Identification of the coupling between skeletal muscle store-operated Ca2+ entry and the inositol trisphosphate receptor

Abstract

Examination of store-operated Ca(2+) entry (SOC) in single, mechanically skinned skeletal muscle cells by confocal microscopy shows that the inositol 1,4,5-trisphosphate (IP(3)) receptor acts as a sarcoplasmic reticulum [Ca(2+)] sensor and mediates SOC by physical coupling without playing a key role in Ca(2+) release from internal stores, as is the case with various cell types in which SOC was investigated previously. The results have broad implications for understanding the mechanism of SOC that is essential for cell function in general and muscle function in particular. Moreover, the study ascribes an important role to the IP(3) receptors in skeletal muscle, the role of which with respect to Ca(2+) homeostasis was ill defined until now.

Figure 1

Ca²⁺ movements across the sealed t-system of a toad skeletal muscle fiber by SOC and Ca²⁺ reloading. Confocal images of the fluorescence of Fluo-5N trapped in the sealed t-system in the presence of standard myoplasmic solution containing <1 nM Ca²⁺ and 1 mM Mg²⁺ (A), 30 s after application of 30 mM caffeine in myoplasmic solution with 0.075 mM Mg²⁺ and <1 nM Ca²⁺ (B) and after 2 (C), 7 (D), and 12 (E) min in myoplasmic solution containing 200 nM Ca²⁺ are shown. Note that depleting the SR of Ca²⁺ with caffeine and low Mg²⁺ also resulted in the loss of Ca²⁺ from the sealed t-system indicating an operational SOC (B) and that Ca²⁺ can be reloaded into the Ca²⁺-depleted t-system (C–E). (Scale bar, 20 μm.)

Figure 2

Summary of the effect of heparin (■) and the addition of IP₃ (□; 10 μM for 10 μg/ml heparin and 100 μM for the 0.1 and 1 mg/ml heparin data points) to the heparin solution on SOC. Note that caffeine completely depletes the SR of Ca²⁺ under all conditions. The data represent means ± SEM from 13–16 cells under both sets of conditions and have been fitted to sigmoidal curves. The two sets of data with and without IP₃ are statistically different (P < 0.05) from each other (two-way ANOVA). The data points for heparin ≥ 0.01 mg/ml are significantly different (P < 0.05) from controls and the initial [Ca²⁺] (one-way ANOVA).

Figure 3

[Ca²⁺] in the t-system 30 s after the following interventions: control fibers after SR Ca²⁺ depletion with caffeine and low Mg²⁺; “high-Ca²⁺ uncoupled” fibers after SR Ca²⁺ depletion with caffeine and low Mg²⁺ (see Results); and fibers preincubated in 10 μM nifedipine after SR Ca²⁺ depletion with caffeine and low Mg²⁺ in the presence of nifedipine and fibers exposed to 500 μM IP₃ in a myoplasmic solution containing 1 mM Mg²⁺ and 200 nM Ca²⁺ (no caffeine). The data represent means ± SEM for three to nine cells per condition. The data points for “uncoupled” fibers are significantly different (P < 0.01) from those for controls and nifedipine (one-way ANOVA), and there is no statistically significant difference (P > 0.5) between controls and nifedipine-treated fibers. The values for IP₃-treated and uncoupled fibers are significantly smaller (P < 0.05) than the initial [Ca²⁺] (1.5 mM) in the t-system.