Measuring Ca²⁺ Pump Activity in Overexpression Systems and Cardiac Muscle Preparations

Abstract

Sarco-/endoplasmic reticulum (SR/ER) Ca²⁺ pumps (SERCAs) build up vital Ca²⁺ gradients across the intracellular SR/ER membrane, helping to control cell function, proliferation, growth, differentiation, and death. We describe two techniques to measure the SERCA activity either in mammalian culture cells overexpressing SERCAs or in muscle tissue containing high levels of endogenous SERCAs. As Ca²⁺ transport is tightly coupled to ATP hydrolysis, it is possible to determine the rate of Ca²⁺-dependent ATP hydrolysis and use it as a measure for SERCA activity or, in a second approach, to quantify ATP-stimulated uptake of radioactive ⁴⁵Ca²⁺. Here, we first provide an overview of the mechanism of Ca²⁺-transport ATPases and show how this can be taken advantage of in protocols for measuring Ca²⁺ pump activity.