How Does the α2δ-1 Subunit Modulate Skeletal CaV1.1 Channels?

Abstract

The pore-forming subunit α1S of voltage-gated L-type calcium channels CaV1.1 forms macromolecular complexes in skeletal muscles by associating with auxiliary subunits α2δ-1, β1a and γ1. To gain insights on the molecular mechanisms of CaV1.1 regulation by the α2δ-1 subunit, known to slow down current activation kinetics (Obermair et al., 2005, J Biol Chem.), we took advantage of the adaptor protein STAC3, which allows for the heterologous expression of CaV1.1 channels (Polster et al., 2015, PNAS). We expressed CaV1.1 complexes (α1S, β1a and STAC3) with or without α2δ-1 subunit in Xenopus oocytes. Using the voltage clamp fluorometry technique, we confirmed that the presence of α2δ-1 slowed current activation kinetic, while leaving channel voltage dependent activation (GV) largely unaffected: Vhalf= 32.9±0.5mV (no α2δ-1), n=6; 32.8±0.3mV (+α2δ-1), n=5. By optically tracking the molecular rearrangements of individual voltage-sensing domain (VSD), we found that the auxiliary subunit α2δ-1 modified the voltage-dependent activation of VSD-IV, shifting the F(V) curve by ∼20mV towards more negative potentials: Vhalf= −7.4±1.9mV (no α2δ-1); −27.0±2.4mV (+α2δ-1) (n=5). The time- and voltage-dependent properties of VSD-III remained mostly unperturbed. As VSD-IV was previously found to be important for CaV1.1 channel activation, the observed α2δ-1-induced shift of VSD-IV voltage dependence without consequence on the GV curve of the channel would suggest that α2δ-1 causes a concurrent modulation of VSDs I and/or II in the opposite direction (under investigation), counterbalancing VSD-IV effect. It is intriguing that the α2δ-1 subunit, which dramatically facilitates the human CaV1.2 voltage activation causing ∼50 mV hyperpolarizing shift of the GV, leaves skeletal CaV1.1 activation curve largely unperturbed. We are using our CaV channel allosteric model to estimate the energetic contribution of each VSD to channel opening in the presence of α2δ-1.