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Solution structure of extracellular loop of human ?4 subunit of BK channel and its biological implication on ChTX sensitivity.

ABSTRACT: Large-conductance Ca2+- and voltage-dependent K+ (BK) channels display diverse biological functions while their pore-forming ? subunit is coded by a single Slo1 gene. The variety of BK channels is correlated with the effects of BK? coexpression with auxiliary ? (?1-?4) subunits, as well as newly defined ? subunits. Charybdotoxin (ChTX) blocks BK channel through physically occluding the K+-conduction pore. Human brain enriched ?4 subunit (h?4) alters the conductance-voltage curve, slows activation and deactivation time courses of BK channels. Its extracellular loop (h?4-loop) specifically impedes ChTX to bind BK channel pore. However, the structure of ?4 subunit's extracellular loop and the molecular mechanism for gating kinetics, toxin sensitivity of BK channels regulated by ?4 are still unclear. To address them, here, we first identified four disulfide bonds in h?4-loop by mass spectroscopy and NMR techniques. Then we determined its three-dimensional solution structure, performed NMR titration and electrophysiological analysis, and found that residue Asn123 of ?4 subunit regulated the gating and pharmacological characteristics of BK channel. Finally, by constructing structure models of BK?/?4 and thermodynamic double-mutant cycle analysis, we proposed that BK? subunit might interact with ?4 subunit through the conserved residue Glu264(BK?) coupling with residue Asn123(?4).

SUBMITTER: Wang Y

PROVIDER: S-EPMC5854672 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Solution structure of extracellular loop of human β4 subunit of BK channel and its biological implication on ChTX sensitivity.

Wang Yanting Y Lan Wenxian W Yan Zhenzhen Z Gao Jing J Liu Xinlian X Wang Sheng S Guo Xiying X Wang Chunxi C Zhou Hu H Ding Jiuping J Cao Chunyang C

Scientific reports 20180315 1

Large-conductance Ca2+- and voltage-dependent K+ (BK) channels display diverse biological functions while their pore-forming α subunit is coded by a single Slo1 gene. The variety of BK channels is correlated with the effects of BKα coexpression with auxiliary β (β1-β4) subunits, as well as newly defined γ subunits. Charybdotoxin (ChTX) blocks BK channel through physically occluding the K+-conduction pore. Human brain enriched β4 subunit (hβ4) alters the conductan ...[more]

PMID: 29545539

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Project description:In most mammalian tissues, Ca2+i/voltage-gated, large conductance K+ (BK) channels consist of channel-forming slo1 and auxiliary (β1-β4) subunits. When Ca2+i (3-20 µM) reaches the vicinity of BK channels and increases their activity at physiological voltages, β1- and β4-containing BK channels are, respectively, inhibited and potentiated by intoxicating levels of ethanol (50 mM). Previous studies using different slo1s, lipid environments, and Ca2+i concentrations-all determinants of the BK response to ethanol-made it impossible to determine the specific contribution of β subunits to ethanol action on BK activity. Furthermore, these studies measured ethanol action on ionic current under a limited range of stimuli, rendering no information on the gating processes targeted by alcohol and their regulation by βs. Here, we used identical experimental conditions to obtain single-channel and macroscopic currents of the same slo1 channel ("cbv1" from rat cerebral artery myocytes) in the presence and absence of 50 mM ethanol. First, we assessed the role five different β subunits (1,2,2-IR, 3-variant d, and 4) in ethanol action on channel function. Thus, two phenotypes were identified: (1) ethanol potentiated cbv1-, cbv1+β3-, and cbv1+β4-mediated currents at low Ca2+i while inhibiting current at high Ca2+i, the potentiation-inhibition crossover occurring at 20 µM Ca2+i; (2) for cbv1+β1, cbv1+wt β2, and cbv1+β2-IR, this crossover was shifted to ∼3 µM Ca2+i Second, applying Horrigan-Aldrich gating analysis on both phenotypes, we show that ethanol fails to modify intrinsic gating and the voltage-dependent parameters under examination. For cbv1, however, ethanol (a) drastically increases the channel's apparent Ca2+ affinity (nine-times decrease in Kd) and (b) very mildly decreases allosteric coupling between Ca2+ binding and channel opening (C). The decreased Kd leads to increased channel activity. For cbv1+β1, ethanol (a) also decreases Kd, yet this decrease (two times) is much smaller than that of cbv1; (b) reduces C; and (c) decreases coupling between Ca2+ binding and voltage sensing (parameter E). Decreased allosteric coupling leads to diminished BK activity. Thus, we have identified critical gating modifications that lead to the differential actions of ethanol on slo1 with and without different β subunits.

Dataset Information

Solution structure of extracellular loop of human ?4 subunit of BK channel and its biological implication on ChTX sensitivity.

Publications

Solution structure of extracellular loop of human β4 subunit of BK channel and its biological implication on ChTX sensitivity.

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