Paramyotonia congenita

ABSTRACT We tested the effects of charge-neutralizing mutations of the eight arginine residues in DIVS4 of the rat skeletal muscle sodium channel (rNav1.4) on deactivation gating from the open and inactivated states. We hypothesized that neutralization of outer or central charges would accelerate the I-to-C transition as measured by recovery delay because these represent a portion of the immobilizable charge. RIQ abbreviated recovery delay as a consequence of reduced charge content. R4Q increased delay, whereas R5Q abbreviated delay, and charge-substitutions at these residues indicated that each effect was allosteric. We also hypothesized that neutralization of any residue in DIVS4 would slow the O-to-C transition with reduction in positive charge. Reduction in charge at Ri, and to a lesser extent at R5, slowed open-state deactivation, while charge neutralizations at R2, R3, R4, R6, and R7 accelerated open-state deactivation. Our findings suggest that arginine residues in DIVS4 in rNav1.4 have differing roles in channel closure from open and inactivated states. Furthermore, they suggest that deactivation in DIVS4 is regulated by charge interaction between the electrical field with the outermost residue, and by local allosteric interactions imparted by central charges.

INTRODUCTION

Voltage-sensitive Na+ channels are responsible for the upstroke of the action potential in excitable cells (Hodgkin and Huxley, 1952; Keynes and Meves, 1993). Depolarization of the membrane potential promotes a transient increase in sodium permeability (activation), which is followed by rapid inactivation (Aldrich et al., 1983). The amino acid sequence of the sodium channel a-subunit predicts four homologous domains containing six transmembrane segments (Noda et al., 1984). The fourth segment (S4) in each domain contains positively charged residues, and several lines of experimentation suggest a role for sodium channel S4 segments as voltage sensors during activation (Stuhmer et al., 1989; Yang and Horn, 1995; Yang et al., 1996; Horn et al., 2000).

Unlike tetrameric potassium channels with identical charge content in S4 segments, sodium channel S4 segments possess unequal charge content. For example, in skeletal muscle sodium channels, DIVS4 contains charged residues at each turn of the proposed a helix (Trimmer et al, 1989). Previous studies have revealed that charged residues within the S4 segments may have domain-specific roles. For example, outer arginine residues in DIVS4 couple activation to fast inactivation (Chahine et al., 1994; Chen et al., 1996; Kontis and Goldin, 1997; Sheets et al., 1999; Horn et al., 2000) and central arginine and neutral residues in this voltage sensor play a role in slow inactivation (Mitrovic et al., 2000).

In sodium channels, a tripeptide IFM motif in the cytoplasmic linker between DIII and DIV is an important component of fast inactivation (Vassilev et al., 1988; West et al., 1992; Kellenberger et al., 1996; Horn et al., 2000). Fast inactivation is accompanied by immobilization of gating charge (Armstrong and Bezanilla, 1977). Immobilization of voltage sensors in DIII and DIV limits recovery from fast inactivation, such that the rate of recovery from fast inactivation is paralleled by recovery of immobilized charge (Cha et al., 1999; KUhn and Greef, 1999; Sheets et al., 1999, 2000). The outermost arginine residue (RI) and two central arginine residues (R4 and RS) in DIVS4 compose at least a portion of immobilizable charge (Ruben et al., 1999 KOM and Greef, 1999; Sheets et al., 1999) and may contribute to a domain-specific role of DIVS4 in fast inactivation.

Inactivated sodium channels must deactivate to become available for activation, with a voltage-sensitive delay before recovery from fast inactivation (Kuo and Bean, 1994). Limited transition through the open state has been described only in Nav1.6 (Raman and Bean, 2001). In hNav1.4, the delay in recovery (here called inactivated-state deactivation) is abbreviated by neutralization of the outermost charged residue in DIIIS4 (K1126C) or DIVS4 (R1448C; Ji et al., 1996; Groome et al., 1999). Reduced charge immobilization in R1448C (Ruben et al., 1999) may explain the abbreviation of recovery delay in this mutation, such that charge immobilization and transmembrane voltage are determinants of the rate of deactivation from the fast-inactivated state.

Charge neutralizing mutations of central residues R4 and RS in DIVS4 affect both charge immobilization and recovery from fast inactivation (Abbruzzese et al., 1998; Kuhn and Greef, 1999; Ruben et al., 1999), as does charge neutralization at RI. These data suggested to us that inactivated-state deactivation might be regulated by outer and central charged residues in DIVS4, with its high proportion of immobilizable charge.

Sodium channels that are opened by brief depolarization return to a closed state in response to hyperpolarization, here called open-state deactivation. The decay of current in this transition is usually best described by a monoexponential function (Rayner et al., 1993; Ji et al., 1996, Kontis et al., 1997; Featherstone et al., 1998), suggesting that a single S4 translocation to the hyperpolarized-favored position is functionally sufficient for channel closure. Domain-specific roles for S4 segments in open-state deactivation are suggested by the results of studies with charge-neutralizing mutations. For example, the O-to-C transition is accelerated by neutralizations of charged residues in DIS4 or DIIS4 (Kontis and Goldin, 1997; Groome et al., 1999). However, the O-to-C transition is slowed by neutralizations of charged residues in DIIIS4 (Kontis and Goldin, 1997; Groome et al., 1999) or of the outermost charged residue in DIVS4 (Ji et al., 1996; Groome et al., 1999), suggesting that deactivation is limited by charge content in DIII and DIV.

In the present study, we tested the hypotheses that open-- state deactivation (regulated by transmembrane electric field) is regulated by similar contributions from each of eight arginine residues in DIVS4, whereas inactivated-state deactivation (regulated by charge immobilization and electric field) is regulated primarily by outer and central residues in this voltage sensor. We find that activation is affected similarly by each of eight charge-neutralizing mutations in DIVS4. In contrast, fast inactivation and deactivation from either the open or inactivated state are differentially affected by these R/Q mutations. Our results suggest that translocations of DIVS4 from the open or inactivated state to a hyperpolarized-favored position are dependent on the electrostatic or allosteric character of charged residues in this voltage sensor. Portions of this work have been reported in abstract form (Groome et al., 2001).

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James Groome,*^ Esther Fujimoto,^ Lisa Walter,* and Peter Ruben^

*Department of Biology, Harvey Mudd College, Claremont, California 91711; and ^Department of Biology, Utah State University, Logan, Utah 84322-5305 USA

Submitted April 25, 2001, and accepted for publication December 7, 2001.

Address reprint requests to Dr. Peter C. Ruben, Dept. of Biology, Utah State University, Logan, UT 84322-5305. Tel.: 435-797-2136; Fax: 435-- 797-1575; E-mail: pruben@biology.usu.edu.

2002 by the Biophysical Society

0006-3495/02/03/1293/15 $2.00

We thank J. Repscher for help with experiments. We thank J. Abbruzzese and Y. Vilin for comments on a draft of this manuscript.

This work was supported by a Harvey Mudd College faculty research grant to J.G., and by Public Health Service Grant R01-NS29204 and a Research grant from the Muscular Dystrophy Association to P.R.

Copyright Biophysical Society Mar 2002
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