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 (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.
Huber
I
I
Institut für Biochemische Pharmakologie, Peter-Mayrstrasse 1, A-6020 Innsbruck, Austria.
Wappl
E
E
Herzog
A
A
Mitterdorfer
J
J
Glossmann
H
H
Langer
T
T
Striessnig
J
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001426:Bacterial Proteins; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004095:Dihydropyridines / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013237:Stereoisomerism; D013379:Substrate Specificity; D013816:Thermodynamics
D001426:Bacterial Proteins; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D004095:Dihydropyridines; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; C107790:prokaryotic potassium channel; C038806:1,4-dihydropyridine