Figure 1.

The I4897T mutation in RyR1 reduces electrically evoked Ca²⁺ release without altering resting cytosolic Ca²⁺ levels. (A) In situ calibration (22) of Indo-1 obtained in intact myotubes. (Left) Three representative experiments in which Indo-1 ratios (F₄₀₅/F₄₈₅) were obtained in Indo-1 AM-loaded myotubes before and after (arrow) establishment of the whole cell configuration by using internal solutions containing different levels of free Ca²⁺. (Right) In situ Indo-1 calibration curve used to determine resting Ca²⁺ levels. This calibration curve was obtained by using four different BAPTA-buffered calibration solutions (closed triangles). The Indo-1 ratio of myotubes dialyzed with the internal solution (except fluo-3 was replaced with Indo-1) used in patch clamp experiments (Figs. 2 and 3) was superimposed on the fitted curve (open triangle). The free Ca²⁺ level of this internal solution (≈60 nM) is nearly identical to that of intact normal myotubes (Fig. 1B) and, thus, should be sufficient for maintaining the loading state of the SR. (B) Resting Ca²⁺ levels determined by using the in situ calibration obtained from normal myotubes (NML), uninjected dyspedic myotubes (DYP), and dyspedic myotubes expressing RyR1 alone (RyR-1), RyR1 and I4897T (RyR-1/I4897T), or I4897T alone (I4897T). (C) Electrically evoked (arrows) Ca²⁺ transients recorded from Indo-1 AM-loaded myotubes in the presence and absence of extracellular Ca²⁺.