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The Journal of Neuroscience, July 27, 2005, 25(30):6984-6996; doi:10.1523/JNEUROSCI.1137-05.2005
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Cellular/Molecular
Interaction via a Key Tryptophan in the I-II Linker of N-Type Calcium Channels Is Required for  1 But Not for Palmitoylated 2, Implicating an Additional Binding Site in the Regulation of Channel Voltage-Dependent Properties
Jérôme Leroy,1
Mark S. Richards,1,2 *
Adrian J. Butcher,1 *
Manuela Nieto-Rostro,1
Wendy S. Pratt,1
Anthony Davies,1 and
Annette C. Dolphin1
1Laboratory of Cellular and Molecular Neuroscience, Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom, and 2School of Crystallography, Birkbeck College, London WC1E 7HX, United Kingdom
The CaV 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  -interacting domain (AID) in the I-II linker of CaV2.2. We showed that the mutation W391 virtually abolishes the binding of CaV1b and CaV2a to the CaV2.2 I-II linker and strongly reduced current density and cell surface expression of both CaV2.2/2 -2/1b and/2a channels. When associated with CaV1b, the W391A mutation also prevented the CaV1b-mediated hyperpolarization of CaV2.2 channel activation and steady-state inactivation. However, the mutated CaV2.2W391A/1b 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, CaV2a 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 CaV2a 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 CaV2a, 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.
Key words: calcium channel; neuron; -interaction domain; subunit; trafficking; G-protein; palmitoylation
Received March 23, 2005;
revised June 13, 2005;
accepted June 15, 2005.
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