Wheat, L; (2008) The role of the N-terminus of the Cav2.2 voltage-dependent calcium channel in the inhibition by G-proteins. Doctoral thesis , UCL (University College London).

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Abstract

The neuronal Cay2.2 calcium channel is inhibited by Gpy subunits of heterotrimeric G-proteins in a voltage-dependent manner. It has been shown previously that an 11 amino acid motif (44-55) in the intracellular N-terminus of Cav2.2al (aiB) is essential for G-protein modulation of currents. Mutation of 2 amino acids, R52 and R54, in this region completely abolishes G-protein inhibition. They may form part of the G0y binding site, or translate binding into a functional response. To investigate the role of the N-terminus of the Cay2.2ai subunit I have expressed functional channels in Xenopus oocytes and recorded currents using the two-electrode voltage-clamp technique. To examine further the role of the N-terminus I have tethered it to the membrane via an N-terminal palmitoylation motif and I have also used an isolated N-terminus. The palmitoylation motif Cav2.2ai showed reduced modulation by G(3y (lower facilitation ratio and reduced GPCR activated inhibition). Other additions on the N-terminus such as GFP or a HA-tag, also reduced modulation. However, only the palmitoylation motif Cay2.2ai showed an increase in the rate of loss of G-protein modulation during depolarisation (faster facilitation rate). This increase in facilitation rate is unique to the palmitoylation motif Cav2.2ai. The increased facilitation rate may be due to a reduction in the affinity of the channel for Gfiy because of the reduced mobility of the N-terminus.

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