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. NO(3)(-) or SCN(-) transport was partially uncoupled from H(+) countertransport but still depended on the proton glutamate. Inserting proton glutamates into CLC channels altered their gating but failed to convert them into Cl(-)/H(+) exchangers. Noise analysis indicated that ClC-5 switches between silent and transporting states with an apparent unitary conductance of 0.5 picosiemens. Our results are consistent with the idea that Cl(-)/H(+) exchange of the endosomal ClC-4 and -5 proteins relies on proton delivery from an intracellular titratable residue at position 268 (numbering of ClC-5) and that the strong rectification of currents arises from the voltage-dependent proton transfer from Glu-268 to Glu-211.
Highlights
CLC6 transport proteins are encoded by a large gene family with members in all phyla [1, 2]
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
It is known that both pores of the double-barreled ClC-0 ClϪ channel can be shut closed simultaneously by a “common gate” that depends on both subunits (10 –13)
Summary
Molecular Biology—We employed the rat ClC-5 cDNA [20], human ClC-4 cDNA [7], Torpedo marmorata ClC-0 [3], and human ClC-1 [21]. Currents were measured using a standard two-electrode voltage clamp at room temperature (20 –24 °C) employing a Turbo Tec amplifier (npi, Tamm, Germany) and a custom acquisition program (GePulse) or pClamp (Molecular Devices). Solution could be quickly exchanged both at the small membrane portion facing the hole and on the much larger rest of the oocyte This is important for comparing currents (which reflect the conductance of the entire membrane) with changes in pHi, which was only measured close to the plasma membrane area that faced the hole. As we observed the difference between the rate of the fluorescence change during the pulse protocol and with the oocytes held at their resting membrane potential, Fluorocyte gave a robust semiquantitative measure for Hϩ transport activity. Data Analysis—Data were analyzed using custom software (Ana), SigmaPlot (SPSS Inc.), Origin (OriginLab Corporation), and pClamp
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