Waheed Shabbir, Anna Stary-Weinzinger, Stanislav Beyl, Katrin Depil, Chonticha Suwattanasophon, Eugen Timin, H. Robert Guy and Steffen Hering
Department of Pharmacology and Toxicology, University of Vienna, Vienna, Austria
Voltage gated calcium channels are expressed in the cell membrane of excitable cells. These channels play critical role in many physiological processes such as excitation-contraction coupling, secretion, and neurotransmission and cell death. L-type calcium channel CaV1.2 in cardiomyocytes are responsible for the long lasting phase of cardiac action potential. Cahnnelopathies in CaV1.2 channels cause many diseases such as angina pectoris and cardiac arrhythmias. Therefore, blocking of CaV1.2 channels are required to treat these diseases. Although Diltaizem a drug from Benziothiazepines (BTZ) group is an established therapy in clinics against cardiovascular diseases for many years, the mechanism of its block of CaV1.2 is still not fully understood. An other enigma is the resistance to diltiazem therapy. We showed previously the diltiazem accesses to its binding site through intracellular route (Shabbir et al 2011). Herein, we identify a novel diltiazem-binding determinant that is necessary for CaV1.2 channel block. The novel determinant was identified with the help of molecular modeling studies. Patch-clamp experiments of transiently transfected CaV1.2 WT and mutant channels were performed and Barium current was measured at room temperature. Diltiazem (Dil) and quaternary Diltiazem (qDil) block were analyzed.
Novel determinant T1143 was substituted by alanine (T1143A). T1143A significantly reduced channel inhibition by qDil and Dil. Convincingly, T1143A did not affect channel activation or inactivation while almost completely diminishing channel block by Dil or qDil. These findinding support the view that T1143 serves as drug binding determinant. Other mutations in this position than T1143A (T1143L/Y/S/N/C/V/E) diminished channel inhibition by qDil but additionally affected channel activation and inactivation and may therefore affect channel block allosterically. Shabbir, W., Beyl, S., Timin, E. N., Schellmann, D., Erker, T., Hohaus, A., Hockerman, G. H., and Hering, S., Interaction of diltiazem with an intracellularly accessible binding site on Ca(V)1.2, Br J Pharmacol 2011, 162(5):1074-82.