Mg²⁺ Modulates Voltage-Dependent Activation in Ether-à-Go-Go Potassium Channels by Binding between Transmembrane Segments S2 and S3

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.