Cl(-) transport in animal cells has fundamental physiological roles and it is mediated by a varie... more Cl(-) transport in animal cells has fundamental physiological roles and it is mediated by a variety of protein families, one of them being the CLC family of ion channels and transporters. Besides their physiological relevance, CLC proteins show peculiar biophysical properties. This review will focus on a member of the CLC protein family, the endosomal Cl(-)/H(+) antiporter ClC-5. ClC-5 mutations cause Dent's disease, a renal syndrome due to defective protein reabsorption in the proximal tubule. This established the critical function of ClC-5 for endocytosis. However, our understanding of ClC-5's molecular role in endosomes and of its biophysical properties has proved elusive in spite of important progress achieved in the last two decades. Early models in which ClC-5 would provide a shunt conductance to enable efficient endosomal acidification conflicted with the antiport activity of ClC-5 that has more recently emerged. Currently, the physiological role of ClC-5 is hotly debated and its biophysical properties are still not fully understood.
Several members of the CLC family are secondary active anion/proton exchangers, and not passive c... more Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent's disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl(-)/1 H+. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO3(-)/H+ antiporter atClCa, led to coupled NO3(-)/H+ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO3(-)/H+ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of approximately 7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.
Cl(-) transport is of fundamental importance in the most diverse physiological contexts and it is... more Cl(-) transport is of fundamental importance in the most diverse physiological contexts and it is mediated by a variety of ion channels and transporters belonging to different protein families. In particular, the recently identified TMEM16 protein family comprises the long sought Ca(2+)-activated Cl(-) channel (CaCC) and the activity of one of its members, TMEM16A, is highly dependent on temperature and is involved in thermal nociception. Among the other protein families mediating Cl(-) transport, CLC proteins are also regulated by temperature although so far the physiological implications of this dependence are unknown.
Cl(-) transport in animal cells has fundamental physiological roles and it is mediated by a varie... more Cl(-) transport in animal cells has fundamental physiological roles and it is mediated by a variety of protein families, one of them being the CLC family of ion channels and transporters. Besides their physiological relevance, CLC proteins show peculiar biophysical properties. This review will focus on a member of the CLC protein family, the endosomal Cl(-)/H(+) antiporter ClC-5. ClC-5 mutations cause Dent's disease, a renal syndrome due to defective protein reabsorption in the proximal tubule. This established the critical function of ClC-5 for endocytosis. However, our understanding of ClC-5's molecular role in endosomes and of its biophysical properties has proved elusive in spite of important progress achieved in the last two decades. Early models in which ClC-5 would provide a shunt conductance to enable efficient endosomal acidification conflicted with the antiport activity of ClC-5 that has more recently emerged. Currently, the physiological role of ClC-5 is hotly debated and its biophysical properties are still not fully understood.
Several members of the CLC family are secondary active anion/proton exchangers, and not passive c... more Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent's disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl(-)/1 H+. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO3(-)/H+ antiporter atClCa, led to coupled NO3(-)/H+ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO3(-)/H+ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of approximately 7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.
Cl(-) transport is of fundamental importance in the most diverse physiological contexts and it is... more Cl(-) transport is of fundamental importance in the most diverse physiological contexts and it is mediated by a variety of ion channels and transporters belonging to different protein families. In particular, the recently identified TMEM16 protein family comprises the long sought Ca(2+)-activated Cl(-) channel (CaCC) and the activity of one of its members, TMEM16A, is highly dependent on temperature and is involved in thermal nociception. Among the other protein families mediating Cl(-) transport, CLC proteins are also regulated by temperature although so far the physiological implications of this dependence are unknown.
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