Abstract
Members of the CLC gene family either function as chloride channels or as anion/proton exchangers. The plant AtClC-a uses the pH gradient across the vacuolar membrane to accumulate the nutrient NO(3)(-) in this organelle. When AtClC-a was expressed in Xenopus oocytes, it mediated NO(3)(-)/H(+) exchange and less efficiently mediated Cl(-)/H(+) exchange. Mutating the "gating glutamate" Glu-203 to alanine resulted in an uncoupled anion conductance that was larger for Cl(-) than NO(3)(-). Replacing the "proton glutamate" Glu-270 by alanine abolished currents. These could be restored by the uncoupling E203A mutation. Whereas mammalian endosomal ClC-4 and ClC-5 mediate stoichiometrically coupled 2Cl(-)/H(+) exchange, their NO(3)(-) transport is largely uncoupled from protons. By contrast, the AtClC-a-mediated NO(3)(-) accumulation in plant vacuoles requires tight NO(3)(-)/H(+) coupling. Comparison of AtClC-a and ClC-5 sequences identified a proline in AtClC-a that is replaced by serine in all mammalian CLC isoforms. When this proline was mutated to serine (P160S), Cl(-)/H(+) exchange of AtClC-a proceeded as efficiently as NO(3)(-)/H(+) exchange, suggesting a role of this residue in NO(3)(-)/H(+) exchange. Indeed, when the corresponding serine of ClC-5 was replaced by proline, this Cl(-)/H(+) exchanger gained efficient NO(3)(-)/H(+) coupling. When inserted into the model Torpedo chloride channel ClC-0, the equivalent mutation increased nitrate relative to chloride conductance. Hence, proline in the CLC pore signature sequence is important for NO(3)(-)/H(+) exchange and NO(3)(-) conductance both in plants and mammals. Gating and proton glutamates play similar roles in bacterial, plant, and mammalian CLC anion/proton exchangers.
Highlights
CLC proteins are found in all phyla from bacteria to humans and either mediate electrogenic anion/proton exchange or function as chloride channels (1)
A third binding site is observed when a certain key glutamate residue, which is located halfway in the permeation pathway of almost all CLC proteins, is mutated to alanine (23). Mutating this gating glutamate in CLC ClϪ channels strongly affects or even completely suppresses single pore gating (23), whereas CLC exchangers are transformed by such mutations into pure anion conductances that are not coupled to proton transport (17, 19, 20)
We have achieved for the first time the functional expression of a plant CLC transporter in animal cells
Summary
AtCIC-n, member n of the CLC family of ClϪ channels and transporters in the plant Arabidopsis thaliana; CIC-n, member n of the CLC family of chloride channel and transporters (in animals); pHi, intracellular pH; pH0, extracellular pH; I(NO3Ϫ) and I(ClϪ), current in the presence of NO3Ϫ and ClϪ, respectively, which in CLC exchangers involves an Hϩ component; WT, wild-type; MES, 2-(N-morpholino)ethanesulfonic acid; BCECF, 2Ј,7Ј-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. When proline replaced the critical serine in Torpedo ClC-0, the relative NO3Ϫ conductance of this model ClϪ channel was drastically increased, and “fast” protopore gating was slowed
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