Interaction of verapamil with lipid membranes and P-glycoprotein. Connecting thermodynamics and membrane structure with functional activity

¹ Biozentrum, University of Basel

^* To whom correspondence should be addressed. E-mail: joachim.seelig{at}unibas.ch.

Submitted on May 23, 2006
Revised on July 5, 2006
Accepted on 17 July 2006

	Abstract

Verapamil and amlodipine are calcium ion influx inhibitors of wide clinical use. They are partially charged at neutral pH and exhibit amphiphilic properties. The non-charged species can easily cross the lipid membrane. We have measured with solid state nmr the structural changes induced by verapamil upon incorporation into phospholipid bilayers and have compared them with earlier data on amlodipine and nimodipine. Verapamil and amlodipine produce a rotation of the phosphocholine headgroup away from the membrane surface and a disordering of the fatty acid chains. We have determined the thermodynamics of verapamil partitioning into neutral and negatively charged membranes with isothermal titration calorimetry. Verapamil undergoes a pK-shift of pK = 1.2 units in neutral lipid membranes and the percentage of the non-charged species increases from 5% to 45%. Verapamil partitioning is increased for negatively charged membranes and the binding isotherms are strongly affected by the salt concentration. The electrostatic screening can be explained with the Gouy-Chapman theory. Using a functional phosphate assay we have measured the affinity of verapamil, amlodipine and nimodipine for P-glycoprotein, and have calculated the free energy of drug binding from the aqueous phase to the active center of Pgp in the lipid phase. By combining the latter results with the lipid partitioning data it was possible, for the first time, to determine the true affinity of the three drugs for the Pgp active center if the reaction takes place exclusively in the lipid matrix.

Key Words: ITC, P-glycoprotein, deuterium nmr, drug binding, thermodynamics, verapamil