Functional complementation of truncated human skeletal-muscle chloride channel (hClC-1) using carboxyl tail fragments.
Crystal structures of bacterial CLC (voltage-gated chloride channel family) proteins suggest the arrangement of permeation pores and possible gates in the transmembrane region of eukaryotic CLC channels. For the extensive cytoplasmic tails of eukaryotic CLC family members, however, there are no equivalent structural predictions. Truncations of cytoplasmic tails in different places or point mutations result in loss of function or altered gating of several members of the CLC family, suggesting functional importance. In the present study, we show that deletion of the terminal 100 amino acids (N889X) in human ClC-1 (skeletal-muscle chloride channel) has minor consequences, whereas truncation by 110 or more amino acids (from Q879X) destroys channel function. Use of the split channel strategy, co-injecting mRNAs and expressing various complementary constructs in Xenopus oocytes, confirms the importance of the Gln879-Arg888 sequence. A split between the two CBS (cystathionine b-synthase) domains (CBS1 and CBS2) gives normal function (e.g. G721X plus its complement), whereas a partial complementation, eliminating the CBS1 domain, eliminates function. Surprisingly, function is retained even when the region Gly721-Ala862 (between CBS1 and CBS2, and including most of the CBS2 domain) is omitted from the complementation. Furthermore, even shorter peptides from the CBS2-immediate post-CBS2 region are sufficient for functional complementation. We have found that just 26 amino acids from Leu863 to Arg888 are necessary since channel function is restored by co-expressing this peptide with the otherwise inactive truncation, G721X.
Wu
Weiping
W
Centre for Advanced Biomedical Studies, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia.
Rychkov
Grigori Y
GY
Hughes
Bernard P
BP
Bretag
Allan H
AH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003541:Cystathionine beta-Synthase / Q000737:chemistry; D004594:Electrophysiology; D015870:Gene Expression; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D009865:Oocytes; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D014981:Xenopus
D000596:Amino Acids; C086321:CLC-1 channel; D018118:Chloride Channels; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003541:Cystathionine beta-Synthase