Stepwise contribution of each subunit to the cooperative activation of BK channels by Ca ²⁺

Abstract

BK channels (Slo1) are widely distributed K+ channels that control Ca2+-dependent processes and cellular excitability. Their activation by intracellular Ca2+ (Ca\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{2+}\end{equation*}\end{document}) is highly cooperative, with Hill coefficients of typically 2–5. To investigate the cooperativity contributed by each of the four α subunits that form the BK channel, we studied single channels comprised of mixtures of functional subunits and subunits with a mutation to disrupt a key site (Ca-bowl) required for activation by low concentrations of Ca\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{2+}\end{equation*}\end{document}. As the number of functional subunits increased, we found a stepwise increase in the Hill coefficient of 0.3–0.8 per functional subunit and a stepwise decrease in the Ca\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{2+}\end{equation*}\end{document} required for half activation (Kd). These results show directly that BK channels can open with 0, 1, 2, 3, or 4 functional Ca-bowls, and that each subunit with a functional Ca-bowl contributes a stepwise increase to both the cooperativity of activation and the apparent Ca2+ affinity. A model with 0–4 high-affinity allosteric activators and four low-affinity allosteric activators was examined. In this model, Ca2+ bindings were independent of one another and the cooperativity arose from the joint action of the allosteric activators on the open–closed equilibrium. Although this model described well the major features of the experimental data, some differences between the observed and predicted results indicated that additional factors not included in the model also contribute to the cooperativity

β1 subunits facilitate gating of BK channels by acting through the Ca 2 + , but not the Mg 2 + , activating mechanisms

“…2A), just as expected for channels with four intact TEA binding sites (38,39). After injecting cRNA for TM channels with the TEA binding site mutated, the observed single-channel currents were Ϸ13 pA in the absence of TEAo and Ϸ9 pA with 1.5 mM TEAo (Fig.…”

“…To check this possibility, we introduced a point mutation to the TEA binding site located at the outer mouth of the TM subunit. The subunit composition of the channels was then identified from the single-channel current amplitude in the presence of 1.5 mM external TEA (TEAo) (38,39). For the conditions of these experiments (ϩ50 mV, symmetrical 158 mM KCl), the singlechannel current amplitude should be Ϸ13 pA without TEAo, and should be reduced to Ϸ9.3, 7.5, 4.0, 1.5, and 0 pA in the presence of 1.5 mM TEAo for channels with 4, 3, 2, 1, and 0 subunits having their TEA binding sites mutated (39).…”

Large conductance Ca2+-activated K+ (BK) channel: Activation by Ca2+ and voltage

“…In support of the model shown in Fig. 6B, Niu and Magleby (2002), using channels with 1, 2, 3 or 4 Ca 2+ bowls (4), determined that the Hill coefficient increased in a stepwise fashion as the number of bowl increased from 1 to 4. This observation is consistent with models like the one shown in Fig.…”

Dual role of protein kinase C on BK channel regulation

“…BK channel α-subunits are assembled as tetramers. To investigate the stoichiometry of BK channel tetramer phosphorylation by PKC, we employed a strategy that allowed us to determine electrophysiologically the subunit composition of each BK channel from the single-channel current amplitude by introducing a mutation to the tetraethylammonium (TEA)-sensitive site (10,14,15). Introducing the point mutation Y 347 V in the subunit pore (Fig.…”