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Abstract
The regulation of Ca2+ release by luminal Ca2+ has been well studied for the ryanodine and IP3 receptors but has been less clear for the NAADP-regulated channel. In view of conflicting reports, we have re-examined the issue by manipulating luminal Ca2+ with the membrane-permeant, low affinity Ca2+ buffer, TPEN, and monitoring NAADP-induced Ca2+ release in sea urchin egg homogenate. NAADP-induced Ca2+ release was almost entirely blocked by TPEN (IC50 17–25μM) which suppressed the maximal extent of Ca2+ release without altering NAADP sensitivity. In contrast, Ca2+ release via IP3 receptors was 3- to 30-fold less sensitive to TPEN whereas that evoked by ionomycin was essentially unaffected. The effect of TPEN on NAADP-induced Ca2+ release was not due to an increase in the luminal pH or chelation of trace metals since it could not be mimicked by NH4Cl or phenanthroline. The fact that TPEN had no effect upon ionophore-induced Ca2+ release also argued against a substantial reduction in the driving force for Ca2+ efflux. We propose that, in the sea urchin egg, luminal Ca2+ is important for gating native NAADP-regulated two-pore channels.
Highlights
Ca2+ release channels on intracellular stores are subject to regulation by second messengers and by additional factors that include accessory proteins, pH and phosphorylation [1,2,3,4,5]
The NAADP receptor has hitherto been reported to be insensitive to cytosolic Ca2+ [1,7,8,9] and local, “trigger” Ca2+ released by NAADP is necessarily amplified by proximal IP3Rs or ryanodine receptors (RyR) which are Ca2+-sensitive [10,11]
By varying the NAADP concentration (Fig. 1D–F), we found that the major effect of TPEN was to reduce the maximal extent of NAADP-induced Ca2+ release (NICR) without substantially altering the affinity of the receptors for NAADP (EC50 (95% confidence intervals) – amplitude: ethanol 32 nM (9–113 nM), TPEN 56 nM (7–47 nM); kinetics: ethanol 79 nM (17–370 nM), TPEN 218 nM (33–1460 nM))
Summary
Ca2+ release channels on intracellular stores are subject to regulation by second messengers and by additional factors that include accessory proteins, pH and phosphorylation [1,2,3,4,5]. The NAADP (nicotinic acid adenine dinucleotide phosphate) receptor has hitherto been reported to be insensitive to cytosolic Ca2+ (or surrogate ions) [1,7,8,9] and local, “trigger” Ca2+ released by NAADP is necessarily amplified by proximal IP3Rs or RyRs which are Ca2+-sensitive [10,11]. Whether these Ca2+-release channel families are regulated by Ca2+ within the lumen of the stores themselves is more controversial [12].
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