Voltage-gated sodium (Na V 1) channels play a critical role in modulating the excitability of sensory neurons, and human genetic evidence points to Na V 1.7 as an essential contributor to pain signaling. Human loss-of-function mutations in SCN9A, the gene encoding Na V 1.7, cause channelopathy-associated indifference to pain (CIP), whereas gain-of-function mutations are associated with two inherited painful neuropathies. Although the human genetic data make Na V 1.7 an attractive target for the development of analgesics, pharmacological proof-of-concept in experimental pain models requires Na V 1.7-selective channel blockers. Here, we show that the tarantula venom peptide ProTx-II selectively interacts with Na V 1.7 channels, inhibiting Na V 1.7 with an IC 50 value of 0.3 nM, compared with IC 50 values of 30 to 150 nM for other heterologously expressed Na V 1 subtypes. This subtype selectivity was abolished by a point mutation in DIIS3. It is interesting that application of ProTx-II to desheathed cutaneous nerves completely blocked the C-fiber compound action potential at concentrations that had little effect on A␤-fiber conduction. ProTx-II application had little effect on action potential propagation of the intact nerve, which may explain why ProTx-II was not efficacious in rodent models of acute and inflammatory pain. Mono-iodo-ProTx-II ( 125 I-ProTx-II) binds with high affinity (K d ϭ 0.3 nM) to recombinant hNa V 1.7 channels. Binding of 125 IProTx-II is insensitive to the presence of other well characterized Na V 1 channel modulators, suggesting that ProTx-II binds to a novel site, which may be more conducive to conferring subtype selectivity than the site occupied by traditional local anesthetics and anticonvulsants. Thus, the 125 I-ProTx-II binding assay, described here, offers a new tool in the search for novel Na V 1.7-selective blockers.Pain relief remains an important, currently unmet, medical need. Voltage-gated sodium channels play a critical role in modulating the excitability of most neurons, including nociceptive sensory neurons signaling pain. Despite the clinical use of systemically administered lidocaine to treat chronic pain since the 1950s (Kugelberg and Lindblom, 1959) and the approval of the weak sodium channel blocker carbamazepine for the treatment of trigeminal neuralgia (Campbell et al., 1966), several oral sodium channel blockers have failed to show efficacy in large clinical trials (Wallace et al., 2002;Vinik et al., 2007). The failure of drugs in the clinic may at least partially be attributed to the narrow therapeutic window of non-subtype-selective sodium channel blockers.Recently, three publications have shown that loss-of-function mutations in the sodium channel subtype Na V 1.7 are the cause for channelopathy-associated insensitivity to pain (CIP) (Cox et al., 2006;Ahmad et al., 2007;Goldberg et al., 2007). Furthermore, genetic linkage analysis has identified gain-of-function mutations in Na V 1.7 as the cause of inherited erythromelalgia (Yang et al., 2004;Han et al., ...

The transmission of pain signals after injury or inflammation depends in part on increased excitability of primary sensory neurons. Nociceptive neurons express multiple subtypes of voltagegated sodium channels (Na V1s), each of which possesses unique features that may influence primary afferent excitability. Here, we examined the contribution of Na V1.9 to nociceptive signaling by studying the electrophysiological and behavioral phenotypes of mice with a disruption of the SCN11A gene, which encodes Na V 1.9. Our results confirm that Na V1.9 underlies the persistent tetrodotoxin-resistant current in small-diameter dorsal root ganglion neurons but suggest that this current contributes little to mechanical thermal responsiveness in the absence of injury or to mechanical hypersensitivity after nerve injury or inflammation. However, the expression of Na V1.9 contributes to the persistent thermal hypersensitivity and spontaneous pain behavior after peripheral inflammation. These results suggest that inflammatory mediators modify the function of NaV1.9 to maintain inflammation-induced hyperalgesia.T he generation and propagation of action potentials in sensory neurons depends on the activity of voltage-gated sodium channels (Na V 1s). The differential expression of Na V 1 subtypes in distinct classes of sensory neurons, combined with their unique biophysical properties, suggest specific roles for each subtype in sensory transmission. Sodium channels in sensory neurons can be classified pharmacologically as sensitive to block by low nanomolar concentrations of tetrodotoxin (TTX) or resistant to Ͼ1 M TTX (1, 2).The contribution of TTX-resistant Na V 1 channel subtypes to the transmission of pain signals is an important area of focus: TTXresistant current carries the majority of charge during action potentials in nociceptive neurons (3), and this current is dynamically regulated in response to injury (4, 5). Na V 1.8, expressed primarily in C-fibers (6), underlies a TTX-resistant current with a high threshold for activation and steady-state inactivation and slow kinetics (7). Comparisons between dorsal root ganglion (DRG) neurons from WT and Na V 1.8 null mutant (Ϫ͞Ϫ) mice suggest that Na V 1.8 contributes the majority of the inward current flowing during action potentials in small-diameter neurons (8). Antisense oligonucleotides directed against Na V 1.8 implicate this channel in both neuropathic (9) and inflammatory (10) pain conditions in rats, although Na V 1.8Ϫ͞Ϫ mice displayed only a mild phenotype (7,11).The functional role of Na V 1.9, another subtype selectively expressed in nociceptors (12), remains poorly defined. The primary sequence of Na V 1.9 predicts that this subtype conducts sodium currents resistant to TTX (13). Indeed, a second TTX-resistant current is present in DRG neurons from Na V 1.8 knockout mice (14). This current has been referred to as the persistent, TTXresistant current because of its negative threshold for activation and depolarized midpoint of inactivation, resulting in a significant windo...

Delayed-rectifier K؉ currents (I DR ) in pancreatic ␤-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. The voltagegated K ؉ channel, K V 2.1, is expressed in ␤-cells, and the biophysical characteristics of heterologously expressed channels are similar to those of I DR in rodent ␤-cells. A novel peptidyl inhibitor of K V 2.1/K V 2.2 channels, guangxitoxin (GxTX)-1 (half-maximal concentration ϳ1 nmol/l), has been purified, characterized, and used to probe the contribution of these channels to ␤-cell physiology. In mouse ␤-cells, GxTX-1 inhibits 90% of I DR and, as for K V 2.1, shifts the voltage dependence of channel activation to more depolarized potentials, a characteristic of gating-modifier peptides. GxTX-1 broadens the ␤-cell action potential, enhances glucose-stimulated intracellular calcium oscillations, and enhances insulin secretion from mouse pancreatic islets in a glucose-dependent manner. These data point to a mechanism for specific enhancement of glucose-dependent insulin secretion by applying blockers of the ␤-cell I DR , which may provide advantages over currently used therapies for the treatment of type 2 diabetes.

Correspondence to Owen McManus: o w e n _ m c m a n u s @ m e r c k . c o m Ion channels are well recognized as important therapeutic targets for treating a number of different pathophysiologies. Historically, however, development of drugs targeting this protein class has been diffi cult. Several challenges associated with molecular-based drug discovery include validation of new channel targets and identifi cation of acceptable medicinal chemistry leads. Proof of concept approaches, focusing on combined molecular biological/pharmacological studies, have been successful. New, functional, high throughput screening (HTS) strategies developed to identify tractable lead structures, which typically are not abundant in small molecule libraries, have also yielded promising results. Automated cell-based HTS assays can be confi gured for many different types of ion channels using fl uorescence methods to monitor either changes in membrane potential or intracellular calcium with high density format plate readers. New automated patch clamp technologies provide secondary screens to confi rm the activity of hits at the channel level, to determine selectivity across ion channel superfamilies, and to provide insight into mechanism of action. The same primary and secondary assays effectively support medicinal chemistry lead development. Together, these methodologies, along with classical drug development practices, provide an opportunity to discover and optimize the activity of ion channel drug development candidates. A case study with voltage-gated sodium channels is presented to illustrate these principles.

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