Whole-Cell Gating-Charge Measurements for Analysis of Allosteric Domain Coupling in Hslo1 BK Channels

Abstract

Large-conductance calcium- and voltage-activated potassium (Slo1 BK) channels are important for smooth muscle contraction and neurotransmitter release. Since BK channel gating is allosteric in nature, the conformations of the domains sensing transmembrane voltage (VSD) and intracellular calcium (CSD) reciprocally influence each other, either directly or via the central pore/gate domain (PGD). Thus, to fully understand the gating of BK channels involving multiple domains, determination of voltage dependence of both conductance (GV) and gating-charge movement (QV) is required. Here we evaluated how VSD-linker segments facing the CSD influence channel gating. Whole-cell gating currents were measured upon BK expression in tsa201 cells. In the absence of intracellular Ca2+, the voltage for half-maximal off-gating charge movement (V0.5,Q) was 159±2 mV (n=6) with a corresponding apparent gating charge (Qapp) of 0.80±0.02 e0. Coexpression of the human β1 subunit or increasing intracellular [Ca2+] to 10 μM left-shifted V0.5,Q by 42 mV and 70 mV, respectively, without significant changes in Qapp. To investigate allosteric domain interactions in BK, we analyzed the effect of combining mutations within S4/S5 and S6/C linkers on GV and QV in the absence of Ca2+. S6/C-linker mutations G327F and K330F shifted the GV by 109 mV and −133 mV and the QV by 7 mV and −10 mV, respectively. S4/S5-linker mutations F223A+L224A and E219R shifted the GV by −22 mV and 120 mV, and the QV by −97 mV and −109 mV, respectively. In the background of G327F or K330F, GVs were further shifted by up to −200 mV only by F223A+L224A. The data suggest that residues F223 and L224 (S4/S5 linker) and G327 and K330 (S6/C linker) are involved in allosteric coupling in BK channels. Supported in part by DFG HE 2993/8 and NIH.