Congruent Pattern of Accessibility within the Pore of a Voltage-Gated Na+ Channel

Abstract

Voltage-gated ion channels open a gate in response to membrane depolarization which allows ions to pass through the transmembrane pore of the channel. MTSET accessibility studies within the Shaker potassium channel revealed that K+ permeation is controlled by an intracellular gate, whereas Cd2+ and Ag+ accessibility measurements in the cyclic nucleotide-gated channel suggests that this process occurs at the selectivity filter. Modification data of cysteine residues introduced into the DIV S6 of a fast-inactivation removed voltage-gated sodium channel (VGSC) by MTSET indicate that sodium channel gating is also regulated by an intracellular gate. However, unlike the Shaker potassium channel, VGSCs are not composed of four identical subunits. Despite sequence similarity between each S6 helix, the multi-domain nature of VGSCs implies that there may be asymmetry within the pore that may have consequences in regard to channel gating. Thus, we sought to determine if analogous positions within the S6 helices of a VGSC act together to form an intracellular gate which occludes the pore while channels are closed. We scanned the MTSET accessibility of substituted cysteines in the pore lining helices of the first three domains (DI-DIII) in the rat skeletal muscle sodium channel. The modification data at these sites, selected on the basis of sequence alignment and MTSET accessibility data of DIV-S6, confirms that DII and DIII follow the same pattern of accessibility as that of DIV; however, the extent of block is quite varied across each domain. Along with DI accessibility data, these findings will be discussed in the context of pore gating in related voltage-gated ion channels.