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Temperature-dependent increase in the calcium sensitivity and acceleration of activation of ANO6 chloride channel variants.

ABSTRACT: Anoctamin-6 (ANO6) belongs to a family of calcium (Ca²⁺)-activated chloride channels (CaCCs), with three splicing variants (V1, V2, and V5) showing plasma membrane expression. Unlike other CaCCs, ANO6 requires a non-physiological intracellular free calcium concentration ([Ca²⁺]_i?>?1??M) and several minutes for full activation under a whole-cell patch clamp. Therefore, its physiological role as an ion channel is uncertain and it is more commonly considered a Ca²⁺-dependent phospholipid scramblase. Here, we demonstrate that physiological temperature (37?°C) increases ANO6 Ca²⁺ sensitivity under a whole-cell patch clamp; V1 was activated by 1??M [Ca²⁺]_i, whereas V2 and V5 were activated by 300?nM [Ca²⁺]_i. Increasing the temperature to 42?°C led to activation of all ANO6 variants by 100?nM [Ca²⁺]_i. The delay t_ime for activation of the three variants was significantly shortened at 37?°C. Notably, the temperature-dependent Ca²⁺-sensitisation of ANO6 became insignificant under inside-out patch clamp, suggesting critical roles of unknown cytosolic factors. Unlike channel activity, 27?°C but not 37?°C (physiological temperature) induced the scramblase activity of ANO6 at submicromolar [Ca²⁺]_i (300?nM), irrespective of variant type. Our results reveal a physiological ion conducting property of ANO6 at 37?°C and suggest that ANO6 channel function acts separately from its scramblase activity.

SUBMITTER: Lin H

PROVIDER: S-EPMC6491614 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Temperature-dependent increase in the calcium sensitivity and acceleration of activation of ANO6 chloride channel variants.

Lin Haiyue H Jun Ikhyun I Woo Joo Han JH Lee Min Goo MG Kim Sung Joon SJ Nam Joo Hyun JH

Scientific reports 20190430 1

Anoctamin-6 (ANO6) belongs to a family of calcium (Ca2+)-activated chloride channels (CaCCs), with three splicing variants (V1, V2, and V5) showing plasma membrane expression. Unlike other CaCCs, ANO6 requires a non-physiological intracellular free calcium concentration ([Ca2+]i > 1 μM) and several minutes for full activation under a whole-cell patch clamp. Therefore, its physiological role as an ion channel is uncertain and it is more commonly considered a Ca<su ...[more]

PMID: 31040335

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Project description:CLC-0, a prototype Cl- channel in the CLC family, employs two gating mechanisms that control its ion-permeation pore: fast gating and slow gating. The negatively-charged sidechain of a pore glutamate residue, E166, is known to be the fast gate, and the swinging of this sidechain opens or closes the pore of CLC-0 on the millisecond time scale. The other gating mechanism, slow gating, operates with much slower kinetics in the range of seconds to tens or even hundreds of seconds, and it is thought to involve still-unknown conformational rearrangements. Here, we find that low intracellular pH (pHi) facilitates the closure of the CLC-0's slow gate, thus generating current inhibition. The rate of low pHi-induced current inhibition increases with intracellular H+ concentration ([H+]i)-the time constants of current inhibition by low pHi = 4.5, 5.5 and 6 are roughly 0.1, 1 and 10 sec, respectively, at room temperature. In comparison, the time constant of the slow gate closure at pHi = 7.4 at room temperature is hundreds of seconds. The inhibition by low pHi is significantly less prominent in mutants favoring the slow-gate open state (such as C212S and Y512A), further supporting the fact that intracellular H+ enhances the slow-gate closure in CLC-0. A fast inhibition by low pHi causes an apparent inverted voltage-dependent activation in the wild-type CLC-0, a behavior similar to those in some channel mutants such as V490W in which only membrane hyperpolarization can open the channel. Interestingly, when V490W mutation is constructed in the background of C212S or Y512A mutation, the inverted voltage-dependent activation disappears. We propose that the slow kinetics of CLC-0's slow-gate closure may be due to low [H+]i rather than due to the proposed large conformational change of the channel protein. Our results also suggest that the inverted voltage-dependent opening observed in some mutant channels may result from fast closure of the slow gate by the mutations.

Dataset Information

Temperature-dependent increase in the calcium sensitivity and acceleration of activation of ANO6 chloride channel variants.

Publications

Temperature-dependent increase in the calcium sensitivity and acceleration of activation of ANO6 chloride channel variants.

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