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Abstract
The regulation of Ca(2+) release by luminal Ca(2+) has been well studied for the ryanodine and IP(3) 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 Ca(2+) with the membrane-permeant, low affinity Ca(2+) buffer, TPEN, and monitoring NAADP-induced Ca(2+) release in sea urchin egg homogenate. NAADP-induced Ca(2+) release was almost entirely blocked by TPEN (IC(50) 17-25μM) which suppressed the maximal extent of Ca(2+) release without altering NAADP sensitivity. In contrast, Ca(2+) release via IP(3) 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 Ca(2+) release was not due to an increase in the luminal pH or chelation of trace metals since it could not be mimicked by NH(4)Cl or phenanthroline. The fact that TPEN had no effect upon ionophore-induced Ca(2+) release also argued against a substantial reduction in the driving force for Ca(2+) efflux. We propose that, in the sea urchin egg, luminal Ca(2+) is important for gating native NAADP-regulated two-pore channels.
Fig. 1. TPEN inhibits NAADP-induced Ca2+ release. (A) Different concentrations of TPEN (or 0.1% (v/v) ethanol vehicle) were preincubated for 2 min prior to addition of a sub-maximal concentration of NAADP (50 nM) followed by 0.5 μM ionomycin. Raw control Δ[Ca2+] was 79 ± 9 nM (NAADP) and 227 ± 18 nM (ionomycin). Summary of the effect of TPEN upon the amplitude (B) or kinetics (C) of NAADP- or ionomycin-induced Ca2+ release shown in (A). (D) Effect of a sub-maximal concentration of TPEN upon NAADP sensitivity. 0.1% (v/v) ethanol (EtOH) or 20 μM TPEN was preincubated for 2 min prior to the addition of different NAADP concentrations. Graphs summarizing the amplitudes (E) or kinetics (F) of the responses in (D). N = 9–16 (A–C), n = 5–9 (D–F). Significance was determined using a Dunnett's test versus 0 μM TPEN (B and C); Student's t test comparing EtOH and TPEN (E and F). NAADP data were fitted as a one-phase exponential decay (B and C) or a Sigmoidal concentration–response (E and F).
Fig. 2. TPEN inhibits IP3-induced Ca2+ release. Different concentrations of TPEN (or 0.1% (v/v) ethanol vehicle) were preincubated for 2 min prior to addition of 4 μM IP3 (A–C) or 0.5 μM ionomycin (D–F). Raw control Δ[Ca2+] was 145 ± 6 nM (IP3) and 259 ± 21 nM (ionomycin). Summary of effect upon the amplitudes (B and E) or kinetics (C and F). The dashed lines (B and C) depict the NAADP curves in Fig. 1 for comparison. Significance was determined using a Dunnett test versus 0 μM TPEN. TPEN had no significant effect upon ionomycin responses (P > 0.05). IP3 data were fit as a one-phase exponential decay (B and C). Data are mean ± SEM of 3–8 experiments. (G–I) Effect of 200 μM TPEN (or 0.1% ethanol vehicle) upon the IP3 concentration–response. Data were normalized to the maximum response with 4 μM IP3 plus ethanol: raw control Δ[Ca2+] was 177 ± 18 nM (amplitude) and 0.578 ± 0.187 U F0/s (kinetics). IP3 data were fit with a Sigmoidal concentration–response (H and I) and ethanol and TPEN compared with a Student's t test (n = 5–12).
Fig. 3. Comparison of the effects of TPEN and NH4Cl on luminal pH or NAADP-induced Ca2+ release. Luminal pH (pHL) was monitored using acridine orange (AO), panels A–C. Cumulative concentration–response curves to TPEN (A) or NH4Cl (B), and summarized in (C), where data were fitted as a Sigmoidal concentration–response. Effect of NH4Cl upon NAADP-induced Ca2+ release (D–F): different concentrations of NH4Cl were preincubated for 2 min prior to addition of sub-maximal NAADP (50 nM) and 0.5 μM ionomycin. Raw control Δ[Ca2+] was 96 ± 10 nM (NAADP) and 249 ± 22 nM (ionomycin). No significant effect of NH4Cl (P > 0.05) upon NAADP (E) and ionomycin (F) responses was observed (Dunnett's test). Data are mean ± SEM of 8–13 determinations. (G and H) For each concentration of TPEN or NH4Cl, the corresponding pHL or Ca2+ signals were plotted to assess the relationship between the two parameters (including data from Fig. 1).
Fig. 4. TPEN does not inhibit the acidic store Ca2+ leak pathway. (A) Nigericin evokes Ca2+ release from the NAADP-sensitive store. Increasing concentrations of nigericin evoke Ca2+ release that progressively depletes the store released by 250 nM NAADP as indicated by the reduced response. (B) Summary of peak data. ‘Total’ refers to the summation of the Ca2+ release evoked by NAADP plus that evoked by nigericin (n = 3). (C and D) Effect of 200 μM TPEN upon the Ca2+ responses to sequential addition of 50 nM NAADP and 20 μM nigericin (nigericin). N = 9. Ethanol vehicle (EtOH). (F) Effect of TPEN upon the Ca2+ leak unmasked by 20 μM nigericin. (G) Summary of the effect of different TPEN concentrations upon the amplitude or kinetics of the nigericin-induced Ca2+ responses (n = 5–6). ns, not significant.
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