Conserved Ca2+-antagonist-binding properties and putative folding structure of a recombinant high-affinity dihydropyridine-binding domain

Sensitivity to 1,4-dihydropyridines (DHPs) can be transferred from L-type (alpha 1C) to non-L-type (alpha 1A) Ca2+ channel alpha 1 subunits by the mutation of nine pore-associated non-conserved amino acid residues, yielding mutant alpha 1A(DHP). To determine whether the hallmarks of reversible DHP binding to L-type Ca2+ channels (nanomolar dissociation constants, stereoselectivity and modulation by other chemical classes of Ca2+ antagonist drugs) were maintained in alpha 1A(DHP), we analysed the pharmacological properties of (+)-[H-3]isradipine-labelled alpha 1A(DHP) Ca2+ channels after heterologous expression. Binding of (+)-isradipine (K-i 7.4 nM) and the non-benzoxadiazole DHPs nifedipine (K-i 86 nM), (+/-)-nitrendipine (K-i 33 nM) and (+/-)-nimodipine (K-i 67 nM) to alpha 1A(DHP) occurred at low nanomolar K-i values. DHP binding was highly stereoselective [25-fold higher affinity for (+)-isradipine]. As with native channels it was stimulated by (+)-cis-diltiazem, (+)-tetrandrine and mibefradil. This suggested that the three-dimensional architecture of the channel pore was maintained within the non-L-type alpha 1A subunit. To predict the three-dimensional arrangement of the DHP-binding residues we exploited the X-ray structure of a recently crystallized bacterial K+ channel (KcsA) as a template. Our model is based on the assumption that the Ca2+ channel S5 and S6 segments closely resemble the KcsA transmembrane folding architecture. In the absence of three-dimensional structural data for the oil subunit this is currently the most reasonable approach for modelling this drug-interaction domain. Our model predicts that the previously identified DHP-binding residues form a binding pocket large enough to co-ordinate a single DHP molecule. It also implies that the four homologous Ca2+ channel repeats are arranged in a clockwise manner.