Synaptotagmin-1 and -7 are functionally overlapping Ca2+ sensors for exocytosis in adrenal chromaffin cells

Abstract

Synaptotagmin-1, the canonical isoform of the synaptotagmin family, is a Ca(2+) sensor for fast synchronous neurotransmitter release in forebrain neurons and chromaffin cells. Even though deletion of synaptotagmin-1 abolishes fast exocytosis in chromaffin cells, it reduces overall secretion by only 20% because of the persistence of slow exocytosis. Therefore, another Ca(2+) sensor dominates release in these cells. Synaptotagmin-7 has a higher Ca(2+) affinity and slower binding kinetics than synaptotagmin-1, matching the proposed properties for the second, slower Ca(2+) sensor. Here, we examined Ca(2+)-triggered exocytosis in chromaffin cells from KO mice lacking synaptotagmin-7, and from knockin mice containing normal levels of a mutant synaptotagmin-7 whose C(2)B domain does not bind Ca(2+). In both types of mutant chromaffin cells, Ca(2+)-triggered exocytosis was decreased dramatically. Moreover, in chromaffin cells lacking both synaptotagmin-1 and -7, only a very slow release component, accounting for approximately 30% of WT exocytosis, persisted. These data establish synaptotagmin-7 as a major Ca(2+) sensor for exocytosis in chromaffin cells, which, together with synaptotagmin-1, mediates almost all of the Ca(2+) triggering of exocytosis in these cells, a surprising result, considering the lack of a role of synaptotagmin-7 in synaptic vesicle exocytosis.

Fig. 1.

Depolarization experiments in the synaptotagmin-7 KO. (A) Expression of synaptotagmin-7 (Syt 7; L and S, long and short splice variants), synaptotagmin-1 (Syt 1), Rab 3, and VCP (loading control) in the brains and adrenal medulla of synaptotagmin-7 KO mice and their WT littermates was analyzed by immunoblotting. Note that only the short synaptotagmin-7 splice variants are expressed in adrenal medulla. (B) Chromaffin cells were stimulated by depolarization trains (18 × 100 ms at 0 mV, the bottom graph shows the current) and exocytosis was monitored by simultaneous capacitance (second graph), and amperometry measurements (third graph, shown is the time integral of the amperometric current). The Ca²⁺ concentration was measured simultaneously by dual-dye microfluorimetry (top graph). In the synaptotagmin-7 KO cells, exocytosis (gray traces, mean of n = 36 cells, N = 7 mice) was slightly depressed when compared with their WT littermates (black traces, n = 41, N = 7). The KO cells displayed a significantly higher Ca²⁺ buildup during the trains (*, P < 0.05), even though resting [Ca²⁺]_i before the beginning of stimulation was unchanged (P > 0.92). (C) (Upper) Mean current during the first depolarization for KO (gray) and WT (black). (Lower) The amplitude of the Ca²⁺ currents was similar in WT and KO cells.

Fig. 2.

Ca²⁺ uncaging reveals changes in several kinetic phases of exocytosis. (A) Flash photorelease of Ca²⁺ (at arrows) resulted in a secretory burst, followed by a sustained component as measured by both capacitance (Middle) and amperometry (Bottom, time-integrated traces). Shown are averaged traces (WT, black traces, n = 72, N = 6; Syt 7 KO, gray traces, n = 62, N = 6). The second flash stimulation (Right) was delivered 90 s after the first one (WT, n = 65, N = 6; KO, n = 51, N = 6). (B) The cubic root of the amperometric charge and the square root of the capacitance increase are both expected to be proportional with vesicle size. The overlapping regression lines from KO and WT measurements indicate no difference in the released amount of catecholamines per unit area of vesicle membrane. (C) Expanded view of the capacitance increase during the first 20 ms after the uncaging flash. The dashed line is the KO trace normalized to WT amplitude at 500 ms after the flash, i.e., after exhaustion of the exocytotic burst (note the split axis). (D–F) Kinetic analysis of capacitance traces. Individual capacitance traces were fitted with a triple-exponential function. Information on pool amplitudes and kinetics was extracted from all cells used to build the average traces in A, except for cells presenting a fast or slow burst amplitude <10 fF, where the pool size is too small for a reliable estimation of the kinetics in the presence of noise (rms noise was typically 2–3 fF). For these cells, only the pool amplitudes were included. This leaves for the fast-burst time constant: WT, n = 63; KO, n = 42; and for the slow burst time constant: WT, n = 66; KO, n = 47. In KO animals, the fast and slow bursts (D) and the sustained component (E) were reduced in size, whereas both the fast time constant and the delay between the UV flash and the beginning of capacitance increase were significantly reduced (F). (G) A weak correlation between fast time constant of release and amplitude was found in both KO and WT cells. (H) Comparing the second flash burst size with the first one reports on the replenishment of the releasable vesicle pools. Replenishment was inhibited in KO cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 3.

Deletion of synaptotagmin-7 does not alter the fusion rate constant in the low-micromolar range of Ca²⁺. (A) (Upper) Measurement from a chromaffin cell stimulated by a Ca²⁺ ramp (blue symbols, right axis). The capacitance signal (red trace, left axis) showed a sigmoid-like increase. The black trace shows the size of the remaining releasable vesicle pool, which was used to calculate normalized release rates in C. (Lower) The second derivative of the capacitance trace, after smoothing, was used to calculate Ca²⁺ threshold values in B. (B) Threshold [Ca²⁺] values ([Ca²⁺] at the point of maximum acceleration of the capacitance trace; WT, black, n = 28, N = 5; Syt 7 KO, gray, n = 31, N = 5). No difference was found between the two populations of cells (P = 0.8). (C) Normalized release rates were deduced from absolute rates by normalization to the remaining pool size at each data point. No difference was observed between WT and KO cells.

Fig. 4.

Ca²⁺ binding to the C2B domain is required for synaptotagmin-7 function. Chromaffin cells from KI mice expressing a C2B domain deficient for Ca²⁺ binding (synaptotagmin-7 KI) present the same exocytosis impairment as synaptotagmin-7 KO cells. (A) Averaged traces recorded from KI mice and WT littermates (n = 23, N = 3; n = 23, N = 3, respectively). (B and C) Kinetic analysis was conducted as for Fig. 2; similar features to that of synaptotagmin-7 KO were found in KI cells (smaller and faster RRP, reduced sustained component), except that in the KI, the size of the slow burst was not significantly reduced. See also Table 1.

Fig. 5.

Synaptotagmin-1 and -7 both determine the secretory-burst components. Mean responses to flash stimulation in Syt-1/Syt-7 double-KO (dKO) cells (Syt 1 −/−, Syt 7 −/−, gray, n = 58, N = 6), control littermates (Syt 1 +/− or +/+, Syt 7 +/−, black, n = 33, N = 4), and Syt 1 KO (Syt 1 −/−, Syt 7 +/−, red trace, n = 26, N = 3) are shown; the residual exocytosis in synaptotagmin 1/7 dKO cells was almost devoid of a secretory burst and was linear in appearance.