IonWorks® HT

Nav Sodium Channel Screening Assay

 
Voltage-gated sodium channel currents depolarize the membrane in excitable cells and are therefore critical for action potential initiation and propagation. As a result, they have been implicated in many pathophysiological states and thus represent important drug discovery targets. Until recently, robust, high-throughput assays that directly measure the function of voltage-gated ion channels were not available. The IonWorks™ HT system can be used for screening and pharmacological studies of the cardiac voltage-gated sodium channel Na_v1.5.

• Na_v1.5 pharmacology
• High throughput IC₅₀ curve generation using the IonWorks HT system
• Z-factor statistical analysis
• Conclusions
• References and recommended reading

Na_v1.5 pharmacology

Na_v1.5 sodium channels are encoded by the SCN5A gene and are predominately expressed in cardiac muscle. Another distinguishing characteristic of Na_v1.5 channels is markedly reduced sensitivity to tetrodotoxin compared to other sodium channel types expressed in skeletal muscle and CNS neurons (> 200-fold). Na_v1.5 currents were elicited by a voltage step from the holding potential of -70 mV to 0 mV for 100 ms. Figure 1 shows representative current recordings from CHL cells expressing Na_v1.5 channels (α subunit) obtained on the IonWorks HT system.

		Figure 1. Nav1.5 currents recorded on the IonWorks HT system. Representative traces of control and full- and partial-antagonist doses are indicated for tetrodotoxin (TTX), tetracaine and lidocaine. Pre-compound traces are shown in black and post-compound traces are shown in orange. Control solution was phosphate-buffered saline (PBS).

high throughput IC₅₀ curve generation using the IonWorks HT system

The IonWorks HT system is ideal for rapidly performing high throughput dose response experiments. Four- to twelve-point drug dilution series can be established in columns or rows of 96-well compound plates. For Na_v1.5 experiments, eight-point dilution series of three sodium channel antagonists were made in columns by performing 1:3 serial dilutions using a multi-channel pipettor. Figure 2 shows the concentrations of TTX, tetracaine and lidocaine in the compound plate; each drug dilution series was replicated in three columns (as indicated by color). Each concentration was diluted an additional 3-fold during the experiment by addition of 3.5 µl drug to 7 µl buffer/cell solution in the PatchPlate™.

		Figure 2. Concentration of drug in compound plate; each compound was replicated in three separate columns, indicated by color. TTX=tetrodotoxin (yellow); Tetra=tetracaine (green); Lido=lidocaine (purple); PBS=Phosphate buffered saline (white). Values are [drug] M.

Concentration-response curves for tetrodotoxin (TTX), tetracaine and lidocaine were fitted to a four parameter logistic equation:

% of control = 100 (1 + ([drug]/IC₅₀)^p)^-1

where IC₅₀ is the concentration of the drug required to inhibit current by 50% and p is the Hill slope. IC₅₀ values for TTX, tetracaine and lidocaine are shown in Figure 3.

		Figure 3. Dose-response curves for IC50 determinations for TTX, tetracaine and lidocaine activity at Nav1.5 channels. Mean ± SD shown; n=12 per data point. IC50 values are shown in the inset.

Z-factor statistical analysis

Evaluation of assay robustness was determined by performing Z-factor statistical analysis of TTX blockade of Na_v1.5 channels. Results from one PatchPlate experiment are summarized in Figure 4. The compound plate in this experiment had ten columns of control solution (PBS) and two columns of sample (1.5 mM TTX). Data was expressed as a percent of the pre-compound current at 0 mV test potential used to elicit sodium currents.

Z-factor was calculated as:

		Figure 4. Z-factor statistical analysis of Nav1.5 currents under control (PBS) and blocked (1.5 mM TTX) conditions. Table indicates values used in calculating Z-factor.

conclusions

The IonWorks HT system can be used for high throughput screening of modulators of voltage-gated sodium channels, and has sufficient fidelity for pharmacology studies with fast currents such as Na_v1.5. In addition, the IonWorks HT system can be used to generate eight-point dose-response curves at n=4 per data point, for up to 100 compounds per day. For more details on experiments described on this page, download our application note entitled Nav1.5 sodium channel assay using IonWorks HT (registration required).

references and recommended reading

Bezanilla, F. and C. M. Armstrong (1977). Inactivation of the sodium channel. I. Sodium current experiments. J. Gen. Physiol. 70(5): 549-66.

Catterall W.A. (2000). From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26(1):13-25.

Lai J., J.C. Hunter and F. Porreca (2003). The role of voltage-gated sodium channels in neuropathic pain. Curr. Opin. Neurobiol. 13(3):291-7.

Schroeder, K., B. Neagle, et al. (2003). Ionworks HT: a new high-throughput electrophysiology measurement platform. J. Biomol. Screen. 8(1): 50-64.

Foundations of Cellular Neurophysiology. Daniel Johnston and Samuel Miao-Sin Wu, The MIT Press, Cambridge, MA, 1995.

Ion Channels and Disease. Frances M. Ashcroft, Academic Press, San Diego, CA, 2000.

Ionic Channels of Excitable Membranes, 3rd edition. Bertil Hille, Sinauer Associates Inc, Sunderland, MA, 2001.