Saviane C et al. (1999), The muscle chloride channel ClC-1 has a double-...

XB-ART-13449

J Gen Physiol 1999 Mar 01;1133:457-68.

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The muscle chloride channel ClC-1 has a double-barreled appearance that is differentially affected in dominant and recessive myotonia.

Saviane C , Conti F , Pusch M .

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Single-channel recordings of the currents mediated by the muscle Cl- channel, ClC-1, expressed in Xenopus oocytes, provide the first direct evidence that this channel has two equidistant open conductance levels like the Torpedo ClC-0 prototype. As for the case of ClC-0, the probabilities and dwell times of the closed and conducting states are consistent with the presence of two independently gated pathways with approximately 1.2 pS conductance enabled in parallel via a common gate. However, the voltage dependence of the common gate is different and the kinetics are much faster than for ClC-0. Estimates of single-channel parameters from the analysis of macroscopic current fluctuations agree with those from single-channel recordings. Fluctuation analysis was used to characterize changes in the apparent double-gate behavior of the ClC-1 mutations I290M and I556N causing, respectively, a dominant and a recessive form of myotonia. We find that both mutations reduce about equally the open probability of single protopores and that mutation I290M yields a stronger reduction of the common gate open probability than mutation I556N. Our results suggest that the mammalian ClC-homologues have the same structure and mechanism proposed for the Torpedo channel ClC-0. Differential effects on the two gates that appear to modulate the activation of ClC-1 channels may be important determinants for the different patterns of inheritance of dominant and recessive ClC-1 mutations.

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Species referenced: Xenopus laevis
Genes referenced: clcn1
GO keywords: voltage-gated chloride channel activity

???displayArticle.disOnts??? myotonia congenita
???displayArticle.omims??? MYOTONIA CONGENITA, AUTOSOMAL DOMINANT [+]

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	Figure 5. Determination of single channel parameters using macroscopic fluctuation analysis. (A) The voltage protocol (top) was applied repeatedly (at least 100 times). Representative individual registrations are shown below (pHi 6.5; scale bars: 0.1 s and 4 pA). (B) Mean current response for the step to −100 mV (top) and corresponding variance (see methods; scale bar: 0.3 pA2). Thick solid lines represent kinetic fits assuming independent single-exponential gating relaxations for the two gates as described in methods. For clarity, averages of 10 data points are shown as circles for the experimental variance. For the fit, no averaging was performed. (C) Comparison of mean values for the single channel current, i, and the stationary open probabilities, Ps and Pf, obtained by the fluctuation analysis (▵: −140 mV, n = 8; −120 mV, n = 11; −100 mV, n = 9) with the values obtained from the amplitude histograms of single-channel recordings (•: −140 mV, n = 4; −120 mV, n = 7; −100 mV, n = 4).
	Figure 6. Fluctuation analysis for WT ClC-1 and the myotonic mutations I290M and I556N. (A) Representative fluctuation analysis for the three channel types. Mean current (top) and variance (bottom) are shown together with the kinetic fits (thick lines; see Fig. 5). (Registrations are at −100 mV, pHi 7.3; horizontal scale bars, 50 ms; vertical scale bars, 200 pA [top] and 5 pA2 [bottom].) (B) Comparison of single channel parameters obtained for WT ClC-1 and mutants I290M and I556N at pHi 7.3.
	Figure 1. Effect of low intracellular pH on macroscopic gating of ClC-1. (A) Families of voltage-clamp traces measured from different inside-out patches in solutions with the indicated pHi using the stimulation protocol shown in the inset. (B) Plot of the apparent steady state open probability at the end of the third segment of stimulation at different voltages measured as the initial tail current for the constant step to −100 mV normalized to the tail current after the prepulse to +60 mV.
	Figure 2. Single ClC-1 channels. (A) Single channel traces at different membrane potentials measured at pHi 6.5. Only short segments of longer registrations are shown. Filtered at 100 Hz for display. For the analysis, data were filtered at 200 Hz (scale bars: 0.2 pA and 0.2 s). (B) Amplitude histogram of a registration at −140 mV and fit with the sum of three equidistant gaussian distributions. (C) Stationary probabilities for the three conductance levels obtained from the gaussian fit (boxes). Circles indicate the best fit assuming two independent channels each with open probability P = 0.31. The “raw” open probability, Praw, obtained by integrating the baseline-subtracted raw current trace [Praw = <I >/(2i)] was Praw = 0.36 for the same data and yielded an even worse fit of the amplitude histogram (not shown). Squares represent the predictions assuming Fig. 3 B for a double-barreled channel with open probabilities Ps = 0.71 and Pf = 0.50, respectively, for the common gate and for the single protopore gate.
	Figure 4. Kinetic analysis of a particularly clean recording. (A) Short segment of the original registration filtered at 200 Hz (top) and the corresponding idealized trace (bottom; scale bars: 0.3 pA and 0.1 s). (B) Dwell time histograms for the three conductance levels (boxes). The dashed line in the top panel is a single exponential fit to the closed-time histogram. Solid lines represent a combined fit of the model shown in Fig. 3 B to the histograms using the maximum-likelihood criterion. Values for the rate constants obtained from the fit are (s−1): α, 34; β, 22; λ, 13; μ, 6. From these, the stationary probabilities Pf = α/(α + β) = 0.59 and Ps = λ/(λ + μ) = 0.68, and the relaxation times τf = 1/ (α + β) = 17 ms and τs = 1/(λ + μ) = 51 ms can be calculated with a ratio τs/τf < 3. The stationary probabilities obtained from the amplitude histogram of the same patch are Pf = 0.48, Ps = 0.74.

References [+] :

Chen, Nonequilibrium gating and voltage dependence of the ClC-0 Cl- channel. 1996, Pubmed