Biophys J, September 2002, p. 1429-1442, Vol. 83, No. 3

A Specific Tryptophan in the I-II Linker Is a Key Determinant of -Subunit Binding and Modulation in Ca_V2.3 Calcium Channels

Département de Physiologie, Membrane Transport Research Group, Université de Montréal, Montréal, Quebec H3C 3J7, Canada

The ancillary  subunits modulate the activation and inactivation properties of high-voltage activated (HVA) Ca²⁺ channels in an isoform-specific manner. The  subunits bind to a high-affinity interaction site, -interaction domain (AID), located in the I-II linker of HVA 1 subunits. Nine residues in the AID motif are absolutely conserved in all HVA channels (QQxExxLxGYxxWIxxxE), but their contribution to -subunit binding and modulation remains to be established in Ca_V2.3. Mutations of W386 to either A, G, Q, R, E, F, or Y in Ca_V2.3 disrupted [³⁵S]3-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 3. 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. -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 Ba²⁺ currents with typical current-voltage properties. Nonetheless, the typical hallmarks of -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 -subunit binding and modulation of HVA Ca²⁺ channels.

Biophys J, September 2002, p. 1429-1442, Vol. 83, No. 3
© 2002 by the Biophysical Society   0006-3495/02/09/1429/14  $2.00

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