Investigating sarcoplasmic reticulum Ca²⁺-ATPase on solid supported membranes: from mechanistic insights to drug interactions

Speaker: Francesco Tadini Buoninsegni , University of Florence

The sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) belongs to the P-type ATPase family of membrane transport proteins. SERCA, which is found in the sarcoplasmic reticulum (SR) of muscle cells, couples ATP hydrolysis to the transport of two Ca²⁺ ions against their electrochemical potential gradient from the cytoplasm to the SR lumen, thus promoting muscle cell relaxation. SERCA maintains a low cytoplasmic Ca²⁺ concentration and contributes to intracellular calcium homeostasis which is essential for cell signaling and survival. Impaired SERCA function and regulation have been related to several diseases, such as cardiac and skeletal muscle diseases, neurodegenerative disorders, diabetes and cancer.

We have used an electrophysiological method based on a biomimetic membrane, the so-called solid supported membrane (SSM), to unravel key features of the SERCA transport mechanism. The SSM method allows label-free direct measurements of charge movements generated by the ATPase following adsorption of the membrane-bound enzyme to the SSM and chemical activation by a substrate concentration jump.

Electrical signals generated by SERCA were measured following Ca²⁺ and ATP jumps on SR vesicles adsorbed to the SSM. The measured charge movements are attributed to distinct electrogenic reaction steps in the SERCA transport cycle, i.e. binding of Ca²⁺ ions to the cytoplasmic side of the ATPase, and translocation and release of Ca²⁺ at the luminal side of the enzyme.

Because of its prominent role in intracellular calcium homeostasis and signaling, SERCA represents an attractive target for a variety of drugs. The effects of pharmacologically relevant compounds on the Ca²⁺ transport activity of SERCA were characterized using the SSM method. We found that these compounds affect calcium transport by SERCA and behave like inhibitors or activators of the enzyme. The molecular mechanism of action of a few selected drugs is discussed.

Finally, SSM-based electrophysiology has also been used to study other prominent members of the P-type ATPase family. In particular, we briefly discuss how the SSM method has contributed to investigating crucial aspects of the transport mechanism of a mammalian copper ATPase and a phospholipid flippase.