Auxiliary voltage-gated calcium channel β subunits (Cavβ) regulate cellular calcium signalling by trafficking pore-forming Cavα1 subunits to the cell surface and modifying channel gating. Cavβs share structural and functional similarity with the membrane-associated guanylate kinase scaffold proteins, having a functional core composed of conserved Src homology 3 (SH3) and guanylate kinase (GK) domains, linked by a flexible “hook” region. While the function of the SH3-GK core is largely known, the roles of the variable N- and C-termini that flank the core are less well understood. Cavβs may function as molecular scaffolds. Thus, the N- and C-termini of specific β isoforms may be involved not only in channel gating but also in membrane targeting and localisation of Cav channels, to enable fine-tuning of calcium signalling within discrete sub-cellular regions. Here, we have systematically examined the influence of the N- and C-termini of Cavβs on the localisation and biophysical properties of neuronal N-type (Cav2.2) channels expressed in COS-7 cells. Using CFP-tagged fusion proteins of all four neuronal β isoforms (β1b, β2a, β3, β4), we initially compared their respective distributions when expressed alone and with Cav2.2. Our findings support other evidence for differential distribution of individual β subunit isoforms. Whilst all four βs were distributed throughout the cell, β2a and β3 exhibited particularly strong plasma membrane expression whereas β4 was localised primarily to the nucleus. Regardless of their differential distributions when expressed alone, all CFP-βs promoted trafficking of Cav2.2 to the cell surface to produce functional channels with biophysical properties identical to those of equivalent wild type β subunits. By construction of CFP-β1b deletion mutants lacking the N- and/or C-termini, we next assessed the role of these variable domains in the localisation and function of Cav2.2. Removal of the N- and/or C-termini reduced surface expression of Cav2.2 although the SH3-GK core alone was nevertheless sufficient to promote membrane trafficking of GFP-Cav2.2. All four constructs produced functional channels although, unexpectedly given imaging data, the current densities were the same for all, suggesting saturation of functional expression. In contrast to other reports, loss of the C-terminus of β1b exerted a profound influence on channel gating, specifically enhancing the rate of current activation and reducing Cav2.2 channel availability. Together, these findings highlight the importance of both the N- and C-termini of β subunits in differential targeting and gating of Cav channels. Experiments are in progress to assess possible additional roles of these hypervariable domains in modulating Cavβ protein-protein interactions.