The Interaction of Voltage-Sensor and Gate in BK Channels

Abstract

To study the molecular basis and mechanism of voltage sensor/gate interaction in BK channels we performed an alanine-scan of the S4-S5 linker and the C-terminal end of S6 in mSlo1. Potassium conductance was measured over a wide range of voltage and calcium and fit to the Horrigan-Aldrich (HA) gating model. Open probability was measured at extreme negative voltages to determine if mutations altered the stability of the gate when voltage sensors are not activated (L in the HA model). Mutations in both linker and S6 were identified that alter L and therefore could be involved in voltage-sensor/gate interaction. The largest inhibitory effects were observed at the ends of S4-S5 linker (Q222A, I233A and K234A) and P319A in S6, each of which decreased L by 10- to 100-fold and shifted V0.5 to more positive voltages. I233A, unlike Q222A or K234A, also produced a decrease in voltage-dependence that is consistent with a decrease in voltage-sensor/gate coupling (D in the HA model). A structural model, based on Kv1.2, suggests inter- or intra-subunit interactions could potentially exist between these sites. Similarly, mutations near the end of S6 (R329A, K330A) that produce an approximate 3 fold increase in L without changing V0.5, may decrease voltage-sensor/gate coupling and could potentially interact with the N-terminal end of the linker where mutations F223A, L227A also produced a moderate increase in L. On the other hand, the largest increases in L were observed with mutations in S6 (P320A, E321A, E324A) that have no obvious interaction partner in S4-S5 and potentially alter the stability of the gate directly. In summary, we have identified several candidates that may be involved in voltage-sensor/gate interaction and will provide a basis for future experiments involving double mutant cycle analysis.