Structural requirement of the calcium-channel subunit alpha(2)delta for gabapentin binding

Gabapentin [Neurontin, 1-(aminomethyl)cyclohexaneacetic acid] is a novel an ticonvulsant drug with a high binding affinity for the Ca2+-channel subunit alpha(2)delta. In this study, the gabapentin-binding properties of wild-ty pe and mutated porcine brain alpha(2)delta proteins were investigated. Remo val of the disulphide bonds between the alpha(2) and the delta subunits did not result in a significant loss of gabapentin binding, suggesting that th e disulphide linkage between the two subunits is not required for binding. Singly expressed alpha(2) protein remained membrane associated. However, al pha(2) alone was unable to bind gabapentin, unless the cells were concurren tly transfected with the expression vector for delta, suggesting that both alpha(2) and delta are required for gabapentin binding. Using internal dele tion mutagenesis, we mapped two regions [amino acid residues 339-365 (Delta F) and 875-905 (Delta J)] within the alpha(2) subunit that are not require d for gabapentin binding. Further, deletion of three other individual regio ns [amino acid residues 206-222 (Delta D), 516-537 (Delta H) and 583-603 (D elta I)] within the alpha(2) subunit disrupted gabapentin binding, suggesti ng the structural importance of these regions. Using alanine to replace fou r to six amino acid residues in each of these regions abolished gabapentin binding. These results demonstrate that region D, between the N-terminal en d and the first putative transmembrane domain of alpha(2), and regions H an d I, between the putative splicing acceptor sites (Gln(511) and Ser(601)), may play important roles in maintaining the structural integrity for gabape ntin binding. Further single amino acid replacement mutagenesis within thes e regions identified Arg(217) as critical for gabapentin binding.