Zifarelli G and Pusch M (2008), The muscle chloride channel ClC-1 is not direct...

XB-ART-37152

J Gen Physiol 2008 Feb 01;1312:109-16. doi: 10.1085/jgp.200709899.

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The muscle chloride channel ClC-1 is not directly regulated by intracellular ATP.

Zifarelli G , Pusch M .

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ClC-1 belongs to the gene family of CLC Cl(-) channels and Cl(-)/H(+) antiporters. It is the major skeletal muscle chloride channel and is mutated in dominant and recessive myotonia. In addition to the membrane-embedded part, all mammalian CLC proteins possess a large cytoplasmic C-terminal domain that bears two so-called CBS (from cystathionine-beta-synthase) domains. Several studies indicate that these domains might be involved in nucleotide binding and regulation. In particular, Bennetts et al. (J. Biol. Chem. 2005. 280:32452-32458) reported that the voltage dependence of hClC-1 expressed in HEK cells is regulated by intracellular ATP and other nucleotides. Moreover, very recently, Bennetts et al. (J. Biol. Chem. 2007. 282:32780-32791) and Tseng et al. (J. Gen. Physiol. 2007. 130:217-221) reported that the ATP effect was enhanced by intracellular acidification. Here, we show that in striking contrast with these findings, human ClC-1, expressed in Xenopus oocytes and studied with the inside-out configuration of the patch-clamp technique, is completely insensitive to intracellular ATP at concentrations up to 10 mM, at neutral pH (pH 7.3) as well as at slightly acidic pH (pH 6.2). These results have implications for a general understanding of nucleotide regulation of CLC proteins and for the physiological role of ClC-1 in muscle excitation.

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Species referenced: Xenopus laevis
Genes referenced: cbs cftr clcn1 tbx2

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	Figure 1. ATP stimulation of currents mediated by ΔR-CFTR. (A) Current recordings from an inside-out patch upon perfusion with control internal solution (left), and with solutions containing ATP as indicated in the panels. Voltage pulses are from −80 to 80 mV in 20-mV increments. Capacity transients are not subtracted. (B) ATP dependence of currents measured at 80 mV normalized to currents measured in 1 mM ATP. Error bars indicate SD (the data point at 1 mM has no error bar because it was used for the normalization). The solid line is the best fit of Eq. 2 with Kd = 38 μM and n = 0.63.
	Figure 2. Effect of ATP on the gating of hClC-1. (A) macroscopic current recordings from an inside-out patch upon perfusion with control internal solution (left), during application of 10 mM ATP (middle), and after washout (right). (B) Voltage dependence of the overall open probability obtained from the macroscopic currents in A as described in Materials and methods. Symbols are: square, control; circle, 10 mM ATP; triangle, wash. Lines are fits of Eq. 1 with the following parameters: control, V1/2 = −89 mV, Pmin = 0.14; 10 mM ATP, V1/2 = −88 mV, Pmin = 0.13; wash V1/2 = −87 mV, Pmin = 0.14. (C) Effect of ATP on the slow gating mechanism of hClC-1. Data are from the same patch and were obtained from tail currents preceded by a 200-μs pulse to 160 mV. Symbols are as in B. Lines are fits of Eq. 1 with the following parameters: control, V1/2 = −82 mV, Pmin = 0.59; 10 mM ATP, V1/2 = −81 mV, Pmin = 0.58; wash V1/2 = −78 mV, Pmin = 0.63.
	Figure 3. Statistical evaluation of the gating parameters V1/2 and Pmin at pH 7.3 obtained in control solution (n = 10) and in solutions containing 1 (n = 3), 5 (n = 5), or 10 mM ATP (n = 5). Mean values of V1/2 (A) and Pmin (B) obtained from the analysis of the overall gating mechanism of hClC-1. Mean values of V1/2 (C) and Pmin (D) obtained from the analysis of isolated slow gating mechanism of hClC-1. The values are not significantly different (P > 0.06 using Student's unpaired t test).
	Figure 4. Effect of ATP on the gating of hClC-1 at pH 6.2. Top panel, macroscopic current recordings from an inside-out patch upon perfusion with control internal solution at pH 7.3 (A), pH 6.2 (B), solution at pH 6.2 with 1 mM ATP (C), and after washout (D). (E) Voltage dependence of the overall open probability obtained from the macroscopic currents in the top panel as described in Materials and methods. Symbols are: square, internal solution at pH 6.2; circle, solution at pH 6.2 with 1 mM ATP. Lines are fits of Eq. 1 with the following parameters: solution at pH 6.2, V1/2 = −94 mV, Pmin = 0.50; solution at pH 6.2 with 1 mM ATP, V1/2 = −95 mV, Pmin = 0.43. (F) Effect of ATP at pH 6.2 on the slow gating mechanism of hClC-1. Data are from the same patch and were obtained from tail currents preceded by a 200-μs pulse to 160 mV. Symbols are as in E. Lines are fits of Eq. 1 with the following parameters: solution at pH 6.2, V1/2 = −98 mV, Pmin = 0.74; solution at pH 6.2 with 1 mM ATP, V1/2 = −93 mV, Pmin = 0.74.
	Figure 5. Statistical evaluation of the gating parameters V1/2 and Pmin obtained with internal solution at pH 6.2 without ATP and with 1 mM ATP. Mean values of V1/2 in the absence and in the presence of 1 mM ATP (n = 36 and n = 21, respectively) (A) and Pmin (n = 28 and n = 9, respectively) (B) obtained from the analysis of the overall gating mechanism of hClC-1. Mean values of V1/2 in the absence and in the presence of 1 mM ATP (n = 8 and n = 5, respectively) (C) and Pmin (n = 19 and n = 13, respectively) (D) obtained from the analysis of isolated slow gating mechanism of hClC-1. The values in the absence and in the presence of 1 mM ATP are not significantly different (P > 0.06 using Student's unpaired t test).

References [+] :

Accardi, Fast and slow gating relaxations in the muscle chloride channel CLC-1. 2000, Pubmed, Xenbase