Disease‐causing mutations C277R and C277Y modify gating of human ClC‐1 chloride channels in myotonia congenita

Abstract

Key points• ClC channels are double-barrelled channels with two ion conduction pathways per individual channel.• Substituting cysteine 277 by serine constitutively opens the common gate suggesting that this residue plays a major role in joint openings/closings of both protopores of ClC-1.• We studied here the functional consequences of two novel myotonia-associated mutations, C277R and C277Y, of human ClC-1 chloride channels.• C277Y not only modified the common gate, but also protopore gating.• C277Y inverts the voltage dependence and reduces the open probabilities of protopore and common gates and thus decreases the absolute open probabilities of homodimeric hClC-1 channels to values below 3%.• C277Y reduces single protopore current amplitudes to about two-thirds of wild-type values, and inverts the anion permeability sequence.• Our results explain the disease-causing effects and provide novel insights into the molecular processes underlying normal and pathologically altered function of muscle chloride channels.Abstract Myotonia congenita is a genetic condition that is caused by mutations in the muscle chloride channel gene CLCN1 and characterized by delayed muscle relaxation and muscle stiffness. We here investigate the functional consequences of two novel disease-causing missense mutations, C277R and C277Y, using heterologous expression in HEK293T cells and patch clamp recording. Both mutations reduce macroscopic anion currents in transfected cells. Since hClC-1 is a double-barrelled anion channel, this reduction in current amplitude might be caused by altered gating of individual protopores or of joint openings and closing of both protopores. We used non-stationary noise analysis and single channel recordings to separate the mutants' effects on individual and common gating processes. We found that C277Y inverts the voltage dependence and reduces the open probabilities of protopore and common gates resulting in decreases of absolute open probabilities of homodimeric channels to values below 3%. In heterodimeric channels, C277R and C277Y also reduce open probabilities and shift the common gate activation curve towards positive potentials. Moreover, C277Y modifies pore properties of hClC-1. It reduces single protopore current amplitudes to about two-thirds of wild-type values, and inverts the anion permeability sequence to I − = NO 3 − > Br − > Cl − . Our findings predict a dramatic reduction of the muscle fibre resting chloride conductance and thus fully explain the disease-causing effects

Molecular determinants of common gating of a ClC chloride channel

“…S1). Further, mutation of several residues in the region of the extracellular junction between helices F and G has profound effects on ClC-1 gating 28,[40][41][42] . It therefore appeared likely that helix G may act as an intermediary for the coordination of common gating between the channel pore and the subunit interface.…”

“…39). Mutations of residue C277 have drastic effects on common gating 40,41 . However, the role of C277 in Zn 2 þ binding is complicated by the fact that common gating is essentially eliminated in C277S mutant channels 39,40 , and therefore reduced Zn 2 þ inhibition may reflect the absence of common gating rather than diminished Zn 2 þ binding.…”

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

“…There are two structurally distinct gating processes, protopore gating that opens and closes individual protopores independently of the adjacent subunit and common gating jointly acting on both pores at once. For such channels, the mean whole-cell current amplitude (I) and the variance 2 depend on the number of channels N, the single channel amplitude of an individual protopore i, as well as on the open probabilities of the protopore and the common gate (P p and P c ) (1,27,38):…”

Reduced Membrane Insertion of CLC-K by V33L Barttin Results in Loss of Hearing, but Leaves Kidney Function Intact

“…For such a channel, the whole cell current amplitude ( I ) depends on the number of channels in the surface membrane ( N ), the single channel pore amplitude ( i ) and the open probabilities of the two protopore gates ( P p ) and the common gate ( P c ) (Accardi and Pusch, 2000; Fischer et al, 2010): $\begin{array}{c}eqnarrayleftI=2N·i·P_p·P_c\end{array}$ Random opening and closing of ion channels produce a Lorentzian type of noise (σ 2 ) that depends on the number of channels as well as its unitary current amplitude and its open probability. For a double-barreled channel σ 2 can be calculated as Fischer et al (2010), Weinberger et al (2012) and Stölting et al (2014a): $\begin{array}{c}eqnarrayleft{\sigma }^2=i·I-\frac{I^2}{N}false(1-\frac{1}{2P_c}false)\end{array}$ In the case of ClC-Ka/barttin channels the common gate was found permanently open using single channel recordings and noise analysis (Fischer et al, 2010), thus simplifying (2) to $\begin{array}{c}eqnarrayleft{\sigma }^2=i·I-\frac{I^2}{2N}\end{array}$ which is equivalent to: $\begin{array}{c}eqnarrayleft\frac{{\sigma }^2}{I}=i-\frac{I}{2N}\end{array}$ Based on an ohmic current-voltage relationship, this relationship can be further simplified to: $\begin{array}{c}eqnarrayleft\frac{{\sigma }^2}{I·false(V-V_{rev}false)}=\gamma -\frac{I}{2N·false(V-V_{rev}false)}\end{array}$ here with V the applied voltage, V rev the reversal potential, N the number of functional channels in the surface membrane and γ the single channel pore conductance (Sesti and Goldstein, 1998). The voltage-independent background noise was recorded at the reversal potential, where ClC-Ka/barttin currents do not contribute, averaged and subtracted from the otherwise measured variance.…”