Molecular basis for differential modulation of BK channel voltage-dependent gating by auxiliary γ subunits

Large conductance Ca2+-and voltage-activated potassium (BK) channels are comprised of pore-forming alpha subunits and various regulatory auxiliary subunits. The BK channel auxiliary gamma ( BK gamma) subunits are a newly identified class of proteins containing an extracellular leucine-rich repeat domain (LRRD), a single transmembrane (TM) segment, and a short cytoplasmic C-terminal tail (C-tail). Although each of the four BK gamma proteins shifts the voltage dependence of BK channel activation in a hyperpolarizing direction, they show markedly different efficacies, mediating shifts over a range of 15-145 mV. Analyses of chimeric BK gamma subunits created by swapping individual structural elements, and of BK gamma deletion and substitution mutants, revealed that differential modulation of BK gating by the four BK gamma subunits depends on a small region consisting of the TM segment and the adjacent intracellular cluster of positively charged amino acids. The gamma 1 and gamma 2 TM segments contributed approximately -100 mV, and the gamma 1 and gamma 3 C-tails contributed approximately. -40 mV, to shifting the voltage dependence of BK channel activation, whereas the gamma 3 and gamma 4 TM segments and the gamma 2 and gamma 4 C-tails contributed much less. The large extracellular LRRDs were mainly functionally interchangeable, although the gamma 1 LRRD was slightly less effective at enhancing (or slightly more effective at attenuating) the shift in BK channel voltage-dependent gating toward hyperpolarizing potentials than those of the other BK gamma subunits. Analysis of mutated BK gamma subunits revealed that juxta-membrane clusters of positively charged amino acids determine the functions of the gamma 1 and gamma 3 C-tails. Therefore, the modulatory functions of BK gamma subunits are coarse-and fine-tuned, respectively, through variations in their TM segments and in the adjacent intracellular positively charged regions. Our results suggest that BK channel modulation by auxiliary gamma subunits depends on intra-and/or juxta-membrane mechanisms.