title: Measuring sarcoplasmic reticulum Ca2+ content, fractional release, and Ca2+ buffering in cardiac myocytes.
abstract: Here, we describe a protocol for the reliable measurement of the amount of Ca(2+) in the sarcoplasmic reticulum (SR) Ca(2+) store of cardiac myocytes. The whole-cell patch-clamp method is used to provide controlled loading of the SR during conditioning depolarizing pulses, followed by rapid application of a high dose of caffeine to release all SR Ca(2+) and to prevent Ca(2+) reuptake by the SR. Simultaneous measurement of membrane currents records Ca(2+) extruded through the Na(+)-Ca(2+) exchanger. The integral of the caffeine-induced Na(+)-Ca(2+) exchange current is then used as a measure of the SR Ca(2+). Derived measurements include the Ca(2+) buffering capacity and measurement of fractional release as an indicator of coupling gain. Caveats, advantages, and disadvantages of this method and alternative methods are discussed.
authors.affiliation: Division of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Belgium.
authors.forename: Niall
authors.initials: N
authors.lastname: Macquaide
authors.affiliation: Division of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Belgium.
authors.forename: Virginie
authors.initials: V
authors.lastname: Bito
authors.affiliation: Division of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Belgium.
authors.forename: Karin R
authors.initials: KR
authors.lastname: Sipido
mesh_terms: D000818:Animals; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018408:Patch-Clamp Techniques / Q000379:methods*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
chemical_list: D002021:Buffers; D002118:Calcium