J Wratten | PubMed Health

PubMed2006

Do voltage-gated calcium channel alpha2delta subunits require proteolytic processing into alpha2 and delta to be functional?

The accessory alpha2delta subunits of voltage-gated calcium channels are type 1 transmembrane proteins that are highly glycosylated and possess multiple disulfide bonds. From studies of the topology and processing of skeletal-muscle alpha2delta-1, it has been shown to be post-translationally cleaved into an alpha2 and a delta subunit, which remain disulfide-bonded. In the present study, we have examined the processing of alpha2delta-2 subunits when stably or transiently expressed, in tsA (temperature-sensitive A)-201, Cos-7 and NG108-15 cells, and compared it with that observed in the cerebellum. Despite showing full functionality and being expressed on the plasma membrane, the vast majority of heterologously expressed alpha2delta-2 is not cleaved into alpha(2)-2 and delta-2, unlike endogenous alpha2delta-2 in the cerebellum. It remains an open question for future research whether alpha2delta-2 is functional in its calcium channel trafficking role in its proteolytically cleaved or non-cleaved state.

Animals↗

PubMed2005

The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of alpha2delta subunits is key to trafficking voltage-gated Ca2+ channels.

All auxiliary alpha2delta subunits of voltage-gated Ca2+ (Ca(V)) channels contain an extracellular Von Willebrand factor-A (VWA) domain that, in alpha2delta-1 and -2, has a perfect metal-ion-dependent adhesion site (MIDAS). Modeling of the alpha2delta-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 alpha2delta-2 subunit that was still processed and trafficked normally when it was expressed alone. However, unlike WT alpha2delta-2, the MIDAS mutant alpha2delta-2 subunit did not enhance and, in some cases, further diminished Ca(V)1.2, -2.1, and -2.2 currents coexpressed with beta1b by using either Ba2+ or Na+ as a permeant ion. Furthermore, expression of the MIDAS mutant alpha2delta-2 reduced surface expression and strongly increased the perinuclear retention of Ca(V)alpha1 subunits at the earliest time at which expression was observed in both Cos-7 and NG108-15 cells. Despite the presence of endogenous alpha2delta subunits, heterologous expression of alpha2delta-2 in differentiated NG108-15 cells further enhanced the endogenous high-threshold Ca2+ currents, whereas this enhancement was prevented by the MIDAS mutations. Our results indicate that alpha2delta subunits normally interact with the Ca(V)alpha1 subunit early in their maturation, before the appearance of functional plasma membrane channels, and an intact MIDAS motif in the alpha2delta subunit is required to promote trafficking of the alpha1 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.

Binding Sites↗