The R-Domain: Identification of an N-terminal Region of the alpha 2 delta-1 Subunit Which is Necessary and Sufficient for its Effects on Ca(v)2.2 Calcium Currents

Voltage-gated calcium channels (Cav) and their associated proteins are pivotal signalling complexes in excitable cell physiology. In nerves and muscle, Cav tailor calcium influx to processes including neurotransmission, muscle contraction and gene expression. Cav comprise a pore-forming 1 and modulatory beta and alpha 2 delta subunits - the latter targeted by anti-epileptic and anti-nociceptive gabapentinoid drugs. However, the mechanisms of gabapentinoid action are unclear, not least because detailed structure-function mapping of the 2 subunit remains lacking. Using molecular biology and electro-physiological approaches we have conducted the first systematic mapping of 2 subunit structure-function. We generated a series of cDNA constructs encoding chimera, from which successive amino acids from the rat alpha(2)delta-1 subunit were incorporated into a Type 1 reporter protein - PIN-G, to produce sequential extensions from the transmembrane (TM) region towards the N-terminus. By successive insertion of a TGA stop codon, a further series of N-to C-terminal extension constructs lacking the TM region, were also generated. Using this approach we have defined the minimal region of alpha(2)delta-1 -we term the R-domain (Rd), that appears to contain all the machinery necessary to support the electro-physiological and trafficking effects of alpha(2)delta-1 on Ca-v. Structural algorithms predict that Rd is conserved across all four alpha(2)delta subunits, including RNA splice variants, and irrespective of phyla and taxa. We suggest, therefore, that Rd likely constitutes the major locus for physical interaction with the alpha(1) subunit and may provide a target for novel Cav therapeutics.