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Structure-function mapping of the voltage-gated calcium channel α2δ-1 subunit.
[Thesis]. Manchester, UK: The University of Manchester; 2016.
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Abstract
Voltage-gated calcium channels (CaV) are key regulators of cellular excitability; they translate electrical information into biochemical responses in excitable cells such as nerve and muscle cells. CaV are separated in three families: CaV1, CaV2 and CaV3. CaV1 and CaV2 typically comprise a pore-forming α1 with auxiliary β and α2δ subunits. The α2δ enhances surface expression and modulates the biophysical properties of CaV. It has been implicated in pain and epilepsy, and the target for anti-epileptic and anti-nociceptive gabapentinoid drugs. Despite its clinical significance, the relationship between the structure and function of this subunit remains poorly understood. Fitzgerald and co-workers recently showed that the N-terminal region of α2δ-1, termed the R domain (Rd), is both necessary and sufficient to replicate the effects of full-length α2δ on CaV2.2 channels. In order to understand the functional role(s) of Rd and the regions downstream of it, the biochemical and cell biological properties of α2δ were explored producing a set of α2δ-truncated proteins, in which the δ protein was inserted into an inert type-1 transmembrane reporter protein (PIN-G). The construct was then extended towards the N-terminal of the α2δ-1 (C- to N- PIN-constructs). Other sets of constructs, lacking the δ protein, were prepared after successive additions of stop codons (TGA) in the α2δ (N- to C- PIN-constructs). The MIDAS motif within the VWA domain of α2δ-1/-2 has been suggested to be critical for trafficking of α2δ to the cell surface. Whilst the present study supports a role for MIDAS in surface expression of α2δ, it is the Rd that appears essential. Mutation of MIDAS reduced expression, whereas the removal of Rd completely abolished the presence of α2δ at the cell surface. Examination of the electrophysiological effects of N- to C- terminal truncated constructs (PIN-Rd, PIN-Rd-VWA and PIN-α2) on CaV2.2/β1b channels revealed that, in contrast to the full functionality of Rd alone, extension to the end of the VWA domain, or the α2 region, abolished typical α2δ-mediated current enhancement. Nevertheless, both constructs increased rate of voltage-dependent inactivation, indicating that they interact with the channel via Rd. Thus, Rd appears to contain all the machinery required to support the electrophysiological and trafficking effects of α2δ. Preliminary work has generated tools that could be used to conduct competition-based assays to identify the extracellular loops of the CaV2.2 α1 subunit that interact with the Rd. Such an approach could be applied to other α1 subtypes to determine discrete α2-Rd interactions, information that is critical for further therapeutic exploitation of α2δ. Finally, the data from this thesis and the existing literature have been used to propose a revised model of how α2δ interacts with CaV.