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 (alpha1C) to non-L-type (alpha1A) Ca(2+) channel alpha1 subunits by the mutation of nine pore-associated non-conserved amino acid residues, yielding mutant alpha1A(DHP). To determine whether the hallmarks of reversible DHP binding to L-type Ca(2+) channels (nanomolar dissociation constants, stereoselectivity and modulation by other chemical classes of Ca(2+) antagonist drugs) were maintained in alpha1A(DHP), we analysed the pharmacological properties of (+)-[(3)H]isradipine-labelled alpha1A(DHP) Ca(2+) 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 alpha1A(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 alpha1A 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 Ca(2+) channel S5 and S6 segments closely resemble the KcsA transmembrane folding architecture. In the absence of three-dimensional structural data for the alpha1 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 Ca(2+) channel repeats are arranged in a clockwise manner.

High affinity interaction of mibefradil with voltage‐gated calcium and sodium channels

“…Taken together these results strongly indicate that mibefradil binds to the same multisubsite domain on the α1 subunit of L‐type Ca 2+ channels that contains the closely associated interaction sites for DHPs, PAAs and BTZs ( Hockerman et al ., 1997 ; Striessnig et al ., 1998 ). This is further supported by our recent finding that mibefradil not only stimulates DHP binding to recombinant α1C and brain L‐type channels (this study) but also to a recombinant DHP binding domain transferred into previously DHP insensitive α1A subunits by site‐directed mutagenesis ( Huber et al ., 2000 ).…”

GluA and GluN receptors regulate the surface density of GluN receptor subunits in cultured neocortical interneurons

“…To study whether L‐type Ca 2+ channels were involved in the reduction of surface NR1 subunits induced by GluA receptors, we examined the effect of nimodipine, which preferentially blocks these channels (Huber et al. 2000).…”

Nanosecond Pulsed Electric Field (nsPEF): Opening the Biotechnological Pandora’s Box

“…Cryo-EM structures have revealed crude structural information about this channel subtype [ 175 , 176 , 177 , 178 ]; however, they do not have enough resolution to gain insight into the structural basis of channel function. Based on the crystallographic structures of potassium channels released in 2005 [ 179 ], several homology models of subunits have been constructed and used to model drug interactions, in particular with L-type channels [ 180 , 181 , 182 ]. While these works have provided some advances in our understanding of subunit regulation of VGCC, it remains to be determined whether the observed interactions are relevant to actual conformations in holochannels or perhaps modified by the presence of transmembrane regions and other intracellular domains.…”