BK channel modulation by positively charged peptides and auxiliary subunits mediated by the Ca²⁺-bowl site

The intracellular C-terminal positively charged regions of the gamma subunits play important roles in BK channel modulation. This work identifies positively charged peptides as BK channel modulators and reveals a role for the Ca2+-bowl site in BK channel modulation by positively charged peptides and the auxiliary gamma subunits. The large-conductance, Ca2+-, and voltage-activated K+ (BK) channel consists of the pore-forming alpha (BK alpha) subunit and regulatory beta and gamma subunits. The gamma 1-3 subunits facilitate BK channel activation by shifting the voltage-dependence of channel activation toward the hyperpolarization direction by about 50-150 mV in the absence of Ca2+. We previously found that the intracellular C-terminal positively charged regions of the gamma subunits play important roles in BK channel modulation. In this study, we found that the intracellular C-terminal region of BK alpha is indispensable in BK channel modulation by the gamma 1 subunit. Notably, synthetic peptide mimics of the gamma 1-3 subunits' C-terminal positively charged regions caused 30-50 mV shifts in BK alpha channel voltage-gating toward the hyperpolarization direction. The cationic cell-penetrating HIV-1 Tat peptide exerted a similar BK channel-activating effect. The BK channel-activating effects of the synthetic peptides were reduced in the presence of Ca2+ and markedly ablated by both charge neutralization of the Ca2+-bowl site and high ionic strength, suggesting the involvement of electrostatic interactions. The efficacy of the gamma subunits in BK channel modulation was reduced by charge neutralization of the Ca2+-bowl site. However, BK channel modulation by the gamma 1 subunit was little affected by high ionic strength and the positively charged peptide remained effective in BK channel modulation in the presence of the gamma 1 subunit. These findings identify positively charged peptides as BK channel modulators and reveal a role for the Ca2+-bowl site in BK channel modulation by positively charged peptides and the C-terminal positively charged regions of auxiliary gamma subunits.