Key result
The W391A mutation in the CaV2.2 I-II linker abolishes binding of CaVβ1b and CaVβ2a, reducing current density, but palmitoylated CaVβ2a still modulates channel properties via a secondary site.
The study reveals that while the primary AID binding site is required for trafficking N-type calcium channels to the plasma membrane, palmitoylated CaVbeta2a can modulate channel properties via a secondary binding site.
Should not yet inform clinical practice; leaves open secondary β-subunit sites as targets for N-type channel modulation in disease models.
The CaVbeta subunits of voltage-gated calcium channels regulate these channels in several ways. Here we investigate the role of these auxiliary subunits in the expression of functional N-type channels at the plasma membrane and in the modulation by G-protein-coupled receptors of this neuronal channel. To do so, we mutated tryptophan 391 to an alanine within the alpha-interacting domain (AID) in the I-II linker of CaV2.2. We showed that the mutation W391 virtually abolishes the binding of CaVbeta1b and CaVbeta2a to the CaV2.2 I-II linker and strongly reduced current density and cell surface expression of both CaV2.2/alpha2delta-2/beta1b and/beta2a channels. When associated with CaVbeta1b, the W391A mutation also prevented the CaVbeta1b-mediated hyperpolarization of CaV2.2 channel activation and steady-state inactivation. However, the mutated CaV2.2W391A/beta1b channels were still inhibited to a similar extent by activation of the D2 dopamine receptor with the agonist quinpirole. Nevertheless, key hallmarks of G-protein modulation of N-type currents, such as slowed activation kinetics and prepulse facilitation, were not observed for the mutated channel. In contrast, CaVbeta2a was still able to completely modulate the biophysical properties of CaV2.2W391A channel and allow voltage-dependent G-protein modulation of CaV2.2W391A. Additional data suggest that the concentration of CaVbeta2a in the proximity of the channel is enhanced independently of its binding to the AID by its palmitoylation. This is essentially sufficient for all of the functional effects of CaVbeta2a, which may occur via a second lower-affinity binding site, except trafficking the channel to the plasma membrane, which requires interaction with the AID region.
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Leroy et al. (2005) studied this question. W391A mutation in CaV2.2 I-II linker vs. Wild-type CaV2.2 was evaluated on Binding of CaVβ1b and CaVβ2a to CaV2.2 I-II linker and current density. The W391A mutation in the CaV2.2 I-II linker abolishes binding of CaVβ1b and CaVβ2a, reducing current density, but palmitoylated CaVβ2a still modulates channel properties via a secondary site.
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