Modulates Voltage-dependent Activation in Ether-à-go-go Potassium Channels by Binding between Transmembrane Segments S2 and S3

Extracellular Mg 2 1 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, Mg 2 1 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 Mg 2 1 -binding site. Two eag-specific acidic residues and three acidic residues found in the S2 and S3 segments of all voltage-dependent K 1 channels were individually mutated in Drosophila eag, mutant channels were expressed in Xenopus oocytes, and the effect of Mg 2 1 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 Mg 2 1 -sensitivity and mimicked the slowing of activation kinetics caused by Mg 2 1 binding to the wild-type channel. These results suggest that Mg 2 1 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 Mg 2 1 , indicating that D284 and D319 do not mediate the slowing of activation caused by Mg 2 1 binding. Mutations at D284 affected the eag gating pathway, shifting the voltage dependence of Mg 2 1 -sensitive, rate limiting transitions in the hyperpolarized direction. Another widely conserved residue, D274 in S2, is not required for Mg 2 1 sensitivity but is in the vicinity of the binding site. We conclude that Mg 2 1 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 1 channels, and suggests a molecular mechanism by which extracellular cations modulate eag activation kinetics. key words: voltage clamp • structural model • kinetics • voltage sensor • metal binding site I N T R O D U C T I O N The Drosophila ether-à-go-go (eag) gene and its homologues encode a distinct subfamily of voltage-gated K 1 channels (Warmke et al., 1991; Brüggemann et al., 1993; Warmke and Ganetzky, 1994; Wei et al., 1996). In most members of the eag family, activation kinetics are dramatically regulated by extracellular Mg 2 1 , an effect that is not seen in other types of voltage-gated K 1 channels (Terlau et al., 1996; Frings et al., 1998; Schönherr et al., 1999; Tang et al., 2000). Analysis of eag ionic and gating currents indicates that Mg 2 1 directly modulates the process of voltage-dependent gating, presumably by binding to a site in or near the voltage sensor (Terlau et al., 1996, Tang et al., 2000). To investigate the mechanism by which Mg 2 1 regulates voltage-dependent activation and to gain novel insights into the structure and function of the voltage sensor, we have identified the Mg 2 1 -binding site in eag channels. In proteins, bound Mg 2 1 ions are often coordinated by the acidic side chains of aspartate and glutamate residues (da Silva and Williams, 1991). Therefore, our analysis focused on acidic amino acids found in and near transmembrane segments S2 through S4, a region that includes essential components of the voltage sensor in K 1 channels (Liman et al., 1991; Papazian et al., 1991, 1995; Perozo et al., 1994; Planells-Cases et al., 1995; Aggarwal and MacKinnon, 1996; Mannuzzu et al., 1996; Seoh et al., 1996; Cha and Bezanilla, 1997; Cha et al., 1999; Glauner et al., 1999). Segments S2 and S3 contain three acidic residues that are conserved among all subfamilies of voltage-gated K 1 channels (Warmke and Ganetzky, 1994; Chandy and Gutman, 1995). Previous work indicates that these residues make important contributions to the biogenesis, structure, and function of the voltage sensor in Shaker K 1 channels (Papazian et al., 1995; Planells-Cases et al., 1995; C.-Y. Tang’s current address is Department of Neurology, UCLA School of Medicine, Los Angeles, CA 90095. Address correspondenceto Diane M. Papazian, Ph.D., Department of Physiology, UCLA School of Medicine, Los Angeles, CA 900951751. Fax: (310) 206-5661; E-mail: [email protected] on Jne 1, 2017 D ow nladed fom Published October 30, 2000