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Inverse effects on gating and modulation caused by a mutation in the M2-M3 Linker of the GABA(A) receptor gamma subunit.

ABSTRACT: M2-M3 linkers are receptor subunit domains known to be critical for the normal function of cysteine-loop ligand-gated ion channels. Previous studies of alpha and beta subunits of type "A" GABA receptors suggest that these linkers couple extracellular elements involved in GABA binding to the transmembrane segments that control the opening of the ion channel. To study the importance of the gamma subunit M2-M3 linker, we examined the macroscopic and single-channel effects of an engineered gamma2(L287A) mutation on GABA activation and propofol modulation. In the macroscopic analysis, we found that the gamma2(L287A) mutation decreased GABA potency but increased the ability of propofol to enhance both GABA potency and efficacy compared with wild-type receptors. Indeed, although propofol had significant effects on GABA potency in wild-type receptors, we found that propofol produced no corresponding increase in GABA efficacy. At the single-channel level, mutant receptors showed a loss in the longest of three open-time components compared with wild-type receptors under GABA activation. Furthermore, propofol reduced the duration of one closed-time component, increased the duration of two open-time components, and generated a third open component with a longer lifetime in mutant compared with wild-type receptors. Taken together, we conclude that although the gamma subunit is not required for the binding of GABA or propofol, the M2-M3 linker of this subunit plays a critical role in channel gating by GABA and allosteric modulation by propofol. Our results also suggest that in wild-type receptors, propofol exerts its enhancing effects by mechanisms extrinsic to channel gating.

SUBMITTER: O'Shea SM

PROVIDER: S-EPMC2730393 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Inverse effects on gating and modulation caused by a mutation in the M2-M3 Linker of the GABA(A) receptor gamma subunit.

O'Shea Sean M SM Williams Carrie A CA Jenkins Andrew A

Molecular pharmacology 20090624 3

M2-M3 linkers are receptor subunit domains known to be critical for the normal function of cysteine-loop ligand-gated ion channels. Previous studies of alpha and beta subunits of type "A" GABA receptors suggest that these linkers couple extracellular elements involved in GABA binding to the transmembrane segments that control the opening of the ion channel. To study the importance of the gamma subunit M2-M3 linker, we examined the macroscopic and single-channel effects of an engineered gamma2(L2 ...[more]

PMID: 19553237

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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

Inverse effects on gating and modulation caused by a mutation in the M2-M3 Linker of the GABA(A) receptor gamma subunit.

Publications

Inverse effects on gating and modulation caused by a mutation in the M2-M3 Linker of the GABA(A) receptor gamma subunit.

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