Allosteric regulation by cytoplasmic Ca2+ and IP3 of the gating of IP3 receptors in permeabilized guinea-pig vascular smooth muscle cells

Abstract

1. The potentiation by Ca2+ of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied by measuring luminal Ca2+ concentrations of the Ca2+ stores using a fluorescent Ca2+ indicator, furaptra, in permeabilized smooth muscle cells. 2. Ca2+ release at 10 microM IP3 was potentiated by an increase in the cytoplasmic Ca2+ concentration in the presence of 10 mM EGTA. This effect was not due to the pharmacological effect of EGTA, because changes in the EGTA concentration at a constant Ca2+ concentration had no effect on the Ca2+ release rate. 3. With an increase in the cytoplasmic Ca2+ concentration from 30 to 630 nM, the Ca2+ release rate at a saturating IP3 concentration increased 110-fold and the EC50 for IP3 increased from 0.07 to 1.0 microM. It was also indicated that the relationship between Ca2+ concentration and Ca2+ release rate was shifted towards higher Ca2+ concentrations at higher IP3 concentrations. 4. These results suggest that IP3 and submicromolar concentrations of Ca2+ allosterically lower the affinity of the IP3 receptor for each other and are both required for IP3 receptor activation. These properties enable the IP3 receptors to detect simultaneous increases in IP3 and Ca2+ concentrations.

Figure 1. IP₃-induced Ca²⁺ release at various cytoplasmic Ca²⁺ concentrations

A, time courses of Ca²⁺ release at the indicated cytoplasmic Ca²⁺ concentrations. Application of 10 μM IP₃ was started at the time point indicated by the arrow. B, the initial rate of Ca²⁺ release was plotted against the cytoplasmic Ca²⁺ concentration (mean ±s.e.m., n = 5).

Figure 2. Extent of Ca²⁺-mediated potentiation of IP₃-induced Ca²⁺ release is independent of EGTA concentration

The total EGTA concentration at 300 nM Ca²⁺ was changed from 10 mM to either 4.5 mM or 14.3 mM for adjustment of the concentration of Ca²⁺-bound or Ca²⁺-unbound EGTA, respectively, to that at 100 nM Ca²⁺. The IP₃ concentration was 100 nM. Means ±s.e.m., n = 5.

Figure 3. Ca²⁺ concentration dependence of IP₃-induced Ca²⁺ release

A, time course of Ca²⁺ release induced by IP₃ at cytoplasmic Ca²⁺ concentrations of 300 nM (left) and 100 nM (right). B, the Ca²⁺ release rate was calculated from experimental results similar to those in A and plotted against the IP₃ concentration. Open and filled circles represent Ca²⁺ release rates at 100 and 300 nM Ca²⁺, respectively. The data were fitted by Hill's equation to give continuous curves. The fitted curve for 100 nM Ca²⁺ was scaled up 6.8-fold (dotted curve). C, the IP₃ concentration required for half-maximal activation of Ca²⁺ release (EC₅₀) was plotted against the cytoplasmic Ca²⁺ concentration. D, the maximal Ca²⁺ release rate was plotted against the Ca²⁺ concentration.

Figure 4. Allosteric activation of IP₃ receptors by Ca²⁺ and IP₃

A, the Ca²⁺ release rate was plotted against the Ca²⁺ and IP₃ concentrations. Experimental data (spheres) are fitted by the equation described in B, as shown by the curved surface. B, a possible mechanism of IP₃ receptor activation. The channel has n Ca²⁺ binding sites and m IP₃ binding sites. K₁ and K₃ represent K_d for Ca²⁺ binding to IP₃-unbound and IP₃-bound forms of the receptor, respectively. K₂ and K₄ represent K_d for IP₃ binding to Ca²⁺-unbound and Ca²⁺-bound forms of the receptor, respectively.