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An S6 mutation in BK channels reveals beta1 subunit effects on intrinsic and voltage-dependent gating.

ABSTRACT: Large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels are exquisitely regulated to suit their diverse roles in a large variety of physiological processes. BK channels are composed of pore-forming alpha subunits and a family of tissue-specific accessory beta subunits. The smooth muscle-specific beta1 subunit has an essential role in regulating smooth muscle contraction and modulates BK channel steady-state open probability and gating kinetics. Effects of beta1 on channel's gating energetics are not completely understood. One of the difficulties is that it has not yet been possible to measure the effects of beta1 on channel's intrinsic closed-to-open transition (in the absence of voltage sensor activation and Ca(2+) binding) due to the very low open probability in the presence of beta1. In this study, we used a mutation of the alpha subunit (F315Y) that increases channel openings by greater than four orders of magnitude to directly compare channels' intrinsic open probabilities in the presence and absence of the beta1 subunit. Effects of beta1 on steady-state open probabilities of both wild-type alpha and the F315Y mutation were analyzed using the dual allosteric HA model. We found that mouse beta1 has two major effects on channel's gating energetics. beta1 reduces the intrinsic closed-to-open equilibrium that underlies the inhibition of BK channel opening seen in submicromolar Ca(2+). Further, P(O) measurements at limiting slope allow us to infer that beta1 shifts open channel voltage sensor activation to negative membrane potentials, which contributes to enhanced channel opening seen at micromolar Ca(2+) concentrations. Using the F315Y alpha subunit with deletion mutants of beta1, we also demonstrate that the small N- and C-terminal intracellular domains of beta1 play important roles in altering channel's intrinsic opening and voltage sensor activation. In summary, these results demonstrate that beta1 has distinct effects on BK channel intrinsic gating and voltage sensor activation that can be functionally uncoupled by mutations in the intracellular domains.

SUBMITTER: Wang B

PROVIDER: S-EPMC2151602 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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An S6 mutation in BK channels reveals beta1 subunit effects on intrinsic and voltage-dependent gating.

Wang Bin B Brenner Robert R

The Journal of general physiology 20061201 6

Large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels are exquisitely regulated to suit their diverse roles in a large variety of physiological processes. BK channels are composed of pore-forming alpha subunits and a family of tissue-specific accessory beta subunits. The smooth muscle-specific beta1 subunit has an essential role in regulating smooth muscle contraction and modulates BK channel steady-state open probability and gating kinetics. Effects of beta1 on channel's gating en ...[more]

PMID: 17130522

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Project description:High voltage-activated Ca2+ (Ca(V)) channels are protein complexes containing pore-forming α1 and auxiliary β and α2δ subunits. The subcellular localization and membrane interactions of the β subunits play a crucial role in regulating Ca(V) channel inactivation and its lipid sensitivity. Here, we investigated the effects of membrane phosphoinositide (PI) turnover on Ca(V)2.2 channel function. The β2 isoform β2e associates with the membrane through electrostatic and hydrophobic interactions. Using chimeric β subunits and liposome-binding assays, we determined that interaction between the N-terminal 23 amino acids of β2e and anionic phospholipids was sufficient for β2e membrane targeting. Binding of the β2e subunit N terminus to liposomes was significantly increased by inclusion of 1% phosphatidylinositol 4,5-bisphosphate (PIP2) in the liposomes, suggesting that, in addition to phosphatidylserine, PIs are responsible for β2e targeting to the plasma membrane. Membrane binding of the β2e subunit slowed Ca(V)2.2 current inactivation. When membrane phosphatidylinositol 4-phosphate and PIP2 were depleted by rapamycin-induced translocation of pseudojanin to the membrane, however, channel opening was decreased and fast inactivation of Ca(V)2.2(β2e) currents was enhanced. Activation of the M1 muscarinic receptor elicited transient and reversible translocation of β2e subunits from membrane to cytosol, but not that of β2a or β3, resulting in fast inactivation of Ca(V)2.2 channels with β2e. These results suggest that membrane targeting of the β2e subunit, which is mediated by nonspecific electrostatic insertion, is dynamically regulated by receptor stimulation, and that the reversible association of β2e with membrane PIs results in functional changes in Ca(V) channel gating. The phospholipid-protein interaction observed here provides structural insight into mechanisms of membrane-protein association and the role of phospholipids in ion channel regulation.

Dataset Information

An S6 mutation in BK channels reveals beta1 subunit effects on intrinsic and voltage-dependent gating.

Publications

An S6 mutation in BK channels reveals beta1 subunit effects on intrinsic and voltage-dependent gating.

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