Fig. 3: Deprotected MASTER-NAADP evokes Ca²⁺ signals in permeabilized Jurkat T cells and increases the open probability of RYR1.

3.5 × 10⁷ Jurkat T cells were permeabilized (80 µg/mL saponin) and transferred to a fluorimeter in 1 mL cuvettes with gentle magnetic stirring. Free Ca²⁺ concentration was measured every 2 s ratiometrically using emission wavelengths of CalRed (ex 488 nm, em 525/650 nm). Ca²⁺ uptake into the ER was stimulated by the addition of creatine phosphate, creatine kinase and adenosine triphosphate, followed by the addition of 15 µg recombinant human HN1L/JPT2 (except for control experiments) and 10 µM glucose 6-phosphate dehydrogenase inhibitor-1 (g6pdi-1). Deprotected MASTER-compounds or natural nucleotides were diluted in bidestillated water and injected using a Hamilton gas-tight 50μL syringe. A Example tracings showing no response to either biological NAADP or deprotected MASTER-compounds without the re-addition of exogenous HN1L/JPT2. Representative tracings out of 4 experiments are shown. B Example tracings showing increases in the free Ca²⁺ concentration after the injection of NAADP or deprotected MASTER-NAADP, but not of the respective NADP controls. A representative out of at least 5 experiments is shown. C, D The delta Ca²⁺ of the different experiments (mean Ca²⁺ concentration during the first 10 s after injection after subtraction of the mean Ca²⁺ concentration the last 10 s before injection) was plotted against the baseline Ca²⁺ (mean Ca²⁺ concentration the last 10 s before injection) and analyzed by linear regression with two-sided F test of the slope using GraphPad Prism 10. The graph show results from the individual experiments (symbols), linear regression (line) with 95% confidence interval (dotted lines) and significance level of the slope being non-zero, i.e. the amplitude of the responses being correlated with the basal Ca²⁺ concentration (ns = not significant, **p < 0.01, ****p < 0.0001, N = 5 experiments for C and N = 8 experiments for D). E Single channel analysis of deMASTER-NAADP with purified HN1L/JPT2 on RYR1. Representative single-channel current traces of purified RYR1 measured under the following conditions: upper tracing: 1 µM free Ca²⁺, middle tracing: 100 nM deMASTER-NAADP, lower tracing: 100 nM deMASTER-NAADP with 10 µM HN1L/JPT2. F Summarized relative open probability for each condition, presented as mean ± SEM, n = biological replications as indicated above the bars. Two-sided, mixed-effects analysis with Holm-Šídák multiple comparison test, with a single pooled variance *p < 0.05. Each p value is adjusted to account for multiple comparisons. Source data and exact p values are provided as a Source Data file.