Elimination of the Slow Gating of Clc-0 Chloride Channel by a Point Mutation

Abstract

The inactivation of the ClC-0 chloride channel is very temperature sensitive and is greatly facilitated by the binding of a zinc ion (Zn2+) from the extracellular side, leading to a Zn2+-induced current inhibition. To further explore the relation of Zn2+ inhibition and the ClC-0 inactivation, we mutated all 12 cysteine amino acids in the channel and assayed the effect of Zn2+ on these mutants. With this approach, we found that C212 appears to be important for the sensitivity of the Zn2+ inhibition. Upon mutating C212 to serine or alanine, the inactivation of the channel in macroscopic current recordings disappears and the channel does not show detectable inactivation events at the single-channel level. At the same time, the channel's sensitivity to Zn2+ inhibition is also greatly reduced. The other two cysteine mutants, C213G and C480S, as well as a previously identified mutant, S123T, also affect the inactivation of the channel to some degree, but the temperature-dependent inactivation process is still present, likewise the high sensitivity of the Zn2+ inhibition. These results further support the assertion that the inhibition of Zn2+ on ClC-0 is indeed due to an effect on the inactivation of the channel. The absence of inactivation in C212S mutants may provide a better defined system to study the fast gating and the ion permeation of ClC-0.

Molecular determinants of common gating of a ClC chloride channel

“…Extracellular Zn 2 þ inhibits ClC-0 and ClC-1 channels by binding to and stabilising a closed state of the common gating mechanism 38,39 . Previous studies suggest that the binding site for Zn 2 þ is at the extracellular surface of helix G, where it intersects helix F in the region of E232 32,39 . Mutations C277S and C278S, at the carboxy-terminal of helix G, greatly reduce Zn 2 þ inhibition of ClC-1 (ref.…”

“…ClC-1 channels have also been shown to transport H þ 6 . In the same experiments Picollo and Pusch 6 did not observe H þ transport by ClC-0 channels, which contained a mutation (C212S) that removes common gating 32 . It therefore seems possible that common gating of ClC channels is an evolutionary vestige of the conformational changes that catalyse coupled anion/H þ transport 30 .…”

Barttin Activates ClC-K Channel Function by Modulating Gating

“…3,23 For most ClC channels, the physiologic significance of these gating processes is unclear. Especially, cooperative gating processes that mediate joint opening and closing of the two identical protopores have attracted much biophysical interest, 22,24 but the functional effect of these processes has yet to be clarified. This study establishes that barttin modifies the function of the ClC-K channel by modifying cooperative slow gating.…”

“…After partial rundown V166E rClC-K1/barttin channels resemble double-barreled channels with fast individual and slow common gating. 21,22 Figure 5 gives single-channel recordings from a human ClC-K isoform, hClCKa/barttin. Representative recordings from a cell-attached patch containing hClC-Ka/barttin showed two equally spaced conductance levels ( Figure 5A).…”

Genetics of Type III Bartter Syndrome in Spain, Proposed Diagnostic Algorithm

“…Alanine 210 is a residue located within the transmembrane G helix. Mutations in many residues within the G helix have been described to cause functional alterations of chloride channels [26-28]. This mutation is present in combination with a novel heterozygous splice-donor mutation c.1756+1G>A.…”