Mg2+ modulates voltage-dependent activation in ether-a-go-go potassium channels by binding between transmembrane segments S2 and S3

Extracellular Mg2+ directly modulates voltage-dependent activation in ether -a-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. Phys iol. 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 r esidues unique to eag family members, located in transmembrane segments S2 and S3, contribute to the Mg2+-binding site. Two eag-specific acidic residu es 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. Neutral ization of eag-specific residues D278 in S2 and D327 in S3 eliminated Mg2+- sensitivity and mimicked the slowing of activation kinetics caused by Mg2binding to the wild-type channel. These results suggest that Mg2+ modulates activation kinetics in wild-type eag by screening the negatively charged s ide chains of D278 and D327. Therefore, these residues are likely to coordi nate the bound ion. In contrast, neutralization of the widely conserved res idues D284 in S2 and D319 in 83 preserved the fast kinetics seen in wild-ty pe 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 affect ed the eag gating pathway, shifting the voltage dependence of Mg2+-sensitiv e, rate limiting transitions in the hyperpolarized direction. Another widel y conserved residue, D274 in 82, is not required for Mg2+ sensitivity but i s in the vicinity of the binding site. We conclude that Mg2+ binds in a wat er-filled pocket between 82 and S3 and thereby modulates voltage-dependent gating. The identification of this site constrains the packing of transmemb rane segments in the voltage sensor of K+ channels, and suggests a molecula r mechanism by which extracellular cations modulate eag activation kinetics .