Determinants of anion-proton coupling in mammalian endosomal CLC proteins.
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.
Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), D-13125 Berlin, Germany.
Anselm A
AA
Zdebik
Giovanni
G
Zifarelli
Eun-Yeong
EY
Bergsdorf
Paolo
P
Soliani
Olaf
O
Scheel
Thomas J
TJ
Jentsch
Michael
M
Pusch
D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions; D018118:Chloride Channels / Q000737:chemistry / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011522:Protons; D051381:Rats; D017386:Sequence Homology, Amino Acid
D000838:Anions; D018118:Chloride Channels; D011522:Protons