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In ether-à-go-go (eag) K(+) channels, extracellular divalent cations bind to the resting voltage sensor and thereby slow activation. Two eag-specific acidic residues in S2 and S3b coordinate the bound ion. Residues located at analogous positions are approximately 4 A apart in the x-ray structure of a Kv1.2/Kv2.1 chimera crystallized in the absence of a membrane potential. It is unknown whether these residues remain in proximity in Kv1 channels at negative voltages when the voltage sensor domain is in its resting conformation. To address this issue, we mutated Shaker residues I287 and F324, which correspond to the binding site residues in eag, to aspartate and recorded ionic and gating currents in the presence and absence of extracellular Mg(2+). In I287D+F324D, Mg(2+) significantly increased the delay before ionic current activation and slowed channel opening with no readily detectable effect on closing. Because the delay before Shaker opening reflects the initial phase of voltage-dependent activation, the results indicate that Mg(2+) binds to the voltage sensor in the resting conformation. Supporting this conclusion, Mg(2+) shifted the voltage dependence and slowed the kinetics of gating charge movement. Both the I287D and F324D mutations were required to modulate channel function. In contrast, E283, a highly conserved residue in S2, was not required for Mg(2+) binding. Ion binding affected activation by shielding the negatively charged side chains of I287D and F324D. These results show that the engineered divalent cation binding site in Shaker strongly resembles the naturally occurring site in eag. Our data provide a novel, short-range structural constraint for the resting conformation of the Shaker voltage sensor and are valuable for evaluating existing models for the resting state and voltage-dependent conformational changes that occur during activation. Comparing our data to the chimera x-ray structure, we conclude that residues in S2 and S3b remain in proximity throughout voltage-dependent activation.

Transfer of ion binding site from ether-à-go-go to Shaker: Mg2+ binds to resting state to modulate channel opening