The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of α 2 δ subunits is key to trafficking voltage-gated Ca 2 + channels

Abstract

All auxiliary α 2 δ subunits of voltage-gated Ca 2+ (Ca V ) channels contain an extracellular Von Willebrand factor-A (VWA) domain that, in α 2 δ-1 and -2, has a perfect metal-ion-dependent adhesion site (MIDAS). Modeling of the α 2 δ-2 VWA domain shows it to be highly likely to bind a divalent cation. Mutating the three key MIDAS residues responsible for divalent cation binding resulted in a MIDAS mutant α 2 δ-2 subunit that was still processed and trafficked normally when it was expressed alone. However, unlike WT α 2 δ-2, the MIDAS mutant α 2 δ-2 subunit did not enhance and, in some cases, further diminished Ca V 1.2, -2.1, and -2.2 currents coexpressed with β1b by using either Ba 2+ or Na + as a permeant ion. Furthermore, expression of the MIDAS mutant α 2 δ-2 reduced surface expression and strongly increased the perinuclear retention of Ca V α1 subunits at the earliest time at which expression was observed in both Cos-7 and NG108–15 cells. Despite the presence of endogenous α 2 δ subunits, heterologous expression of α 2 δ-2 in differentiated NG108–15 cells further enhanced the endogenous high-threshold Ca 2+ currents, whereas this enhancement was prevented by the MIDAS mutations. Our results indicate that α 2 δ subunits normally interact with the Ca V α1 subunit early in their maturation, before the appearance of functional plasma membrane channels, and an intact MIDAS motif in the α 2 δ subunit is required to promote trafficking of the α1 subunit to the plasma membrane by an integrin-like switch. This finding provides evidence for a primary role of a VWA domain in intracellular trafficking of a multimeric complex, in contrast to the more usual roles in binding extracellular ligands in other exofacial VWA domains.