Assessing Ca²⁺-Removal Pathways in Cardiac Myocytes

Abstract

The decline of an intracellular calcium ([Ca²⁺]_i) transient during a single excitation–contraction coupling (ECC) cycle reflects the combined activity of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) pump and the sarcolemmal Na⁺–Ca²⁺ exchanger (NCX), along with minor contributions of the plasma membrane Ca²⁺-ATPase and mitochondrial Ca²⁺ uniporter, in removing Ca²⁺ from the cytosol. A traditional approach for assessing the individual components is to fit the decline of the [Ca²⁺]_i transient evoked during electrical stimulation with an exponential. This reflects mostly the SERCA-dependent rate of uptake, which can be properly deduced after correcting for a component of NCX removal. As NCX function is an important determinant of the membrane potential as well as the Ca²⁺ balance, we present here several detailed protocols for assessing NCX function. As the reversal potential and the amplitudes of the current are highly dependent on the prevailing concentrations of Na⁺ and Ca²⁺, we show how NCX function can be assessed under highly controlled conditions, with Ca²⁺ and Na⁺ clamped, as well as under more physiological conditions, with freely changing Ca²⁺ and Na⁺.