NIH Research Festival
FARE Award Winner
Voltage-gated K+ (Kv) channels are homotetramers. Each subunit is formed by six transmembrane segments, S1 to S6. The permeation pathway is formed by S5, the P-region and S6 from each subunit, that assemble around a central canal through which K+ permeate. In response to voltage changes, the voltage sensor domain (S1-S4) undergoes conformational changes that are coupled to the opening and closing of an intracellular gate located at the inner end of S6. It is known, that a cysteine at position 476 in S6 (near the gate) traps the mutant channel in the open state when Cd2+ is added to the intracellular solution. This behavior is caused by a metal bridge between the cysteine at 476 of one subunit and a native histidine at 486 in an adjacent subunit. We constructed a Shaker Kv concatemer that has all 4 subunits linked at the DNA level. When all four V476 are mutated to cysteine, intracellular Cd2+, locked the channels open as in wild-type channels. We are presently developing concatemer channels with one, two and three metal bridges to assess one by one their contribution to the opening of the channels. Because the metal bridges can be disrupted by a H486Y mutation, this approach should allow us to discern the assembly of a functional concatemer channel in the membrane.
Scientific Focus Area: Biomedical Engineering and Biophysics
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