Functional study of cytoplasmic loops of human skeletal muscle chloride channel, hClC-1.
The 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.
Ma
Linlin
L
Sansom Institute, School of Pharmacy and Medical Sciences, University of South Australia, North Terrace, Adelaide, SA 5000, Australia.
Rychkov
Grigori Y
GY
Bretag
Allan H
AH
D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D018482:Muscle, Skeletal / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D014162:Transfection
C086321:CLC-1 channel; D018118:Chloride Channels; D000409:Alanine