The cloning and functional characterisation of a family of human stargazin-like genes

1) Stargazin, the product of the gene mutated in the epileptic mouse Stargazer, is hypothesised to be the first example of a neuronal voltage-dependent calcium channel (VDCC) γ subunit. The aim of this thesis project was to identify, clone and characterise the functional properties of a family of human stargazin-like genes when expressed as part of a VDCC complex. 2) A family of five human stargazin-like genes were identified and cloned, including the human orthologue of stargazin itself. 3) The γ2, γ3 and γ4 subunits are closely related to each other and are encoded by four exon genes. The γ5 and γ7 subunits display much lower homology to stargazin and are encoded by five exon genes, indicating they form a distinct subfamily of stargazin-like proteins. 4) The human γ2, γ3, γ4, γ5 and γ7 subunit proteins share a conserved predicted tetra- spanning transmembrane topology. However, whilst the γ2, γ3 and γ4 subunits localise specifically to the plasma membrane upon heterologous expression, γ7 is expressed throughout the cell with little insertion into the membrane. 5) The γ2, γ3, γ4 and γ7 subunits are selectively expressed in brain with varying distributions in the different brain regions. More detailed distribution and cellular localisation data for the γ2, γ3, and γ4 subunits in human cerebellum and hippocampus are reported. 6) Co-expression of either the γ2 or γ4 subunits failed significantly to modulate the biophysical properties of Cav2.1/β4 VDCCs in the absence or presence of an α2δ subunit. However, N-type current through Cav2.2 channels was almost abolished when co-expressed transiently with γ7 in either Xenopus oocytes or COS-7 cells. Furthermore, γ7 has this effect by causing a large reduction in expression of Cav2.2 protein rather than by interfering with trafficking or biophysical properties of the channel