We examined [Ca²⁺]_i and L‐type Ca²⁺ channel current (I_Ca) in single cardiac myocytes to determine how the intracellular protein phospholamban (PLB) influences excitation‐contraction (E–C) coupling in heart. Wild type (WT) and PLB‐deficient (KO) mice were used. Cells were patch clamped in whole–Cell mode while [Ca²⁺]_i was imaged simultaneously using the Ca²⁺ indicator fluo‐3 and a confocal microscope.

Although I_Ca was similar in magnitude, the decay of I_Ca was faster in KO than in WT cells and the [Ca²⁺]_i transient was larger and decayed faster. Furthermore, the E–C coupling ‘gain’ (measured as Δ[Ca²⁺]_i/I_Ca) was larger in KO cells than in WT cells.

Spontaneous Ca²⁺ sparks were three times more frequent and larger in KO cells than in WT myocytes but, surprisingly, the time constants of decay were similar.

SR Ca²⁺ content was significantly greater in KO than in WT cells. When the SR Ca²⁺ content in KO cells was reduced to that in WT cells, Ca²⁺ sparks in these ‘modified’ (KO') cells decayed faster. E–C coupling gain, [Ca²⁺]_i transient amplitude and the kinetics of decay of I_Ca were similar in KO' and WT cells.

We conclude that SR Ca²⁺ content influences (1) I_Ca, (2) the amplitude and kinetics of Ca²⁺ sparks and [Ca²⁺]_i transients, (3) the sensitivity of the RyRs to triggering by [Ca²⁺]_i, (4) the amount of Ca²⁺ released, (5) the magnitude of the E–C coupling ‘gain’ function, and (6) the rate of Ca²⁺ re‐uptake by the SR Ca²⁺‐ATPase. In KO cells, the larger [Ca²⁺]_i transients and Ca²⁺ sparks speed up I_Ca inactivation. Finally, we conclude that PLB plays an important regulatory role in E–C coupling by modulating SR Ca²⁺‐ATPase activity, which establishes the SR Ca²⁺ content and consequently influences the characteristics of local and global Ca²⁺ signalling.