Lísal J and Maduke M (2008), The ClC-0 chloride channel is a 'broken' Cl-/H+...

XB-ART-38339

Nat Struct Mol Biol 2008 Aug 01;158:805-10. doi: 10.1038/nsmb.1466.

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The ClC-0 chloride channel is a 'broken' Cl-/H+ antiporter.

Lísal J , Maduke M .

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Ion channels have historically been viewed as distinct from secondary active transporters. However, the recent discovery that the CLC 'chloride channel' family is made up of both channels and active transporters has led to the hypothesis that the ion-transport mechanisms of these two types of membrane proteins may be similar. Here we use single-channel analysis to demonstrate that ClC-0 channel gating (opening and closing) involves the transmembrane movement of protons. This result indicates that ClC-0 is a 'broken' Cl(-)/H(+) antiporter in which one of the conformational states has become leaky for chloride ions. This finding clarifies the evolutionary relationship between the channels and transporters and conveys that similar mechanisms and analogous protein movements are used by both.

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	Figure 2. Slow-gate closures recorded from patches with identical chloride gradients and different proton gradients. For each patch, the first five asymmetrical slow-gate closures are shown. For both patches, the chloride concentration was 120 mM (symmetrical) and the membrane voltage was held at −90 mV. In a, a one-pH-unit gradient (pHint 7.5, pHext 8.5) was imposed; in b, a two-pH-unit gradient (pHint 7.0, pHext 9.0) was imposed. The corresponding energies for the chloride and proton electrochemical gradients (ΔGCl and ΔGH) are noted.
	Figure 3. The gating asymmetry ratio J+/J− as a function of free energy released from chloride transport, ΔGCl, and proton transport, ΔGH. The black solid lines show the maximal expected gating asymmetry for the case in which the gating is powered by chloride transport (a) or proton transport (b). Error bars represent the 95% confidence intervals. In b, the different colors code for different values of pHext (black, 7.5; red, 8.5; blue, 9.5) and the different symbols represents different voltages (circles, −50 mV; squares, −58 mV; triangles, −70 mV; diamonds −90 mV). Thus, the data points represented by the same symbols (but different colors) have the same electrochemical gradients for chloride ions (but different electrochemical gradients for protons).
	Figure 4. A possible model for non-equilibrium gating in ClC-0. The diagrams represent ClC-0 dimers with chloride pathways (green) and proton pathways (orange). Both pathways are controlled by the fast gate (dark blue) at the extracellular side. Only the chloride pathway is gated by the slow gate (black) at the intracellular side. The slow gates within individual subunits of the dimer are coupled (gray ‘spring’), such that these gates always open and close simultaneously. The number in the corner of each diagram indicates the number of open chloride pores (that is, the conductance level). The red spheres represent protons binding inside the translocation pathway. The proton electrochemical gradient is inward (movement of protons from outside to inside is energetically downhill) and hence J+/J− > 1. In this framework, the simple assumption that binding of protons to the intracellular site favors slow-gate opening results in cycling in the J+ direction. Experimentally, this is observed as channels more often entering the inactivated state (left-hand column) from the one-pore open state and returning from inactivation to the two-pore open state. (Note: transitions to the inactivated state directly from the level-1 state (right, middle) cannot be distinguished experimentally from those that occur from the level-0 state (right, below).) The slow gate opens more frequently when the fast gates are open because the protons can enter and bind to the activation site; the slow gate closes more frequently when the fast gates are closed because the protons escape down their electrochemical gradient.

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