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 alpha1 with auxiliary β and alpha2delta subunits. The alpha2delta 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 alpha2delta-1, termed the R domain (Rd), is both necessary and sufficient to replicate the effects of full-length alpha2delta 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 alpha2delta were explored producing a set of alpha2delta-truncated proteins, in which the delta protein was inserted into an inert type-1 transmembrane reporter protein (PIN-G). The construct was then extended towards the N-terminal of the alpha2delta-1 (C- to N- PIN-constructs). Other sets of constructs, lacking the delta protein, were prepared after successive additions of stop codons (TGA) in the alpha2delta (N- to C- PIN-constructs). The MIDAS motif within the VWA domain of alpha2delta-1/-2 has been suggested to be critical for trafficking of alpha2delta to the cell surface. Whilst the present study supports a role for MIDAS in surface expression of alpha2delta, it is the Rd that appears essential. Mutation of MIDAS reduced expression, whereas the removal of Rd completely abolished the presence of alpha2delta at the cell surface. Examination of the electrophysiological effects of N- to C- terminal truncated constructs (PIN-Rd, PIN-Rd-VWA and PIN-alpha2) 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 alpha2 region, abolished typical alpha2delta-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 alpha2delta. Preliminary work has generated tools that could be used to conduct competition-based assays to identify the extracellular loops of the CaV2.2 alpha1 subunit that interact with the Rd. Such an approach could be applied to other alpha1 subtypes to determine discrete alpha2-Rd interactions, information that is critical for further therapeutic exploitation of alpha2delta. Finally, the data from this thesis and the existing literature have been used to propose a revised model of how alpha2delta interacts with CaV.
|Date of Award||1 Aug 2016|
- The University of Manchester
|Supervisor||Liz Fitzgerald (Supervisor) & David Eisner (Supervisor)|
- calcium channel
- auxiliary subunit