Rychkov GY et al. (1998), Permeation and block of the skeletal muscle chl...

XB-ART-14786

J Gen Physiol 1998 May 01;1115:653-65.

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Permeation and block of the skeletal muscle chloride channel, ClC-1, by foreign anions.

Rychkov GY , Pusch M , Roberts ML , Jentsch TJ , Bretag AH .

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A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl- as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO3-, BrO3-); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl- at the regulatory binding site but impair Cl- passage through the channel pore (Br-, NO3-, ClO3-, I-, ClO4-, SCN-). The permeability sequence for rClC-1, SCN- approximately ClO4- > Cl- > Br- > NO3- approximately ClO3- > I- > BrO3- > HCO3- > methanesulfonate approximately cyclamate approximately glutamate, was different from the sequence determined for blocking potency and ability to shift the Popen curve, SCN- approximately ClO4- > I- > NO3- approximately ClO3- approximately methanesulfonate > Br- > cyclamate > BrO3- > HCO3- > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be approximately 4.5 A. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl- and SCN- or ClO4- suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.

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Species referenced: Xenopus
Genes referenced: clcn1 nbl1 sri uqcc6

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	Figure 2. Kinetics of whole-cell currents in Sf9 cells expressing rClC-1 when impermeant or poorly permeant anions replace 95% of Clâ in the external solution. Currents were elicited by the standard activation protocol from a holding potential of â30 mV with a prepulse to +40 mV followed by steps from â120 to +80 mV in 20-mV increments. Since experiments were performed on different cells, currents have been normalized to the peak inward current in Clâ solution at â120 mV. Peak inward currents from cell to cell ranged between â5 and â10 nA.
	Figure 3. Effect of cyclamate and methanesulfonate on current kinetics and apparent Popen curves. Currents were elicited by the standard activation protocol (as in Fig. 2) and 95% of external Clâ was replaced by (A) cyclamate or (B) methanesulfonate. Apparent Popen curves (C) are plotted for control (â), methanesulfonate (â¢) and cyclamate (â). Voltages are corrected for liquid junction potentials (see methods).
	Figure 4. Membrane conductance of Sf9 cells expressing rClC-1 when various permeant anions replace Clâ in the external solution. Relative conductance has been plotted against mole fraction of foreign anion as for Fig. 1.
	Figure 5. Kinetics of whole-cell currents in Sf9 cells expressing rClC-1 when various permeant anions replace 100% of Clâ in the external solution. Currents were elicited by the standard activation protocol as for Fig. 2.
	Figure 6. Effects of replacing Clâ with SCNâ in the external solution. Amplitudes of outward current in Clâ solution and in SCNâ solution were found to be closely correlated (A). Anomalous mole fraction effects were apparent for both permeability and conductance (B). Permeability changes are indicated by the shift in reversal potential (â). Relative conductance (â¦) in this case was determined from the chord conductance measured at +80 mV. Both permeability and relative conductance are plotted against mole fraction of SCNâ.
	Figure 7. Nonstationary noise analysis performed on outside-out patches from Xenopus oocytes expressing hClC-1 in the presence of Clâ, NO3â, Iâ, or Brâ. Variances (top) and means (middle) of current records (n = 150) are shown for standard voltage protocols incorporating a prepulse to +60 mV followed by a 70-ms test pulse to â140 mV. (bottom) Varianceâmean current plots are fitted with parabolas according to the equation: Ï2 = Ï02â+ iI â Iââ2/n, where Ï02 is baseline noise variance, i is single channel current, I is mean current, and n is number of channels. The results shown were obtained from a single patch. Fitted values for the parabolas shown are: (A) for chloride, i = 0.26 pA; (B) for nitrate, i = 0.1 pA; (C) for 50% iodide/50% chloride, i = 0.077 pA; (D) for bromide, i = 0.21 pA.
	Figure 8. Relationship between Clâ conductance and concentration in rClC-1. In this case, Clâ was replaced by glucose in the external solution but by glutamate in the internal solution. From Fig. 1, it can be seen that the effects of glucose or glutamate replacement in the external solution are almost identical. See text for methods employed in normalization of data.
	Figure 9. Kinetics of currents in inside-out patches from Xenopus oocytes expressing hClC-1 when Brâ and Iâ are substituted for Clâ in the internal solution. A shows control currents in the patch to which 100% Brâ was then applied (B), while C shows control currents in the patch to which 50% Iâ was later applied (D). Voltage protocol: after a 50-ms prepulse to +60 mV, the membrane potential was stepped for 150 ms from â140 to +80 mV in 20-mV increments, followed by a step to â100 mV for 50 ms.
	Figure 10. Dependence of relative permeability of rClC-1 expressed in Sf9 cells on the apparent ionic diameter of various anions. Apparent ionic diameters, d, were calculated from the Einstein-Stokes relation, d = 183.6/Î»Â°, where Î»Â° is the limiting conductance for the ion (Robinson and Stokes, 1959). Relative permeability for Fâ was determined from the biionic potential with 160 mM of Fâ in the internal solution and 170 mM of Clâ in the external solution.

References [+] :

Astill, Characteristics of skeletal muscle chloride channel C1C-1 and point mutant R304E expressed in Sf-9 insect cells. 1996, Pubmed