Effect of the H29D mutation on cytosolic Ca²⁺ induced Ca²⁺ release in HEK293 cells.

<p>Stable, inducible HEK293 cell lines expressing RyR2 WT (A) or the H29D mutant (B) were transfected with the FRET-based ER luminal Ca²⁺-sensing protein D1ER and their expression were induced using tetracycline. The transfected and induced cells were permeabilized with saponin, washed and perfused with intracellular-like medium plus increasing levels of free cytosolic Ca²⁺ (0.1, 0.2, 0.4, 1.0 and 10 μM) to induce Ca²⁺ release. FRET recordings from representative cells (total 51–93 cells each) are shown. To minimize the influence by CFP/YFP cross-talk, we used relative FRET measurements for calculating the steady state ER Ca²⁺ level (defined in panel A). Dash lines (F_{0.1 –}F₁₀) indicate the steady state FRET levels after perfusion with each Ca²⁺ concentration (0.1, 0.2, 0.4, 1.0 or 10 μM). The maximum FRET signal F_max is defined as the FRET level after tetracaine application. The minimum FRET signal F_min is defined as the FRET level after caffeine application. Data shown are mean ± SEM (n = 4–5).