A Molecular Switch between the Outer and the Inner Vestibules of the Voltage-gated Na+ Channel

Gregory M Lipkind,Markus Mille,Hannes Todt,Xaver Koenig,Walter Sandtner,Touran Zarrabi,Peter Lukacs,Harry A Fozzard,Rene Cervenka,Karlheinz Hilber

doi:10.1074/jbc.m110.132886

Gregory M Lipkind, Markus Mille + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m110.132886

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Abstract

Voltage-gated ion channels are transmembrane proteins that undergo complex conformational changes during their gating transitions. Both functional and structural data from K+ channels suggest that extracellular and intracellular parts of the pore communicate with each other via a trajectory of interacting amino acids. No crystal structures are available for voltage-gated Na+ channels, but functional data suggest a similar intramolecular communication involving the inner and outer vestibules. However, the mechanism of such communication is unknown. Here, we report that amino acid Ile-1575 in the middle of transmembrane segment 6 of domain IV (DIV-S6) in the adult rat skeletal muscle isoform of the voltage-gated sodium channel (rNaV1.4) may act as molecular switch allowing for interaction between outer and inner vestibules. Cysteine scanning mutagenesis of the internal part of DIV-S6 revealed that only mutations at site 1575 rescued the channel from a unique kinetic state (“ultra-slow inactivation,” IUS) produced by the mutation K1237E in the selectivity filter. A similar effect was seen with I1575A. Previously, we reported that conformational changes of both the internal and the external vestibule are involved in the generation of IUS. The fact that mutations at site 1575 modulate IUS produced by K1237E strongly suggests an interaction between these sites. Our data confirm a previously published molecular model in which Ile-1575 of DIV-S6 is in close proximity to Lys-1237 of the selectivity filter. Furthermore, these functional data define the position of the selectivity filter relative to the adjacent DIV-S6 segment within the ionic permeation pathway.

Highlights

Ion channels are transmembrane proteins that regulate the flow of ions across the cell membrane
Terminal end of the pore helix resulting in a structural change that leads to a non-conductive conformation of the selectivity filter [8, 9]
To explain the involvement of both the outer and the inner vestibule in IUS, we proposed a model in which the mutation K1237E produces a conformational change of the outer vestibule, which is transmitted to the internal vestibule through the DIV-S6 segment [12, 14, 15]

Summary

Introduction

Ion channels are transmembrane proteins that regulate the flow of ions across the cell membrane. These structural data for the first time confirmed that in Kϩ channels some forms of inactivation encompass an interaction between the outer vestibule (P-loops) and the inner helices (corresponding to the S6 segment of voltage-gated Naϩ channels).

Results

Conclusion