Modulation of Nav1.5 Channels, IonWorks Barracuda Plus System


Sodium channels play a critical role in the initiation and transmission of signals in excitable cells. They also represent the primary molecular targets of numerous drugs including local anesthetics, antiarrhythmics and anticonvulsants. Over the years the investigation of the structure, function and modulation of sodium channels have been greatly facilitated by neurotoxins such at tetrodotoxin (TTX) and saxitoxin (STX).

The immensely diverse library of neurotoxins evolved to be highly selective molecules against ion channels and therefore represents a novel source of molecules for treating ion channel-related diseases. In fact a synthetic form of conotoxin (ziconotide), a blocker of N-type calcium channel was approved for clinical management of chronic pain.

Despite the great potential of developing peptide toxins as drug candidates, the high-throughput electrophysiological characterization of neurotoxins has been challenging. This is largely due to physicochemical properties of the toxins, presence of multiple binding sites, relatively slower binding kinetics, and statedependent binding affinities.

In this study we evaluated the modulation of Nav1.5 channels by a number of peptide neurotoxins on the IonWorks Barracuda® Plus Automated Patch Clamp System. The combination of sophisticated protocol editing, long and stable assay window, and high throughput of IonWorks Barracuda Plus System make it an ideal platform for screening peptide modulators of ion channels in the drug discovery environment.


  • Extended assay window for sophisticated pharmacology analysis
  • Throughput of 1,100 to 6,000 data points/hour
  • Low running cost of any automated electrophysiology platform
  • Software amenable to rapid assay development and screening

Materials and methods

Chinese hamster lung (CHL) cells stably expressing the human SCNA5 (Nav1.5) gene were used. All peptide toxins (Jingzhaotoxin II, ProTx-II, and µ-Conotoxin PIIIA) were obtained from Alomone labs (Jerusalem, Israel) and freshly dissolved in external buffer containing 0.3% BSA. All experiments were performed in the Population Patch Clamp (PPC) mode. Internal buffer contained (in mM): 100 K+ gluconate, 30 KCl, 3.2 MgCl2, 5.0 EGTA, 5.0 HEPES, pH 7.25 with KOH; External buffer was Phosphate Buffered Saline (PBS, Gibco Cat #14040).


Validation of Nav1.5 assay protocol

To evaluate both the voltage- and timedependent modulation of Nav1.5 channels by neurotoxins, we designed a voltage protocol using a 30-pulse, 10 Hz train. The cells were held at -100mV, with 20ms depolarizing steps to -35mV (Figure 1A). This voltage protocol was repeated 3 times for baseline recordings and 10 times after the compound (or buffer) additions. A wait time of three minutes was used between scans (Figure 1B). As shown in Figure 1, the peak current amplitudes remained stable within each scan, and in multiple scans for up to 35 minutes.

Figure 1. Stable recordings of Nav1.5 currents for over 30 minutes. A) The voltage protocol and the elicited currents. P1 and P30 refer to the currents evoked at pulse 1 and pulse 30 of the depolarizing train. B) Diagram of the experiment procedure, where the appropriate concentrations of compounds or buffer were applied at 1X concentration directly to the cells. C) The amplitude of Nav1.5 currents were plotted as vertical bars for each scan, and for multiple scans collected at different time points, where time 0 refers to the beginning of the experiment. The arrow represents the time of compound application.

Modulation of Nav1.5 by neurotoxins

Neurotoxins are known to modulate sodium channels in multiple ways. For example, they can inhibit channel conductance by blocking the pore, modify the gating or voltage-dependence of the channels. In this study, we examined three types of toxins (Jingzhaotoxin II, ProTx-II, and µ-Conotoxin PIIIA) on the IonWorks Barracuda Plus System. As shown in Figure 2, Jingzhaotoxin II significantly enhanced the Nav1.5 currents by inhibiting channel inactivation; whereas ProTx-II significantly blocked channel conductance. As a negative control, ω-conotoxin PIIIA, a selective Nav1.4 channel blocker, did not affect the Nav1.5 currents.

Figure 2. Different modalities of Nav1.5 modulation by neurotoxins. Plate view of the Nav1.5 channel currents (P1) at baseline scan 3 (A), and post-compound scan 6 (B). Each well received 1X “push-through” applications of toxins (500 nM Jingzhaotoxin II, 200 nM ProTx-II, or 100nM ω-conotoxin PIIIA) or buffer (with 0.3% DMSO). C) The overlay of Nav1.5 current traces from a representative recording well before (black) and after (red) application of Jingzhaotoxin II. The peak and the persistent values of the currents are defined as the absolute values of the min and max currents within 5 ms of channel activation.

Characterization of time- and voltagedependence of modulation

To examine the on-rate of neurotoxins for Nav1.5 channels, the amplitude of currents (both the peak and the persistent components) were plotted as a function of time (Figure 3). The data demonstrated the relatively slower progression to peak effects for both Jingzhaotoxin II (t = 4.5 minutes) and ProTx-II (t = 2.4 minutes). These results are in agreement with published data collected with manual patch clamp method.

In addition, ProTx-II induced block was gradually relaxed as shown in the peak currents within each scan from P1 to P30 (Figure 3C). This reversal of inhibition by repetitive depolarizations suggests voltage-dependent dissociation of toxin from the channels, which is consistent with that reported by Sokolov et, al.

Figure 3. Analysis of on-rate and voltage-dependence of neurotoxins. The peak (A, C) and the persistent (B, D) components of the Nav1.5 currents are plotted as a function of time. The arrow in each panel represents the time of compound application. The vertical bars represent the amplitude of the currents.

Reversal of ProTx-II inhibition by strong depolarization

To further investigate the reversal of ProTxII inhibition by strong depolarization, a voltage protocol was designed to first apply a conditioning depolarization to 0 mV of increasing duration, followed by a 50 ms holding potential -100 mV to allow recovery from fast inactivation and then by a test pulse to -35 mV to measure ionic currents. As shown in Figure 4, the amplitude of channel currents increased because of the depolarizing pulses, and reversal of ProTx-II inhibition followed an exponential time course with a time constant of 67 ms.

Figure 4: Reversal of ProTx-II inhibition by incremental depolarization. A) After the channels were inhibited by 200 nM ProTx-II, the kinetic profile of current recovery (measured at the test voltage of -35mV) as a function of prolonged conditioning step; B) current remains stable in wells from the same experiment, where only the buffer was added.


In this study, we designed and validated a high-throughput electrophysiology assay to evaluate the modulation of sodium channels by neurotoxins. Our data from the IonWorks Barracuda Plus System are consistent with those reported in the literature. The assay protocol, in combination with a prolonged assay window, provides the complexity and the throughput necessary for the screening of neurotoxins in a drug discovery environment.


  1. Sokolov S, et al. Inhibition of sodium channel gating by trapping the domain II voltage sensor with protoxin II. Mol Pharmacol. 2008 Mar;73(3):1020-8.
  2. Wang M, et al. Jingzhaotoxin-II, a novel tarantula toxin preferentially targets rat cardiac sodium channel. Biochem Pharmacol. 2008 Dec 15;76(12):1716-27.

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