Figure 2. Inhibition of IP₃R by 1-8-14 peptide.

(A) Typical recording of the free Ca²⁺ concentration within the endoplasmic reticulum of a population of permeabilized DT40-IP₃R1 cells showing Ca²⁺ uptake after addition of MgATP (1.5 mM), release of Ca²⁺ after addition of IP₃ (10 μM, with 1 μM thapsigargin to inhibit Ca²⁺ re-uptake) and inhibition of that release by 1-8-14 peptide (3 mM, present throughout as indicated, upper trace). Results are means±S.E.M. for three replicates from a single experiment. (B) Concentration-dependent release of intracellular Ca²⁺ stores by IP₃ alone or after pre-incubation for 2.5 min with 1-8-14 peptide (3 mM). Inhibition by 1-8-14 peptide at each IP₃ concentration is also shown (%). 1-8-14 peptide caused a significant decrease in the maximal response (P<0.001) without significantly changing the sensitivity to IP₃. (C and D) Permeabilized cells pre-incubated for 10–20 min with the indicated concentrations of peptide were stimulated with IP₃ (10 μM, in the continued presence of peptide). Results show the Ca²⁺ content of the stores before addition of IP₃, and the Ca²⁺ release evoked by IP₃. (E) Permeabilized cells were incubated alone or with 1-8-14 peptide (3 mM) for 10–20 min, washed and then resuspended in CLM. Ca²⁺ release by IP₃ (10 μM) was then measured after a further incubation for 10–20 min with the indicated concentrations of 1-8-14 peptide. The Ca²⁺ release evoked by IP₃ with and without peptide is shown for naive cells and after the pre-treatment with 3 mM peptide. The results establish that the effects of 1-8-14 peptide are fully reversible. (F) Permeabilized cells pre-incubated with or without 1-8-14 peptide (1 mM) for 10–20 min were stimulated with a maximally effective concentration of IP₃ in the continued presence of peptide in CLM with the indicated free Ca²⁺ concentration. Results show the inhibition of IP₃-evoked Ca²⁺ release (%) by 1-8-14 peptide at each free Ca²⁺ concentration. Results in (B)–(F) are means±S.E.M. (n≥3). *P<0.05, **P<0.01 and ***P<0.001.