Electrical activity in nerve, skeletal muscle, and heart requires finely tuned activity of voltage-gated Na+ channels that open and then enter a nonconducting inactivated state upon depolarization. Inactivation occurs when the gate, the cytoplasmic loop linking domains III and IV of the α subunit, occludes the open pore. Subtle destabilization of inactivation by mutation is causally associated with diverse human disease. Here we show for the first time that the inactivation gate is a molecular complex consisting of the III-IV loop and the COOH terminus (C-T), which is necessary to stabilize the closed gate and minimize channel reopening. When this interaction is disrupted by mutation, inactivation is destabilized allowing a small, but important, fraction of channels to reopen, conduct inward current, and delay cellular repolarization. Thus, our results demonstrate for the first time that physiologically crucial stabilization of inactivation of the Na+ channel requires complex interactions of intracellular structures and indicate a novel structural role of the C-T domain in this process.

Mutations in the sodium channel genes SCN1A and SCN2A have been identified in monogenic childhood epilepsies, but SCN3A has not previously been investigated as a candidate gene for epilepsy. We screened a consecutive cohort of 18 children with cryptogenic partial epilepsy that was classified as pharmacoresistant because of nonresponse to carbamazepine or oxcarbazepine, antiepileptic drugs that bind sodium channels. The novel coding variant SCN3A-K354Q was identified in one patient and was not present in 295 neurological normal controls. Twelve novel SNPs were also detected. K354Q alters an evolutionarily conserved amino acid in the pore domain of SCN3A. Functional analysis of this mutation in the backbone of the closely related gene SCN5A demonstrated an increase in persistent current that is similar in magnitude to epileptogenic mutations of SCN1A and SCN2A. This observation of a potentially pathogenic mutation of SCN3A (Nav1.3) indicates that this gene should be further evaluated for its contribution to childhood epilepsy.

Amino acids located in the outer vestibule of the voltage-gated Na؉ channel determine the permeation properties of the channel. Recently, residues lining the outer pore have also been implicated in channel gating. The domain (D) IV P-loop residue alanine 1529 forms a part of the putative selectivity filter of the adult rat skeletal muscle (1) Na ؉ channel. Here we report that replacement of alanine 1529 by aspartic acid enhances entry to an ultraslow inactivated state. Ultra-slow inactivation is characterized by recovery time constants on the order of ϳ100 s from prolonged depolarizations and by the fact that entry to this state can be reduced by binding to the pore of a mutant -conotoxin GIIIA, suggesting that ultra-slow inactivation may reflect a structural rearrangement of the outer vestibule. The voltage dependence of ultra-slow inactivation in DIV-A1529D is U-shaped, with a local maximum near ؊60 mV, whereas activation is maximal only above ؊20 mV. Furthermore, a train of brief depolarizations produces more ultra-slow inactivation than a single maintained depolarization of the same duration. These data suggest that ultra-slow inactivation emanates from "partially activated" closed states and that the P-loop in DIV may undergo a conformational change during channel activation, which is accentuated by DIV-A1529D. Upon depolarization voltage-gated Naϩ channels first open and then enter one or more inactivated states. These inactivated states can be separated by their contribution to the time course of recovery. Upon repolarization from brief (millisecond time scale) depolarizations recovery is characterized by a single kinetic phase with a time constant of a few milliseconds ("fast inactivation"). If adult rat skeletal muscle (1) Na ϩ channels, heterologously expressed in Xenopus oocytes, are inactivated for Ն20 ms and then repolarized, the channels recover from inactivation with three distinct time constants, implying that there are at least three distinct inactivated states. These time constants are in the order of several ms (fast inactivation

Membrane-impermeant quaternary derivatives of lidocaine (QX222 and QX314) block cardiac Na ؉ channels when applied from either side of the membrane, but they block neuronal and skeletal muscle channels poorly from the outside. To find the molecular determinants of the cardiac external QX access path, mutations of adult rat skeletal muscle ( 1) and rat heart (rH1) Na ؉ channels were studied by two-electrode voltage clamp in Xenopus oocytes. Mutating the 1 domain I P-loop Y401, which is the critical residue for isoform differences in tetrodotoxin block, to the heart sequence (Y401C) allowed outside QX222 block, but its mutation to brain type (Y401F) showed little block. 1-Y401C accelerated recovery from block by internal QX222. Block by external QX222 in 1-Y401C was diminished by chemical modification with methanethiosulfonate ethylammonium (MTSEA) to the outer vestibule or by a double mutant ( 1-Y401C͞F1579A), which altered the putative local anesthetic binding site. The reverse mutation in heart rH1-C374Y reduced outside QX314 block and slowed dissociation of internal QX222. Mutation of 1-C1572 in IVS6 to Thr, the cardiac isoform residue (C1572T), allowed external QX222 block, and accelerated recovery from internal QX222 block, as reported. Blocking efficacy of outside QX222 in 1-Y401C was more than that in 1-C1572T, and the double mutant ( 1-Y401C͞C1572T) accelerated internal QX recovery more than 1-Y401C or 1-C1572T alone. We conclude that the isoform-specific residue (Tyr͞Phe͞Cys) in the P-loop of domain I plays an important role in drug access as well as in tetrodotoxin binding. Isoform-specific residues in the IP-loop and IVS6 determine outside drug access to an internal binding site.V oltage-gated Na ϩ channels are membrane proteins responsible for generation of action potentials in nerve, skeletal muscle, and heart. The Na ϩ channel ␣-subunit consists of four homologous domains (I-IV), each containing six transmembrane segments (S1-S6) (1, 2). P-loops connecting S5 and S6 in each domain form the Na ϩ channel outer vestibule (3-7). Na ϩ channel isoforms from various tissues are highly homologous (2, 8). However, cardiac channels are relatively insensitive to tetrodotoxin (TTX) compared with brain and skeletal muscle channels (9, 10). This isoform difference is explained by an isoformspecific residue (Tyr͞Phe͞Cys) in the P-loop of domain I (IP-loop) (11-14) (Fig. 1). An aromatic residue at this position renders the channel TTX sensitive.The Na ϩ channel is also the site of action of local anesthetic antiarrhythmic drugs. Two conserved aromatic residues (Phe and Tyr) in IVS6 are involved in the local anesthetic binding site (15)(16)(17), and isoform differences in local anesthetic affinity are much less than in toxin affinity (18). In contrast, access of local anesthetic drugs differs markedly between Na ϩ channel isoforms. Quaternary membrane-impermeant derivatives of lidocaine such as QX314 and QX222 do not block nerve channels when applied to the outside (19,20), but outside QX314 blocks the cardiac chann...

Activation of G protein-gated inwardly rectifying potassium (GIRK) channels leads to a hyperpolarization of the neuron’s membrane potential, providing an important component of inhibition in the brain. In addition to the canonical G protein-activation pathway, GIRK channels are activated by small molecules but less is known about the underlying gating mechanisms. One drawback to previous studies has been the inability to control intrinsic and extrinsic factors. Here we used a reconstitution strategy with highly purified mammalian GIRK2 channels incorporated into liposomes and demonstrate that cholesterol or intoxicating concentrations of ethanol, i.e., >20 mM, each activate GIRK2 channels directly, in the absence of G proteins. Notably, both activators require the membrane phospholipid PIP2 but appear to interact independently with different regions of the channel. Elucidating the mechanisms underlying G protein-independent pathways of activating GIRK channels provides a unique strategy for developing new types of neuronal excitability modulators.