Interaction of SR Ca2+-ATPase with drugs

Sarcoplasmic Reticulum (SR) Ca2+-ATPase is an integral membrane protein with a central role in cellular calcium homeostasis. It is found in the SR of muscle cells and it pumps two calcium ions, against their electrochemical gradient, from the cytoplasm into the lumen of the SR, using the energy released after the hydrolysis of an ATP molecule. In such way SR Ca2+-ATPase promotes muscle relaxation. Failure in the functioning of this protein can generate relevant diseases. Drugs can correct such failures, restoring the normal calcium pumping by the protein. We are currently investigating the interaction of some drugs with Ca2+-ATPase: clotrimazole and miconazole, two antimycotic drugs, and curcumin, a molecule with antioxidant and antitumoral properties. We are making use of a rapid solution exchange technique: the protein is first adsorbed on a modified gold surface (the SSM: Solid Supported Membrane) and then it is activated by a rapid concentration jump of an appropriate substrate (e.g. ATP, calcium, etc.). If at least one electrogenic step is involved in the reactions following such activation, a transient current can be measured in the external electrical circuit. The acquisition of such type of signals under different experimental conditions, together with their subsequent elaboration, can give important kinetic information about protein functioning and its modulation by drugs. In our case, while clotrimazole seems to be a pure blocker of the pump, binding to it before ATP in the enzymatic cycle, the others two molecules show a more complicate behaviour, affecting both calcium binding and general pumping kinetic.