Analysis of carboxyl tail function in the skeletal muscle Cl- channel hClC-1.
Human ClC-1 (skeletal muscle Cl- channel) has a long cytoplasmic C-tail (carboxyl tail), containing two CBS (cystathionine beta-synthase) domains, which is very important for channel function. We have now investigated its significance further, using deletion and alanine-scanning mutagenesis, split channels, GST (glutathione transferase)-pull-down and whole-cell patch-clamping. In tagged split-channel experiments, we have demonstrated strong binding between an N-terminal membrane-resident fragment (terminating mid-C-tail at Ser(720) and containing CBS1) and its complement (containing CBS2). This interaction is not affected by deletion of some sequences, suggested previously to be important, particularly in channel gating. Contact between CBS1 and CBS2, however, may make a major contribution to assembly of functional channels from such co-expressed complements, although the possibility that C-tail fragments could, in addition, bind to other parts of the membrane-resident component has not been eliminated. We now show such an interaction between a membrane-resident component terminating at Ser(720) (but with CBS1 deleted) and a complete C-tail beginning at Leu(598). Channel function is rescued in patch-clamped HEK-293T (human embryonic kidney) cells co-expressing these same fragments. From our own results and those of others, we conclude that the CBS1-CBS2 interaction is not sufficient, in itself, for channel assembly, but rather that this might normally assist in bringing some part of the CBS2/C-tail region into appropriate proximity with the membrane-resident portion of the protein. Previously conflicting and anomalous results can now be explained by an hypothesis that, for split channels to be functional, at least one membrane-resident component must include a plasma membrane trafficking signal between Leu(665) and Lys(680).
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
Hughes
Bernard P
BP
Bretag
Allan H
AH
D000595:Amino Acid Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D018118:Chloride Channels / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating; D018482:Muscle, Skeletal / Q000378:metabolism*; D017434:Protein Structure, Tertiary
C086321:CLC-1 channel; D018118:Chloride Channels