Cloning and Deletion Mutagenesis of the α2δ Calcium Channel Subunit from Porcine Cerebral Cortex

Abstract

The anti-epileptic, anti-hyperalgesic, and anxiolytic agent gabapentin (1-(aminomethyl)-cyclohexane acetic acid or Neurontin) has previously been shown to bind with high affinity to the α2δ subunit of voltage-dependent calcium channels (Gee, N. S., Brown, J.P., Dissanayake, V. U. K., Offord, J., Thurlow, R., and Woodruff, G.N. (1996) J. Biol. Chem. 271, 5768-5776). We report here the cloning, sequencing, and deletion mutagenesis of the α2δ subunit from porcine brain. The deduced protein sequence has a 95.9 and 98.2% identity to the rat and human neuronal α2δ sequences, respectively. [3H]Gabapentin binds with a K(D) of 37.5 ± 10.4 nM to membranes prepared from COS-7 cells transfected with wild-type porcine α2δ cDNA. Six deletion mutants (B-G) that lack the δ polypeptide, together with varying amounts of the α2 component, failed to bind [3H]gabapentin. C-terminal deletion mutagenesis of the δ polypeptide identified a segment (residues 960-994) required for correct assembly of the [3H]gabapentin binding pocket. Mutant L, which lacks the putative membrane anchor in the sequence, was found in both membrane-associated and soluble secreted forms. The soluble form was not proteolytically cleaved into separate α2 and δ chains but still retained a high affinity (K(D) = 30.7 ± 8.1 nM) for [3H]gabapentin. The production of a soluble α2δ mutant supports the single transmembrane model of the α2δ subunit and is an important step toward the large-scale recombinant expression of the protein.