Vol. 289, Issue 3, 1472-1479, June 1999

Distinctions in the Molecular Determinants of Charged and Neutral Dihydropyridine Block of L-Type Calcium Channels¹

L. Lacinová^{2 5}, R. H. An³, J. Xia³, H. Ito², N. Klugbauer², D. Triggle⁴, F. Hofmann² and R. S. Kass³

Department of Pharmacology, College of Physicians & Surgeons of Columbia University, New York, New York

We investigated block of the _1Cb subunit of L-type calcium channels by dihydropyridines (DHPs) in which a permanently charged or neutral head group was linked to the active DHP moiety by a spacer chain containing ten methylene (CH₂) groups. We compared the sensitivity of channel modulation by the charged (DHPch) and neutral (DHPn) forms to specific _1Cb mutations in domains IIIS5, IIIS6, and IVS6, which had previously been shown to reduce channel modulation by the neutral DHP (+)-isradipine. The effects of these mutations were studied on channel block recorded from polarized (80 mV) and depolarized (40 mV) holding potentials (HPs). We found that channel block by DHPn was markedly reduced at both HPs by each mutation studied. In contrast, channel block by DHPch was only modestly reduced by mutations in IIIS6 and IVS6 for block from either 40 mV or 80 mV. Replacement of IIIS5 Thr1061 by Tyr, which abolished block by DHPn in an HP-independent manner, had little effect on channel block by DHPch recorded from 40 mV. However, this mutation markedly reduced DHPch block of currents recorded from a 80 mV HP. Inhibition of current by DHPch was not markedly use-dependent, in contrast with block by verapamil, another charged calcium channel blocker. These results suggest that the presence of a permanently charged head group restricts the access of the attached DHP moiety to a subset of interaction residues on the _1C subunit in a voltage-dependent manner. Furthermore, these restricted interactions confer distinct functional properties upon the charged DHP molecules.