Measuring Sarcoplasmic Reticulum Ca2+ Content, Fractional Release, and Ca2+ Buffering in Cardiac Myocytes

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.