Regulation of ClC-2 gating by intracellular ATP

Abstract

ClC-2 is a voltage-dependent chloride channel that activates slowly at voltages negative to the chloride reversal potential. Adenosine triphosphate (ATP) and other nucleotides have been shown to bind to carboxy-terminal cystathionine-ß-synthase (CBS) domains of ClC-2, but the functional consequences of binding are not sufficiently understood. We here studied the effect of nucleotides on channel gating using single-channel and whole-cell patch clamp recordings on transfected mammalian cells. ATP slowed down macroscopic activation and deactivation time courses in a dose-dependent manner. Removal of the complete carboxy-terminus abolishes the effect of ATP, suggesting that CBS domains are necessary for ATP regulation of ClC-2 gating. Single-channel recordings identified long-lasting closed states of ATP-bound channels as basis of this gating deceleration. ClC-2 channel dimers exhibit two largely independent protopores that are opened and closed individually as well as by a common gating process. A seven-state model of common gating with altered voltage dependencies of opening and closing transitions for ATP-bound states correctly describes the effects of ATP on macroscopic and microscopic ClC-2 currents. To test for a potential pathophysiological impact of ClC-2 regulation by ATP, we studied ClC-2 channels carrying naturally occurring sequence variants found in patients with idiopathic generalized epilepsy, G715E, R577Q, and R653T. All naturally occurring sequence variants accelerate common gating in the presence but not in the absence of ATP. We propose that ClC-2 uses ATP as a co-factor to slow down common gating for sufficient electrical stability of neurons under physiological conditions.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-013-1286-0) contains supplementary material, which is available to authorized users.

Tryptophan Scanning Mutagenesis Identifies the Molecular Determinants of Distinct Barttin Functions

“…Moreover, whereas niflumic acid potentiates hClC-Ka/ barttin currents in oocytes, such an effect is absent in mammalian cells (25). These differences in channel properties in separate expression systems suggest that additional factors, such as yet unidentified subunits or modifying factors (37), might alter gating of CLC-K/barttin channels. If there were additional subunits of the CLC-K/barttin complex, it appears possible that some of the side chains identified in tryptophan scanning mutagenesis might interact with these proteins.…”

Carboxyl-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking

“…In this study, the effects on the subcellular distribution were only studied by confocal imaging, and none of the truncated hClC-1 exhibited major changes on this parameter (48). For hClC-2, ATP binding to the CBS domains results in longer slow gate closures (51), and removal of the full carboxyl terminus accelerates hClC-2 gating by preventing slow gate closures (50). Multiple partial carboxyl-terminal truncations and deletions constitutively locked hClC-2 in a closed conformation, without any effect on surface membrane insertion (50).…”

Metabolic Energy Sensing by Mammalian CLC Anion/Proton Exchangers

“…Cytosolic adenine nucleotides regulate the ion currents of the CLC channels ClC-1 and ClC-2 by altering their voltage-dependent activation [21–23]. Similar to CLC channels, the here-investigated CLC transporters also exhibit pronounced voltage dependence [24,19,17].…”