The high sensitivity of voltage-gated ion channels to changes in membrane potential implies that the process of channel opening is accompanied by large charge movements. Previous estimates of the total charge displacement, q, have been deduced from the voltage dependence of channel activation and have ranged from 4 to 8 elementary charges (e0). A more direct measurement of q in Drosophila melanogaster Shaker 29-4 potassium channels yields a q value of 12.3 e0. A similar q value is obtained from mutated Shaker channels having reduced voltage sensitivity. These results can be explained by a model for channel activation in which the equilibria of voltage-dependent steps are altered in the mutant channels.

Significance Voltage-gated sodium (Na v ) channels contribute to physiological and pathophysiological electrical signaling in nerve and muscle cells. Because Na v channel isoforms exhibit tissue-specific expression, subtype selective modulation of this channel family provides important drug development opportunities. However, most available Na v channel modulators are unable to distinguish between Na v channel subtypes, which limits their therapeutic utility because of cardiac or nervous system toxicity. This study describes a new class of subtype selective Na v channel inhibitors that interact with a region of the channel that controls voltage sensitivity. This interaction site may enable development of selective therapeutic interventions with reduced potential for toxicity.

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A leucine heptad repeat is well conserved in voltage-dependent ion channels. Herein we examine the role of the repeat region in Shaker K+ channels through substitution of the leucines in the repeat and through coexpression of normal and truncated products. In contrast to leucine-zipper DNA-binding proteins, we find that the subunit assembly of Shaker does not depend on the leucine heptad repeat. Instead, we report that substitutions of the leucines in the repeat produce large effects on the observed voltage dependence of conductance voltage and prepulse inactivation curves. Our results suggest that the leucines mediate interactions that play an important role in the transduction of charge movement into channel opening and closing.The Shaker gene family (Sh) encodes proteins that produce voltage-dependent K+-selective currents (1-3). Like other voltage-dependent ion channels, Sh channels open and close an aqueous pore by undergoing cotnformational transitions in response to changes in membrane potential. This gating behavior includes the movement of a charged component or voltage sensor (4, 5). Interestingly, mutagenesis of charged residues in the S4 domain (a proposed transmembrane segment that contains four to eight basic residues and is found in virtually all voltage-dependent ion channels including Sh) of the rat II Na' channel showed that it exhibits some of the properties expected for a voltage sensor (6). However, the voltage-dependent gating mechanism remains unclear; in particular, it is not known how movement of the voltage sensor(s) is transduced into the conformational transitions that result in opening and closing of the channel pore.Functional Sh channels are likely to be tetramers since Na+ and Ca2" channels are composed of four homology domains, each roughly equivalent to a single K+ channel subunit (7). Although recent work has shown that Sh channels are multimeric (8), the sites of subunit interaction are unknown. Sh channels contain a conserved leucine heptad repeat (five leucines long) that overlaps two proposed transmembrane segments (S4 and S5); similar motifs are found in Na+ and Ca2+ channels (9) (Fig. 1). Ion-channel leucine heptad repeats are preceded by, and partially overlap, the basic S4 domain (Fig. 1) Deduced amino acid alignments of the leucine-heptadrepeat region. Basic residues in S4 (stars) and leucine residues in the heptad repeat (boxed) were aligned. Amino acids identical to Sh are indicated by dashes. Proposed transmembrane segments S4 and S5 are indicated. Sh (10) is the Drosophila Shaker channel. Other K+ channel sequences are a rat Sh homolog (RCK1) (2), two human Sh homologs (HukI and HukII) (11), and Shab, Shaw, and Shal, which represent other Drosophila K+ channel genes (3). Na and Ca sequences are from the second homology domains of the rat Ila Na' channel and the skeletal muscle dihydropyridine receptor (12), respectively.Since Sh K+ channels are multimeric it suggests that the leucine heptad repeat might act as a site for subunit assembly. In addition, the c...