Ma, L.Rychkov, G.Bretag, A.2009-10-272009-10-272009The International Journal of Biochemistry and Cell Biology, 2009; 41(6):1402-14091357-27251878-5875http://hdl.handle.net/2440/51484The membrane-resident domain of chloride channels and transporters of the CLC family is composed of 18 alpha-helices (designated A to R) connected sequentially by extracellular and intracellular loops, whose functional characteristics are generally unclear. To study the relevance of the intracellular loops linking helices D and E, F and G, H and I and J and K, alanine-exchange mutagenesis, split channel strategy, GST (glutathione transferase)-pull-down methods and whole-cell patch-clamp recordings were used. We investigated the possible roles of these loops in binding to the cytoplasmic, carboxyl tail (C-tail) of the protein, as well as their physiological roles in channel function. Although other interacting positions are conceivable, our results indicate that there is unlikely to be significant binding between the C-tail and any one of these individual cytoplasmic loops. Particular loops might, however, be essential for some channel characteristics. For example, alanine-exchange mutation of the loop linking helix D to E eliminated channel currents; of the loop linking helix H to I caused a significant shift of the open probability of fast gating (P(o)(f)), towards more positive voltages; and of the loop linking helix J to K locked the common gating of hClC-1 open. Therefore, the gating mechanisms of hClC-1 might not only involve the helices and the C-tail, but also involve some of the loops.enMuscle, SkeletalCells, CulturedCytoplasmHumansAlanineChloride ChannelsBlotting, WesternTransfectionGene ExpressionProtein Structure, TertiaryStructure-Activity RelationshipJournal article002009041110.1016/j.biocel.2008.12.0060002646693000232-s2.0-6034911339339109Rychkov, G. [0000-0002-2788-2977]