A specific tryptophan in the I-II linker is a key determinant of beta-subunit binding and modulation in Ca(V)2.3 calcium channels.
The ancillary beta subunits modulate the activation and inactivation properties of high-voltage activated (HVA) Ca(2+) channels in an isoform-specific manner. The beta subunits bind to a high-affinity interaction site, alpha-interaction domain (AID), located in the I-II linker of HVA alpha1 subunits. Nine residues in the AID motif are absolutely conserved in all HVA channels (QQxExxLxGYxxWIxxxE), but their contribution to beta-subunit binding and modulation remains to be established in Ca(V)2.3. Mutations of W386 to either A, G, Q, R, E, F, or Y in Ca(V)2.3 disrupted [(35)S]beta3-subunit overlay binding to glutathione S-transferase fusion proteins containing the mutated I-II linker, whereas mutations (single or multiple) of nonconserved residues did not affect the protein-protein interaction with beta3. The tryptophan residue at position 386 appears to be an essential determinant as substitutions with hydrophobic (A and G), hydrophilic (Q, R, and E), or aromatic (F and Y) residues yielded the same results. beta-Subunit modulation of W386 (A, G, Q, R, E, F, and Y) and Y383 (A and S) mutants was investigated after heterologous expression in Xenopus oocytes. All mutant channels expressed large inward Ba(2+) currents with typical current-voltage properties. Nonetheless, the typical hallmarks of beta-subunit modulation, namely the increase in peak currents, the hyperpolarization of peak voltages, and the modulation of the kinetics and voltage dependence of inactivation, were eliminated in all W386 mutants, although they were preserved in part in Y383 (A and S) mutants. Altogether these results suggest that W386 is critical for beta-subunit binding and modulation of HVA Ca(2+) channels.
Berrou
L
L
Département de Physiologie, Membrane Transport Research Group, Université de Montréal, Montréal, Quebec H3C 3J7, Canada.
Klein
H
H
Bernatchez
G
G
Parent
L
L
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism; D020908:Calcium Channels, R-Type; D027682:Cation Transport Proteins; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014364:Tryptophan / Q000737:chemistry*; D014981:Xenopus / Q000196:embryology / Q000378:metabolism
C423009:CACNA1E protein, human; D015220:Calcium Channels; D020908:Calcium Channels, R-Type; D027682:Cation Transport Proteins; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D004247:DNA; D005982:Glutathione Transferase