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 Ca²⁺ in the sarcoplasmic reticulum (SR) Ca²⁺ 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 Ca²⁺ and to prevent Ca²⁺ reuptake by the SR. Simultaneous measurement of membrane currents records Ca²⁺ extruded through the Na⁺–Ca²⁺ exchanger. The integral of the caffeine-induced Na⁺–Ca²⁺ exchange current is then used as a measure of the SR Ca²⁺. Derived measurements include the Ca²⁺ 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.