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. 2018 Jul 25;7:e38981. doi: 10.7554/eLife.38981

Figure 5. Replacing the CpxII C-terminus with SNAP25-SN1 (residues 44 to 77) largely restores synchronous exocytosis.

(A) Cartoon illustrates CpxII domains and the structure of the tested mutants. (B) Expression of the Cpx1-100LC largely restores synchronous secretion reaching the response level of wt cells. The Cpx1-100helix supports exocytosis like Cpx1-100 (compare with Figure 1B). Mean [Ca2+]i levels (top) and corresponding CM signals (bottom) of wt cells (n = 11), CpxII ko cells (n = 58), and ko cells expressing CpxII (n = 68) or its mutants (Cpx1-100LC, n = 41; Cpx1-100helix, n = 20). Flash, t = 0.5 s. (C) Normalized CM (as shown in B) scaled to the wt response (1 s after flash). (D) Cpx1-100LC (green) restores RRP and SRP almost like CpxII (blue). (E) The time constants of the EB components (τRRP and τSRP), and the exocytotic delay are similarly changed for CpxII and its chimera (color coding as in A). (F–H) The Cpx1-100LC clamps tonic secretion (F) of CpxII ko cells at similar [Ca]i (G) almost like CpxII (F, H). ANOVA followed by Tukey-Kramer post-hoc test. *p<0.05; **p<0.01; ***p<0.001. Error bars indicate mean ± SEM.

Figure 5.

Figure 5—figure supplement 1. The Cpx1-115SC arrests tonic release and fully rescues synchronous secretion like CpxII.

Figure 5—figure supplement 1.

(A) Cartoon of the tested truncated and chimera mutants of CpxII. (B) Cpx1-115SC but not Cpx1-115helix restores synchronous release like CpxII. Mean [Ca2+]i levels (top) and corresponding capacitance responses (bottom) of wt (black, n = 11), CpxII ko (red, n = 43), CpxII ko expressing CpxII (blue, n = 46), Cpx1-115 (autumn orange, n = 28), Cpx1-115SC (purple, n = 33), Cpx1-115helix (dark green, n = 19). Flash at t = 0.5 s. (C) Amplitudes of RRP and SRP, the rate of sustained release (SR, fF/s). (D–F) The time constants of the exocytotic burst components (τRRP, τSRP) and the exocytotic delay. C-terminal truncated or mutated CpxII variants restore exocytosis timing like CpxII (see also Dhara et al., 2014) (G) Cpx1-115SC clamps premature secretion as CpxII. Averaged tonic capacitance responses (same group of cells as in B) over 120 s at submicromolar [Ca2+]i infusion (arrow, UV-flash). (H) Mean rate of ΔCM before the flash. ANOVA followed by Tukey-Kramer post-hoc test. **p<0.01; ***p<0.001. Error bars indicate mean ± SEM.