Differential Stability of CaVβ2a and CaVβ3 in a CaV1.2 Calcium Channel Complex

Abstract

In voltage-gated calcium channels, auxiliary CaVβ subunits function in membrane targeting of the channel and modulation of its gating properties. Structural studies have revealed that the high affinity CaVα1/β interaction relies on a conserved CaVβ-binding sequence in the I-II loop of the CaVα1 subunit, called the α-interaction domain (AID). We have previously shown that in skeletal muscle triads the β1a subunit forms stable complexes with both CaV1.1 and CaV1.2, while β2a forms dynamic complexes (Campiglio et al., JCS 2013). To further test the reversibility of the CaV1.2/β2a interaction, here we injected an AID peptide into Xenopus oocytes expressing CaV1.2/β2a/α2δ-1 complexes and examined the effect of the competing peptide on current inactivation. Unexpectedly, the AID peptide had no effect on the currents of wildtype CaV1.2/β2a/α2δ-1 channel complexes. However, when a CaV1.2 AID residue known to decrease the interaction with β2a by 1000-fold was mutated (Y437A), injection of AID peptide changed the inactivation properties of the channels similar to properties of channels expressed without a β subunit. These results suggest that under our experimental conditions binding of the wild-type CaV1.2/β2a pair is too strong for peptide competition. In contrast, in wild-type CaV1.2 channel complexes containing the β3 subunit injection of the AID peptide caused a strong effect on inactivation properties, indicating efficient competition. Consistent with a lower stability of CaV1.2/β3 complexes compared to CaV1.2/β2a, β3 also showed a significantly reduced extent of co-clustering with CaV1.2 in the triads of dysgenic myotubes. Ongoing FRAP experiments will reveal whether diminished co-clustering of β3 correlates with higher mobility and dynamic β subunit exchange in a native calcium channel signaling complex. Funding: FWF P23479 and W1101 (BEF), NIH R01 HL080050 (DLM).