Determinants of Anion-Proton Coupling in Mammalian Endosomal CLC Proteins Export

@article{citeulike:5400496, 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-4E281A and ClC-5E268A 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. [IMG]f1.gif" ALT="Formula" BORDER="0"> 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. 10.1074/jbc.M708368200}, author = {Zdebik, Anselm A. and Zifarelli, Giovanni and Bergsdorf, Eun-Yeong and Soliani, Paolo and Scheel, Olaf and Jentsch, Thomas J. and Pusch, Michael}, citeulike-article-id = {5400496}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M708368200}, citeulike-linkout-1 = {http://www.jbc.org/cgi/content/abstract/283/7/4219}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/18063579}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=18063579}, day = {15}, doi = {10.1074/jbc.M708368200}, journal = {J. Biol. Chem.}, keywords = {channel, clc}, month = {February}, number = {7}, pages = {4219--4227}, posted-at = {2009-08-09 08:44:37}, priority = {2}, title = {Determinants of Anion-Proton Coupling in Mammalian Endosomal CLC Proteins}, url = {http://dx.doi.org/10.1074/jbc.M708368200}, volume = {283}, year = {2008} }

TY - JOUR ID - citeulike:5400496 L3 - citeulike-article-id:5400496 N2 - 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-4E281A and ClC-5E268A 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. [IMG]f1.gif" ALT="Formula" BORDER="0"> 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. 10.1074/jbc.M708368200 IS - 7 JF - J. Biol. Chem. EP - 4227 TI - Determinants of Anion-Proton Coupling in Mammalian Endosomal CLC Proteins VL - 283 SP - 4219 KW - channel KW - clc AU - Zdebik, Anselm AU - Zifarelli, Giovanni AU - Bergsdorf, Eun-Yeong AU - Soliani, Paolo AU - Scheel, Olaf AU - Jentsch, Thomas AU - Pusch, Michael PY - 2008/02/15/ UR - http://dx.doi.org/10.1074/jbc.M708368200 ER -