Interaction via a Key Tryptophan in the I-II Linker of N-Type Calcium Channels Is Required for beta 1 But Not for Palmitoylated beta 2, Implicating an Additional Binding Site in the Regulation of Channel Voltage-Dependent Properties

• Published in: The Journal of neuroscience 2005-07, Vol.25 (30), p.6984-6996
• Main Authors: Leroy, Jerome, Richards, Mark S, Butcher, Adrian J, Nieto-Rostro, Manuela, Pratt, Wendy S, Davies, Anthony, Dolphin, Annette C
• Format: Article
• Language: English
• ISSN: 0270-6474; 1529-2401
• DOI: 10.1523/JNEUROSCI.1137-05.2005

The Ca sub(V) beta 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 Ca sub(V)2.2. We showed that the mutation W391 virtually abolishes the binding of Ca sub(V) beta 1b and Ca sub(V) beta 2a to the Ca sub(V)2.2 I-II linker and strongly reduced current density and cell surface expression of both Ca sub(V)2.2/ alpha 2 delta -2/ beta 1b and/ beta 2a channels. When associated with Ca sub(V) beta 1b, the W391A mutation also prevented the Ca sub(V) beta 1b-mediated hyperpolarization of Ca sub(V)2.2 channel activation and steady-state inactivation. However, the mutated Ca sub(V)2.2W391A/ beta 1b channels were still inhibited to a similar extent by activation of the D sub(2) 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, Ca sub(V) beta 2a was still able to completely modulate the biophysical properties of Ca sub(V)2.2W391A channel and allow voltage-dependent G-protein modulation of Ca sub(V)2.2W391A. Additional data suggest that the concentration of Ca sub(V) beta 2a 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 Ca sub(V) beta 2a, 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.