CONSTRUCTION OF A HIGH-AFFINITY RECEPTOR-SITE FOR DIHYDROPYRIDINE AGONISTS AND ANTAGONISTS BY SINGLE AMINO-ACID SUBSTITUTIONS IN A NON-L-TYPE CA2+ CHANNEL

The activity of L-type Ca2+ channels is increased by dihydropyridine ( DHP) agonists and inhibited by DHP antagonists, which are widely used in the therapy of cardiovascular disease. These drugs bind to the pore -forming al subunits of L-type Ca2+ channels. To define the minimal re quirements for DHP binding and action, we constructed a high-affinity DHP receptor site by substituting a total of nine amino acid residues from DHP-sensitive L-type alpha(1) subunits into the S5 and S6 transme mbrane segments of domain III and the S6 transmembrane segment of doma in IV of the DHP-insensitive P/Q-type alpha(1A) subunit. The resulting chimeric alpha(1A/DHPS) subunit bound DHP antagonists with high affin ity in radioligand binding assays and was inhibited by DHP antagonists with high affinity in voltage clamp experiments. Substitution of thes e nine amino acid residues yielded 86% of the binding energy of the L- type alpha(1C) subunit and 92% of the binding energy of the L-type alp ha(1S) subunit for the high-affinity DHP antagonist PN200-110. The act ivity of chimeric Ca2+ channels containing alpha(1A/DHPS) was increase d 3.5 +/- 0.7-fold by the DHP agonist (-)Bay K8644. The effect of this agonist was stereoselective as in L-type Ca2+ channels since (i) Bay K8644 inhibited the activity of alpha(1A/DHPS.) The results show concl usively that DHP agonists and antagonists bind to a single receptor si te at which they have opposite effects on Ca2+ channel activity. This site contains essential components from both domains III and IV, consi stent with a domain interface model for binding and allosteric modulat ion of Ca2+ channel activity by DHPs.