Assessing Ca²⁺-removal pathways in cardiac myocytes.
The decline of an intracellular calcium ([Ca(2+)]i) transient during a single excitation-contraction coupling (ECC) cycle reflects the combined activity of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) pump and the sarcolemmal Na(+)-Ca(2+) exchanger (NCX), along with minor contributions of the plasma membrane Ca(2+)-ATPase and mitochondrial Ca(2+) uniporter, in removing Ca(2+) from the cytosol. A traditional approach for assessing the individual components is to fit the decline of the [Ca(2+)]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 Ca(2+) 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 Ca(2+), we show how NCX function can be assessed under highly controlled conditions, with Ca(2+) and Na(+) clamped, as well as under more physiological conditions, with freely changing Ca(2+) and Na(+).
Division of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Belgium.
Virginie
V
Bito
Division of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Belgium.
Karin R
KR
Sipido
Division of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Belgium.
Niall
N
Macquaide
D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D003584:Cytological Techniques / Q000379:methods*; D055724:Electrophysiological Phenomena; D053858:Metabolic Networks and Pathways; D032383:Myocytes, Cardiac / Q000737:chemistry* / Q000378:metabolism
D002118:Calcium