorylation, accelerating the kinetics of channel opening and modulating the voltage dependence of current activation2.
HCN mediates a Na+/K+ conductance (Ih) which contributes to the establishment of the resting membrane potential3. It is not surprising, therefore, that HCN channels play an important role in the regulation of neuronal excitability and firing, along with pacemaking. Disruption of HCN function slows down heart rate, and represents a potential target for the treatment of neuronal disorders such as epilepsy4 and neuropathic pain5.
This article reviews the data acquired using the SyncroPatch 384PE demonstrating the modulation and pharmacology of hHCN2 expressed in HEK cells. Along with this, these cells heterologously express a light-sensitive bacterial phospho-adelynate cyclase (bPAC).
Two techniques were used for the activation of the cAMP pathway as a means to regulate the HCN2 channel opening kinetics. Firstly, the SyncroPatch 384PE’s internal perfusion system was used to directly apply cAMP to the intracellular environment. Secondly, the cAMP pathway was activated by optically stimulating bPAC.
A voltage-dependent block of Ih with ZD7288 and Cs+ was also demonstrated. Ivabradine, a drug used to manage symptoms related to stable heart-related chest pain and heart failure, blocked the channel with an IC50 of 0.1 mM, in good agreement with the literature6&7.
The currents mediated by hHCN2 were activated by plasma membrane hyperpolarization. Figure 1 shows the responses of the currents to decreasing voltage steps for an exemplar HEK cell expressing hHCN2 and the related current-voltage plot for an average of 346 cells.
The example trace shows typical slow activation upon hyperpolarization. The voltage eliciting half maximal activation (Vhalf) was -89.2 mV.
Figure 1. Activation of HCN2 expressed in HEK cells on the SyncroPatch 384PE by hyperpolarizing voltage steps. A. Raw traces from an exemplar cell expressing hHCN2 recorded on the SyncroPatch 384PE. Shown are current responses to decreasing voltage steps from -30 to -130 mV. B. Current-voltage plot for an average of 346 cells. Shown are mean of steady-state current amplitudes ± S.E.M.
When cAMP was applied in the internal solution, current amplitude was not increased further, but rather affected current kinetics, decreasing the time constant (tau) to reach peak current by 31% ± 1.1% for an average of 204 cells.
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 modulation of tau by 8 ms when cAMP was applied.
In these cells engineered by Axxam S.p.A, the intracellular cAMP concentration in these cells can also be increased by light stimulation of bPAC.
Figure 3 shows the reversible modulation of Ih current kinetics following blue light excitation (λ = 470 to 495 nM for 0.5 Hz), decreasing the tau of activation to a similar degree compared with intracellular perfusion of cAMP.
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 (8 cells on the left, black traces) and no light (red traces (left) and red and black traces (right)). B. Online analysis plot showing tau versus time for the same cells shown in A.
In order to study the current kinetics and voltage-dependent effect of compounds in parallel, voltage steps of increasing amplitudes (-30 mV, -90 mV, -110 mV, -130 mV) were applied within one sweep.
A single concentration of either ZD7288, ivabradine, or Cs+ was applied to each well. The concentration-response curves were calculated by combining the results across the plate (Figure 4).
The IC50 of ZD7288 and Cs+ vary moderately at different voltages (Cs+-90= 124 µM, 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 the voltage dependent blocking effect of these compounds on the current. No such effect was found for ivabradine (IC50 ~ 6 µM).
For ZD7288 and Cs+, a cumulative concentration-response curve was also carried out (data not shown), leading to similar IC50s as measured from the single-point concentration-response curves.
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 the left panel) and full block with 2 mM Cs+ (blue traces). B. The concentration-response curves were calculated across the plate. The data collected were normalized to the reference current and fitted with a standard Hill-equation. Average concentration-response curves were calculated for the -90 mV, -110 mV and -130 mV voltage steps.
Figure 5. The graphical user interface of the screening and data analysis software used on the SyncroPatch384PE. The screenshot depicts raw data traces of hHCN2- expressing HEK cells as recorded on one NPC-384 patch-clamp chip. A single point concentration-response curve for Cs+, ZD7288 and ivabradine was performed on individual wells. One control application of physiological solution was made followed by one concentration of compounds on each well and increasing concentrations over the plate. Multi-hole chips were used where 4 holes were present per well. The data of the 384 well plate representation in the upper left part are color-coded for easy assessment of data. Depending on the seal resistance, pictures are green (Rmemb > 300 MΩ), 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).
Figure 6. Timeline of an experiment on the SyncroPatch384PE. The completion of 1 experiment on the SyncroPatch384 patch-clamp chip (384 wells) for a 1 - 4 point concentration-response curve on hHCN2-mediated currents took approximately 10-40 min.
Figure 5 shows a screenshot of the SyncroPatch 384 software during an experiment. A color-coded overview (based on seal resistance in this example) of all 384 wells gives the user a good impression of the success rate of the experiment.
The user can easily toggle between raw traces and online analysis. In the example displayed, raw traces are selected and the graphs depict control responses to physiological buffer (red traces), followed by block with Cs+, ZD7288 or ivabradine, (black traces) and full block with 2 mM Cs+ (blue traces).
Theexperiment shows that time- and state-dependent effects can be studied simultaneously. The cells remained stable for more than 30 minutes and compound could be incubtation for up to 8 minutes per concentration to make sure that the complete effect of the compound was achieved.
An individual well can be highlighted to monitor progression of the experiment and is shown enlarged at the bottom of the screen.
In conclusion, the SyncroPatch 384PE can be used to record Ih expressed in HEK cells with good success rates (89 % of wells were included in analysis of pharmacological experiments).
The SyncroPatch 384PE’s internal perfusion feature was utilized to modulate HCN2 using internal cAMP and alterations to the current kinetics could be monitored online.
This continuous monitoring of analysis parameters such tau is important for differentiating compound effects from loss of seal or increase in leak current and can be useful for assay development.
Modulation of HCN2 by cAMP was also triggered by optical stimulation of bPAC, using the light stimulation tool of the SyncroPatch 384PE. The HCN2-mediated current response was inhibited by Cs+, ZD7288, and ivabradine in a concentration-dependent manner as expected, with an IC50 within the range found in the literature6,7.
The SyncroPatch 384PE is a high throughput and highly reliable automated patch clamp device for recording hHCN2 mediated currents. User-friendly software, excellent success rates, single or multiple additions of compounds to each well, perfusion of internal solution, light stimulation and easy analysis result in high quality at an increased throughput with an affordable cost per data point.
HEK293 cells co-expressing the Photoactivated Adenylyl Cyclase from Beggiatoa bacterium (bPAC) and hHCN2 were developed and kindly supplied by Axxam S.p.A., Milan; (https://axxam.com/).
Cells were cultured and harvested accoding to Nanion's standard cell culture protocol.
Whole cell patch clamp recordings were performed according to Nanion's standard procedure for the SyncroPatch 384PE using multi-hole (four holes per well) chips. Perforated patch recordings were performed.
The internal solution was supplemented with 2 or 3 mM ATP and 2 mM cAMP, either present from the beginning or washed in during the recording using the Internal Exchange function of the SyncroPatch 384PE.
Ih currents were evoked by stepping from a Vhold of -30 mV in -20 mV increments to -130 mV for 2 s. Blue light excitation (λ=470- 495nM) was utilized to stimulate bPAC and consequently trigger the cellular cAMP pathway.
hHCN2-mediated currents were calculated as the difference of the inward current at the beginning and the end of the voltage step. A mono-exponential decay
fit was used to determine the time constant (tau) of the current activation.
References and Further Reading
- Ludwig et al., 1999. EMBO J., 18 (9): 2323-9.
- Ludwig et al., 1998. Nature. 393: 587-591
- Pape. 1996. Annu. Rev. Physiol. 58, 299–327.
- Dyhrfjeld-Johnson et al., 2009. Front. Neurosci. 3:25–33
- Emery et al., 2011. Science 333 1462–1466.
- Stieber et al., 2005. J. Biol. Chem., 280 (41): 34635-43.
- Stieber et al., 2006. Mol. Pharmacol., 69 (4): 1328-37.
This information has been sourced, reviewed and adapted from materials provided by Nanion Technologies GmbH.
For more information on this source, please visit Nanion Technologies GmbH.