Butcher, A.; (2006) Protein interaction studies of the voltage-dependent calcium channel beta subunit. Doctoral thesis , University of London.

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Abstract

Voltage dependent calcium channels (VDCCs) are heteromultimeric membrane proteins which open to allow Ca entry upon depolarisation of the plasma membrane. The p subunit of voltage-dependent calcium channels interacts with the alpha interaction domain (AID) in the I-II linker of the pore forming al subunit and regulates trafficking and biophysical properties of these channels. A surface plasmon resonance binding assay has been developed to measure the interaction between the I-II linkers of Cav 1.3, Cav 2.1 and Cay2.2 and CavP lb, Cavp2a and CavP3 and the dissociation constants (Kd) for these interactions have been calculated and were found to be between 12.1 and 20nM. The assay was then extended to measure the interaction between the I-II linkers of Cav 13 and Cav 2.2 and G protein py subunits. The dissociation constant for G Py binding to the I-II linker of Cav2.2 was calculated to be 62.2nM whereas no interaction was detected between the I-II linker of Cavl.3 and GPy. The effects of specific mutations within AID on CavP subunit binding and calcium channel trafficking were measured using surface plasmon resonance and cell surface biotinylation assays. Mutation of tryptophan within AID abolished CavP binding and reduced the amount of Cav2.2 channels in the plasma membrane whereas mutation of tyrosine reduced the affinity of CavP for AID by 24 fold but had no effect on the biophysical properties or trafficking of Cay2.2 expressed in tsA 201 cells. Activation of phosphoinositide 3-kinase (PI3 kinase) promotes translocation of VDCC's to the plasma membrane, this mechanism involves protein kinase B and is specific for calcium channels containing the CavP2a subunit. Co-expression of PI3 kinase resulted in increased phosphorylation on serine 574 of CavP2a and protein kinase B was found to co-precipitate with Cavp2a. Finally, a proteomic approach was used to identify proteins which may interact with CavP subunits. Ca2+/calmodulin dependent kinase II was found to interact specifically with the C-terminus of CavP lb.

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