Determinants of Anion-Proton Coupling in Mammalian Endosomal CLC Proteins

Abstract

Many proteins of the CLC gene family are Cl ؊ channels, whereas others, like the bacterial ecClC-1 or mammalian ClC-4 and -5, mediate Cl ؊ /H ؉ exchange. Mutating a "gating glutamate" (Glu-224 in ClC-4 and Glu-211 in ClC-5) converted these exchangers into anion conductances, as did the neutralization of another, intracellular "proton glutamate" in ecClC-1. We show here that neutralizing the proton glutamate of ClC-4 (Glu-281) and ClC-5 (Glu-268), but not replacing it with aspartate, histidine, or tyrosine, rather abolished Cl ؊ and H ؉ transport. Surface expression was unchanged by these mutations. Uncoupled Cl ؊ transport could be restored in the ClC-4 E281A and ClC-5 E268A proton glutamate mutations by additionally neutralizing the gating glutamates, suggesting that wild type proteins transport anions only when protons are supplied through a cytoplasmic H ؉ donor. Each monomeric unit of the dimeric protein was found to be able to carry out Cl ؊ /H ؉ exchange independently from the transport activity of the neighboring subunit. CLC 6 transport proteins are encoded by a large gene family with members in all phyla (1, 2). Because the founding member of this gene family, ClC-0, from the electric organ of Torpedo (3), is a chloride channel, all CLC genes were believed to encode anion channels. This is undoubtedly true for mammalian ClC-1, -2, and -K, which belong to the same homology branch as ClC-0. However, the bacterial CLC protein ecClC-1 turned out to be an electrogenic Cl Ϫ /H ϩ exchanger (4). Our studies then revealed that endosomal ClC-4 and -5, which reach the plasma membrane to some degree, are Cl Ϫ /H ϩ exchangers as well (5, 6). The fact that several members of the gene family function as ion channels, whereas others carry out stoichiometrically coupled ion exchange, provides unprecedented opportunities to elucidate the structural basis for these different transport modes.The linear I/V relationship of ecClC-1 allowed the estimation of a 2:1 stoichiometry of transport from reversal potentials (4). Unlike ecClC-1, ClC-4 and -5 mediate strongly outwardly rectifying currents (7), precluding a precise determination of their coupling ratio from reversal potentials. Comparing Cl Ϫ and H ϩ transport rates yielded estimates for the Cl Ϫ /H ϩ stoichiometry between 1 and 5 (5, 6). The biological consequences of endosomal CLCs being Cl Ϫ /H ϩ antiporters rather than Cl Ϫ channels are intriguing (8). These proteins are thought to facilitate endosomal/lysosomal acidification by neutralizing proton pump currents, a process important for endocytotic trafficking and lysosomal function. Indeed, ClC-5 is crucial for renal endocytosis and is mutated in a human disorder associated with proteinuria and kidney stones (9).It remains unclear whether Cl Ϫ /H ϩ exchange depends on the dimeric structure of CLC proteins. It is known that both pores of the double-barreled ClC-0 Cl Ϫ channel can be shut closed simultaneously by a "common gate" that depends on both subunits (10 -13). Similarly, it may be that Cl Ϫ /H ϩ flux coupling ...

ClC-3 Is an Intracellular Chloride/Proton Exchanger with Large Voltage-Dependent Nonlinear Capacitance

“…Moreover, we measured unitary transport rates for ClC-3 that are comparable to the unitary transport rates of ClC-4 and ClC-5. 38,39 The nonstationary noise analysis revealed also that ClC-3, similarly to ClC-5, but not ClC-4, exhibits low transport probability.…”

“…Similar values were reported previously for the two other isoforms ClC-4 and ClC-5. 28,38,39,44 Nonstationary noise analysis can also be used to determine the number of proteins in the membrane and the apparent open probability of the investigated channels. 45 Unfortunately, in the case of ClC-3, the current variances depended linearly on the current amplitudes ( Figure 3c).…”

The ClC-3 Cl−/H+ Antiporter Becomes Uncoupled at Low Extracellular pH

“…As a control for nonspecific effects of pH we performed these experiments using either Cl Ϫ or SCN Ϫ as the extracellular anion. ClC antiporters become much less coupled in the presence of non-halide anions (23,32,35). Thus the E rev of SCN Ϫ currents would be expected to be much less sensitive to pH.…”

“…The loss of rectification in response to neutralization of the gating glutamate suggests that rectification may result from the voltage dependence of gating. It has also been proposed that protons may much more readily reach the fast gate glutamate from the intracellar side and must be moved there against an energy barrier by voltage (35).…”

Molecular determinants of common gating of a ClC chloride channel

“…S1), suggesting equivalent interactions to those identified here. One possible candidate is ClC-5; however, like ClC-ec1 (refs 56,57), individual ClC-5 monomers appear to competently mediate coupled Cl À /H þ transport without cooperative interactions with the opposing subunit 58 . Like ClC-1 common gating however 46 , ClC-5 function is regulated by intracellular adenosine nucleotides 59 .…”