Structural Basis of Nav1.7 Inhibition by a Gating-Modifier Spider Toxin.

Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable ∼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.

A novel NMDA receptor positive allosteric modulator that acts via the transmembrane domain.

Ionotropic glutamate receptors (iGluRs) mediate fast excitatory neurotransmission and are key nervous system drug targets. While diverse pharmacological tools have yielded insight into iGluR extracellular domain function, less is known about molecular mechanisms underlying the ion conduction gating process within the transmembrane domain (TMD). We have discovered a novel NMDAR positive allosteric modulator (PAM), GNE-9278, with a unique binding site on the extracellular surface of the TMD. Mutation of a single residue near the Lurcher motif on GluN1 M3 can convert GNE-9278 modulation from positive to negative, and replacing three AMPAR pre-M1 residues with corresponding NMDAR residues can confer GNE-9278 sensitivity to AMPARs. Modulation by GNE-9278 is state-dependent and significantly alters extracellular domain pharmacology. The unique properties and structural determinants of GNE-9278 reveal new modulatory potential of the iGluR TMD.

Pannexin-1 is blocked by its C-terminus through a delocalized non-specific interaction surface.

The Pannexin-1 (Panx1) channel is known to become activated under a variety of physiological conditions resulting in the release of medium-sized molecules such as ATP and amino acids 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 from the intracellular side. We sought to 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 fails to disrupt the ability of the c-terminus to keep the channel closed. Surprisingly, even replacing the entire c-terminal gating peptide with a scrambled peptide of the same length maintains 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.