Functional and Molecular Interaction of Phenylalkylamines and Dihydropyridines with the Model Calcium Channel CavAb

Abstract

Mammalian voltage-gated calcium channels (mCavs) are molecular targets of phenylalkylamines (PAAs) and dihydropyridines (DHPs), which are widely used antiarrhythmic and antihypertension drugs that block mCavs in a frequency- and voltage-dependent manner. The two distinct receptor sites for these drugs in mCavs have been elucidated through extensive photoaffinity labeling and mutagenesis studies, revealing key residues along the S5 and S6 transmembrane segments of domains III and IV of mCavs (Hockerman et al., 1997). We have recently reported the crystal structure of CavAb, which is a highly calcium-selective mutant of the prokaryotic sodium channel NavAb (Tang et al., 2014). Even though CavAb is separated evolutionarily from mCavs by more than one billion years, we found that PAAs and DHPs interact with CavAb in a similar state-dependent manner. The PAAs tested have Kd's in the range of 250 nM to 800 nM, and verapamil shows low-affinity block of the resting state and high-affinity use-dependent block of activated and/or inactivated states. Mutations of T206 in the center of segment S6 reduced PAA affinity by 20-fold. In the case of DHPs, nimodipine showed voltage-dependent block of activated CavAb with a Kd of 194 nM, but no resting-state block was observed in the tested concentration range. DHPs shift the voltage dependence of inactivation up to 100 mV negatively. This indicates that DHP interaction with CavAb is state-dependent and greatly favors the inactivated state, similar to mCavs. Mutation of I199 in the outer region of segment S6, which is critical for binding DHPs in mCavs, reduced binding affinity 40-fold. Our results reveal surprising similarity of drug action on CavAb and mCavs and thereby support use of CavAb as a molecular model to determine the three-dimensional structure of these important drug receptor sites.