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Figure 1. State-dependent and Use-dependent hNav1.7. Aa - Ad: Pre-conditioning enhances hNav1.7 sensitivity to lidocaine and carbamazepine. Block of hNav1.7 channels with lidocaine and carbamazepine was measured using stimulus voltage pattern shown in panel Aa, holding potential -80 mV. The pulse pattern was repeated twice, once before and and again 5 minutes after a test article (TA) addition and peak current amplitudes at the 1st test pulse (TP1; panel Ab), the 11th test pulse (TP11; panel Ac) and the 12th test pulse (TP12; panel Ad) were measured. B-C: Concentration-response relationships. IC50 values for lidocaine-induced block (panel Ba) were 548 µM for the tonic block, 49 µM for the block at 10 Hz stimulation frequency (frequency-dependent block) and 8 µM for the inactivated state block. Data presented as Mean ± SD (n = 3 – 5). The IC50 values for carbamazepine-induced block (panel Bb) were: >300 µM for the tonic block, 156 D:TTX IC50 12 -14 nM. (Both figures: ChanTest.)  

Ion channels are integral membrane proteins that conduct transmembrane ionic currents responsible for cell signaling, excitability, and volume control. The diversity of ion channels, their involvement in a broad spectrum of physiologic mechanisms, and their accessibility on the cell surface make them attractive drug targets. Yet, because of the difficulties in adapting conventional patch-clamp assays to high-throughput drug discovery paradigms, and the lack of selectivity among structurally-related ion channel isoforms, they are undeveloped targets. Recent technological advances in cell-based ion channel assays that enable high- and medium-throughput assessment of functional selectivity achieved through state-dependent modulation of channel function have focused renewed attention on ion channels in drug discovery.

Characterization of the state- and use-dependent effects of modulators is critical to differentiating beneficial interactions from adverse ones. For instance, in voltage-gated Na+ channels, use-dependent block during repetitive cycles of channel opening and closing can be advantageous in blunting disease-related hyperexcitability. On the other hand, tonic blockade of channels in a resting state or ultra-fast open channel block can result in undesirable impediments to normal impulse conduction in unaffected tissue. Moreover, gating modulators that increase channel activity by interfering with channel closing or by shifting the voltage-dependence of channel activation to more negative potentials can promote hyper-excitation with potential to cause seizures, arrhythmia, motor, or sensory disturbances, depending upon the affected tissue.

Traditionally, experimental control of membrane potential and stimulation relied on single-cell, manual patch clamp techniques unsuited to modern drug development. The emergence of automated patch clamp technologies has dramatically expanded the possibilities in ion channel drug research. Cell lines that stably express recombinant, human ion channels are commercially available together with test methods that allow detection of desirable state-dependent antagonism as well as undesirable agonist effects.

This article will present an example of the usefulness of medium-throughput automated patch clamp in assessing voltage-gated Na+ channels for functional selectivity arising from state-dependent effects of blockers and gating modifiers.

Characterization of Na+ channel antagonists and agonists 
Voltage-gated Na+ channels that trigger and propagate action potentials in excitable tissues are important both as therapeutic targets (e.g., pain, cardiac arrhythmia, seizure, and neurodegenerative diseases), and safety anti-targets (cardiac conduction block, pesticide toxicity). Under physiological conditions, Na+ channels undergo voltage-dependent gating transitions; from the non-conducting closed-resting state at polarized membrane potentials, to the conducting open-activated state, followed quickly by a non-conducting open-inactivated state (fast inactivation) at depolarized potentials. Deeper levels of inactivation (slow inactivation) are reached during prolonged depolarization.

Pathological conditions, such as seizure, are triggered in neurons by high-frequency action potential discharge (hyper-excitation) leading to partial depolarization of affected tissues. Treatment with Na+ channel blockers that exhibit depolarization-dependent inhibition dampens excitability and prevents the spread of hyperexcitation to normal areas. The apparent voltage-dependence of channel blockade is attributed to preferential drug binding to one or more voltage-dependent channel gating states. State-dependent drug-binding within the cytoplasmic mouth of the channel pore is responsible for the therapeutic and functional selectivity of channel blockers. According to the modulated receptor hypothesis,1,2 heightened affinity for the resting state results in tonic suppression of action potential activation, increased activation threshold, and slowed conduction velocity. In contrast, increased binding to the open-inactivated state with rapid dissociation at rest typically results in stabilization of a drug-bound inactivated state, and use-dependent suppression of repetitive firing rates or suppression of channel activity in partially depolarized cells. The rate of drug dissociation from the inactivated state is a critical factor for optimizing therapeutic applications. For example, the anti-arrhythmic or anesthetic effects of blockers appear to be associated with relatively slow dissociation that promotes use-dependent block at low stimulus frequencies (<10 Hz), whereas high-affinity block of the open-inactivated state with relatively fast dissociation from the resting state promotes antiepileptic drug actions by suppressing high-frequency discharge (30 to 100 Hz) without interfering with normal activity.3

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Figure 2. Pyrethroid-induced gating modification in Nav1.8 A: Bioallethrin, a type I pyrethroid promotes slow development of inactivation and rapid tail currents. B: Esfenvalerate, a type II pyrethroid produces a persistent inward current, extreme slowing of inactivation, and extreme slowing of deactivation. Type II compounds are typically more toxic than type I to cause hyperexcitability. 

We have used Na+ channel-expressing cell lines and reference compounds to optimize assays suitable for moderate-throughput screening regimens in IonWorks Quattro (Molecular Devices, Sunnyvale, Calif.), an automated electrophysiology platform capable of screening ~2000 compounds/day. Nav channel clone selection and initial validation were performed as described previously.4 Figure 1 illustrates a Nav1.7 channel assay performed in stably transfected Chinese hamster ovary (CHO) cells. In this assay, channels are activated by a pulse pattern consisting of a series of depolarizing steps delivered at precise intervals to evaluate state- and use-dependent effects. For Nav1.7 the maximum depolarization-evoked current amplitude occurs at a membrane potential of ~0 mV, whereas the midpoint of steady-state inactivation was -60 mV, and complete relief of inactivation could occurred at -120 mV. Thus, an effective pulse protocol (Figure 1 Aa) for detecting both state- and use-dependent inhibition of peak test pulse currents consists of three phases of stimulation. A train of short test pulses (TP1 – TP10) at a 10 Hz-frequency, preceded by a preconditioning hyperpolarization to -120 mV allows measurement of tonic block of channels in the resting state (TP1) and serves as conditioning for use-dependent inhibition assessed at TP11. TP11 also provides a long inactivating conditioning pulse to assess inactivation-dependent inhibition evaluated by TP 12 after a brief sojourn at -60 mV to allow partial recovery of unblocked channels from inactivation.

The current traces shown in Figure 1 Ab-d illustrate the changes in lidocaine block under this test pulse regimen. Channels in the resting state (TP1, Fig. 1Ab) were the least sensitive to block. Block levels achieved by repetitive stimulation (TP11, Fig. 1Ac) showed use-dependent augmentation. The most effective channel inhibition was reached by prolonged depolarization (TP12, Fig. 1Ad) that corresponds to inactivation-state block. The concentration dependence of the three forms of lidocaine block is presented in Fig. 1B, where the leftmost curve (inverted triangles) represents inactivated state block. Use-dependence at 10 Hz produced intermediate block potency (triangles) and tonic block of resting channels (circles) was the least potent. This pattern of augmented block potency with repetitive stimulation or long depolarization is typical of local anesthetic sodium channel blockers. In contrast, anticonvulsants that block Na+ channels, such as carbamazepine (Fig. 1C) typically show less use-dependent block augmentation during repetitive stimulation, but strong increase in potency for blocking inactivated channels.

Lidocaine and carbamazepine have been shown to interact with a site in the cytoplasmic mouth of the channel pore that undergoes conformational changes during voltage-dependent gating.3 By contrast, the pufferfish toxin tetrodotoxin (Figure 1D), produces block that is insensitive to changes in membrane potential or channel gating, consistent with the evidence that the binding site is at the extracellular end of the pore and not influenced by gating-induced conformation changes. Compounds such as tetrodotoxin are extremely potent neurotoxins, killing by preventing normal Na+ channel activity.

Besides Na+ channel blockers, a variety of channel agonists have been identified that can cause adverse neurologic or cardiac effects. Examples include pyrethrins, derived from chrysanthemum flowers, and peptide toxins components of scorpions and jelly-fish venoms. Agonists typically potentiate Na+ flux, not by increasing single channel conductance, but by stabilizing the open state. This modifies channel gating such that transitions from open to closed or open to inactivated states become very slow, and the voltage dependence of activation gating shifts toward the resting membrane potential. As shown in Figure 2, synthetic pyrethroid agonists have distinctive effects on Na+ channel currents recorded in IonWorks Quattro. In Figure 2A, bioallethrin at 50 ?M exerts its effect by slowing the rate of inactivation such that the human Nav1.8 channels remain open longer to maintained depolarization. It is noteworthy that the analysis metrics required to quantify agonist effects in this software are quite different from those used for antagonists. We find that integrating the test pulse current rather than measuring current amplitude provides a useful index of agonist activity. Esfenvalerate, a more potent neurotoxin, (Figure 2B) produces near complete removal of inactivation during the test depolarization and slows the rate of channel closing upon repolarization to produce remarkably long “tail” currents. Both effects promote hyperexcitability in insect Na+ channels that accounts for their insecticidal effects and, at much higher concentrations, human neurotoxicity from environmental exposure.5

Coupled with the enhanced throughput capabilities of automated patch clamp systems, these results provide evidence that drug discovery, development, and safety screening projects can benefit from functional assessments of the pharmacological and toxicological properties of voltage-gated targets, such as Na+ channels, that previously had been accessible only through low-throughput manual patch clamp. Moreover, we anticipate that these methods will provide new insights into the functional pharmacologic and toxicologic selectivity of human ion channel isoforms and an efficient means of comparing these properties with those of homologous channels in animal species used in traditional in vivo drug research.

About the Author
Glenn Kirsch is the head of pharmacological sciences at ChanTest. His area of research interest is the pharmacology and molecular physiology of ion channels in excitable membranes, with focus on neuronal and cardiac channels.

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