Natural substitutions at highly conserved T1-domain residues perturb processing and functional expression of squid Kv1 channels.
Shaker-type K-channel alpha-subunits (SqKv1A, B, D) expressed in neurons of the squid stellate ganglion differ in the length of their N-termini and in the species of amino acid present at several points in the T1 domain, an intracellular region involved in the tetramerization process during channel assembly. Heterologous expression of wild-type SqKv1A, B, and D in Xenopus oocytes reveals large differences in the level of both functional channels (assayed by whole-oocyte voltage clamp) and total channel protein (assayed by immunoblotting). Functional expression is poorest with SqKv1A and by far the best with SqKv1D. Biophysical properties of the three SqKv1 channels are essentially identical (assayed by cell-attached patch clamp). Site-directed mutagenesis was used to determine whether the observed differences in expression level are impacted by two residues in the T1 domain at which SqKv1A and B (but not D) differ from the consensus sequences found in many other taxa. In SqKv1A, glycine is substituted for arginine in an otherwise universally conserved sequence (FFDR in the T1(B) subdomain). In SqKv1B, glycine replaces serine in a sequence that is conserved within the Kv1 subfamily (SGLR in the T1(A) subdomain). Restoration of the consensus amino acid at these positions largely accounts for the observed differences in expression level. Analysis of the glycosylation state of aberrant versus restored alpha-subunits suggests that the anomalous amino acids in SqKv1A and B exert their influence during early steps in channel processing and assembly which take place in the endoplasmic reticulum (ER).
Liu
T I
TI
Neurosciences Program, Hopkins Marine Station, Stanford University, Pacific Grove, California 93950, USA.
Lebaric
Z N
ZN
Rosenthal
J J
JJ
Gilly
W F
WF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D016384:Consensus Sequence / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics*; D049832:Decapodiformes; D006031:Glycosylation; D015151:Immunoblotting; D008845:Microinjections; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics* / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D018075:RNA, Complementary / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014981:Xenopus
D015221:Potassium Channels; D020033:Protein Isoforms; D018075:RNA, Complementary