The S4 segment comprises part of the voltage sensor in Shaker K+ channels. We have used a strategy similar to intragenic suppression, but without a genetic selection, to identify electrostatic interactions of the S4 segment that may be important in the mechanism of voltage-dependent activation. The S4 neutralization mutations K374Q and R377Q block maturation of the protein, suggesting that they prevent proper folding. K374Q is specifically and efficiently rescued by the second site mutations E293Q and D316N, located in putative transmembrane segments S2 and S3, respectively. These results suggest that K374, E293, and D316 form a network of strong, local, electrostatic interactions that stabilize the structure of the channel. Some other double mutant combinations result in inefficient suppression, identifying weak, presumably long-range electrostatic interactions. A simple structural hypothesis is proposed to account for the effects of the rescued double mutant combinations on the relative stabilities of open and closed channel conformations.

Potassium channel mutations have been described in episodic neurological diseases. We report that K+ channel mutations cause disease phenotypes with neurodevelopmental and neurodegenerative features. In a Filipino adult-onset ataxia pedigree, the causative gene maps to 19q13, overlapping the SCA13 disease locus described in a French pedigree with childhood-onset ataxia and cognitive delay. This region contains KCNC3 (also known as Kv3.3), encoding a voltage-gated Shaw channel with enriched cerebellar expression. Sequencing revealed two missense mutations, both of which alter KCNC3 function in Xenopus laevis expression systems. KCNC3(R420H), located in the voltage-sensing domain, had no channel activity when expressed alone and had a dominant-negative effect when co-expressed with the wild-type channel. KCNC3(F448L) shifted the activation curve in the negative direction and slowed channel closing. Thus, KCNC3(R420H) and KCNC3(F448L) are expected to change the output characteristics of fast-spiking cerebellar neurons, in which KCNC channels confer capacity for high-frequency firing. Our results establish a role for KCNC3 in phenotypes ranging from developmental disorders to adult-onset neurodegeneration and suggest voltage-gated K+ channels as candidates for additional neurodegenerative diseases.

In voltage-dependent Shaker K+ channels, charged residues E293 in transmembrane segment S2 and R365, R368, and R371 in S4 contribute significantly to the gating charge movement that accompanies activation. Using an intragenic suppression strategy, we have now probed for structural interaction between transmembrane segments S2, S3, and S4 in Shaker channels. Charge reversal mutations of E283 in S2 and K374 in S4 disrupt maturation of the protein. Maturation was specifically and efficiently rescued by second-site charge reversal mutations, indicating that electrostatic interactions exist between E283 in S2 and R368 and R371 in S4, and between K374 in S4 and E293 in S2 and D316 in S3. Rescued subunits were incorporated into functional channels, demonstrating that a native structure was restored. Our data indicate that K374 interacts with E293 and D316 within the same subunit. These electrostatic interactions mediate the proper folding of the protein and are likely to persist in the native structure. Our results raise the possibility that the S4 segment is tilted relative to S2 and S3 in the voltage-sensing domain of Shaker channels. Such an arrangement might provide solvent access to voltage-sensing residues, which we find to be highly tolerant of mutations.

A recently proposed model for voltage-dependent activation in K+ channels, largely influenced by the KvAP X-ray structure, suggests that S4 is located at the periphery of the channel and moves through the lipid bilayer upon depolarization. To investigate the physical distance between S4 and the pore domain in functional channels in a native membrane environment, we engineered pairs of cysteines, one each in S4 and the pore of Shaker channels, and identified two instances of spontaneous intersubunit disulfide bond formation, between R362C/A419C and R362C/F416C. After reduction, these cysteine pairs bound Cd2+ with high affinity, verifying that the residues are in atomic proximity. Molecular modeling based on the MthK structure revealed a single position for S4 that was consistent with our results and many other experimental constraints. The model predicts that S4 is located in the groove between pore domains from different subunits, rather than at the periphery of the protein.

Extracellular Mg2+ directly modulates voltage-dependent activation in ether-à-go-go (eag) potassium channels, slowing the kinetics of ionic and gating currents (Tang, C.-Y., F. Bezanilla, and D.M. Papazian. 2000. J. Gen. Physiol. 115:319-337). To exert its effect, Mg2+ presumably binds to a site in or near the eag voltage sensor. We have tested the hypothesis that acidic residues unique to eag family members, located in transmembrane segments S2 and S3, contribute to the Mg2+-binding site. Two eag-specific acidic residues and three acidic residues found in the S2 and S3 segments of all voltage-dependent K+ channels were individually mutated in Drosophila eag, mutant channels were expressed in Xenopus oocytes, and the effect of Mg2+ on ionic current kinetics was measured using a two electrode voltage clamp. Neutralization of eag-specific residues D278 in S2 and D327 in S3 eliminated Mg2+-sensitivity and mimicked the slowing of activation kinetics caused by Mg2+ binding to the wild-type channel. These results suggest that Mg2+ modulates activation kinetics in wild-type eag by screening the negatively charged side chains of D278 and D327. Therefore, these residues are likely to coordinate the bound ion. In contrast, neutralization of the widely conserved residues D284 in S2 and D319 in S3 preserved the fast kinetics seen in wild-type eag in the absence of Mg2+, indicating that D284 and D319 do not mediate the slowing of activation caused by Mg2+ binding. Mutations at D284 affected the eag gating pathway, shifting the voltage dependence of Mg2+-sensitive, rate limiting transitions in the hyperpolarized direction. Another widely conserved residue, D274 in S2, is not required for Mg2+ sensitivity but is in the vicinity of the binding site. We conclude that Mg2+ binds in a water-filled pocket between S2 and S3 and thereby modulates voltage-dependent gating. The identification of this site constrains the packing of transmembrane segments in the voltage sensor of K+ channels, and suggests a molecular mechanism by which extracellular cations modulate eag activation kinetics.