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The voltage-gated sodium channel Nav1.7 is one of the most validated pain targets, as gain-of-function mutations cause excessive pain, whereas loss-of-function mutations produce insensitivity to pain. Targeting Nav1.7 holds great promise in treating pain, yet presents a tantalizing challenge, as few Nav1.7 selective chemical scaffolds exist. So far millions of compounds have been screened by using fluorescence membrane potential (MP) assays, but very few selective hits have been identified. Here we show that the conventional MP approach, which uses Nav1.7 WT channel and the activator veratridine, is intrinsically flawed: it is biased toward non-selective pore blockers which compete with veratridine, and fails to detect highly potent, selective compounds such as the aryl sulfonamide PF-771. By using a mutant channel and an alternative activator, we developed a novel method that not only robustly detects known Nav1.7 blockers, but also decreases the number of non-selective pore blocker hits. We conducted a high-throughput screen using this methodology and identified novel Nav1.7-selective blockers.

Pannexin 1 (Panx1) is a member of a family of large-pore ion channels distantly related to invertebrate gap junction channels, the innexins. Activation of Panx1 occurs under a variety of physiological processes, but the molecular mechanism of such activation has not in general been clearly established. Recently it was shown that Panx1 could be activated by direct cleavage near the c-terminus by caspase-3. This occurs during apoptosis and leads to the release of large intracellular molecules such as ATP, which act as “find-me” signals enabling nearby phagocytes to quickly clear dying cells. The C-terminal domain peptide seems to function as a tethered pore blocker and is thought to have a specific interaction with structural elements within the pore to inhibit the channel. However, the basis for this structural interaction is poorly understood. Here we dissect those interactions using progressive alanine substitutions and long alanine repeats within the region distal to the caspase cleavage site. Our results suggest that any specific interaction between the pore and the C-terminus is minimal since replacement of the amino acid residues in the region adjacent to the caspase cleavage site with alanines does not produce a constitutively open channel. The most important feature of the C-terminal inhibitory peptide appears instead to simply be its length. We therefore propose that the c-terminal peptides loop back into the pore to reach a constricted region, thereby blocking the pore by sterically interfering with ion permeation. Peptides not long enough to reach this constriction within the pore are consequently unable to block the channel while longer peptides are able to block the pore in a promiscuous fashion.

The Pannexin-1 (Panx1) channel is known to become activated under a variety of physiological conditions resulting in the release of large molecules such as ATP from the cell. The detailed molecular mechanism of activation of the channel resulting in the opening of the Pannexin pore is poorly understood. The best-studied gating mechanism is caspase-3/7-mediated cleavage and truncation of the c-terminus. In the absence of caspase-cleavage, the c-terminal peptide maintains the channel in the closed state, possibly by directly plugging the pore. We sought to better understand in detail the part of the c-terminus necessary for this interaction by alanine-scanning and truncation mutagenesis of the c-terminal gating peptide. These experiments demonstrate that no single amino acid side-chain is necessary for this interaction. In fact, replacing blocks of 10-12 amino acids in different parts of the c-terminal peptide with alanines failed to disrupt the ability of the c-terminus to keep the channel blocked. Surprisingly, even replacing the entire c-terminal gating peptide with a scrambled peptide of the same length could maintain the interaction in some cases. Further analysis revealed that the interaction surface, while delocalized, is located within the amino-terminal two-thirds of the c-terminal peptide. Such a delocalized and potentially low-affinity interaction surface is allowed due to the high effective concentration of the c-terminal peptide near the inner vestibule of the pore and likely explains why this region is poorly conserved between species. This type of weak interaction with a tethered gating peptide may be required to maintain high-sensitivity to caspase-dependent activation.