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Mechanisms of noncovalent ? subunit regulation of NaV channel gating.

ABSTRACT: Voltage-gated Na+ (NaV) channels comprise a macromolecular complex whose components tailor channel function. Key components are the non-covalently bound ?1 and ?3 subunits that regulate channel gating, expression, and pharmacology. Here, we probe the molecular basis of this regulation by applying voltage clamp fluorometry to measure how the ? subunits affect the conformational dynamics of the cardiac NaV channel (NaV1.5) voltage-sensing domains (VSDs). The pore-forming NaV1.5 ? subunit contains four domains (DI-DIV), each with a VSD. Our results show that ?1 regulates NaV1.5 by modulating the DIV-VSD, whereas ?3 alters channel kinetics mainly through DIII-VSD interaction. Introduction of a quenching tryptophan into the extracellular region of the ?3 transmembrane segment inverted the DIII-VSD fluorescence. Additionally, a fluorophore tethered to ?3 at the same position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD. Together, these results provide compelling evidence that ?3 binds proximally to the DIII-VSD. Molecular-level differences in ?1 and ?3 interaction with the ? subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting NaV channel regulation of cell excitability.

SUBMITTER: Zhu W

PROVIDER: S-EPMC5560778 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Mechanisms of noncovalent β subunit regulation of NaV channel gating.

Zhu Wandi W Voelker Taylor L TL Varga Zoltan Z Schubert Angela R AR Nerbonne Jeanne M JM Silva Jonathan R JR

The Journal of general physiology 20170807 8

Voltage-gated Na+ (NaV) channels comprise a macromolecular complex whose components tailor channel function. Key components are the non-covalently bound β1 and β3 subunits that regulate channel gating, expression, and pharmacology. Here, we probe the molecular basis of this regulation by applying voltage clamp fluorometry to measure how the β subunits affect the conformational dynamics of the cardiac NaV channel (NaV1.5) voltage-sensing domains (VSDs). ...[more]

PMID: 28720590

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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.

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Mechanisms of noncovalent ? subunit regulation of NaV channel gating.

Publications

Mechanisms of noncovalent β subunit regulation of Na<sub>V</sub> channel gating.

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