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

Abstract

Sensitivity to 1,4-dihydropyridines (DHPs) can be transferred from L-type (α1C) to non-L-type (α1A) Ca2+ channel α1 subunits by the mutation of nine pore-associated non-conserved amino acid residues, yielding mutant α1ADHP. 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 α1ADHP, we analysed the pharmacological properties of (+)-[3H]isradipine-labelled α1ADHP Ca2+ channels after heterologous expression. Binding of (+)-isradipine (Ki 7.4 nM) and the non-benzoxadiazole DHPs nifedipine (Ki 86 nM), (±)-nitrendipine (Ki 33 nM) and (±)-nimodipine (Ki 67 nM) to α1ADHP occurred at low nanomolar Ki 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 α1A 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 α1 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.