The immediately releasable pool of mouse chromaffin cell vesicles is coupled to P/Q-type calcium channels via the synaptic protein interaction site

Abstract

It is generally accepted that the immediately releasable pool is a group of readily releasable vesicles that are closely associated with voltage dependent Ca(2+) channels. We have previously shown that exocytosis of this pool is specifically coupled to P/Q Ca(2+) current. Accordingly, in the present work we found that the Ca(2+) current flowing through P/Q-type Ca(2+) channels is 8 times more effective at inducing exocytosis in response to short stimuli than the current carried by L-type channels. To investigate the mechanism that underlies the coupling between the immediately releasable pool and P/Q-type channels we transiently expressed in mouse chromaffin cells peptides corresponding to the synaptic protein interaction site of Cav2.2 to competitively uncouple P/Q-type channels from the secretory vesicle release complex. This treatment reduced the efficiency of Ca(2+) current to induce exocytosis to similar values as direct inhibition of P/Q-type channels via ω-agatoxin-IVA. In addition, the same treatment markedly reduced immediately releasable pool exocytosis, but did not affect the exocytosis provoked by sustained electric or high K(+) stimulation. Together, our results indicate that the synaptic protein interaction site is a crucial factor for the establishment of the functional coupling between immediately releasable pool vesicles and P/Q-type Ca(2+) channels.

Figure 1. IRP exocytosis is tightly coupled to Ca²⁺ source. A.

(i) Summary of capacitance increases after stimulation with depolarizing pulses of different lengths, between 5 and 200 ms, in control condition (with EGTA 0.5 mM in the internal solution). Note that there is a clearly defined initial component, which saturates approximately between 30 and 50 ms pulses. (ii) The black circles in the figure on the right is an expanded representation of this initial component (n = 116), while the gray squares represent experiments performed with BAPTA (0.5 mM) as exogenous internal buffer (n = 53). The results obtained with EGTA were fitted with a single exponential function of the form A. (1−e^−t/τ) (A = 33±1 fF, τ = 15±0.8 ms, R = 0.998) which is represented by the continuous black line. The black dashed lines represent the confidence intervals (95%). B. The paired pulse protocol, composed of two 10 ms depolarization pulses separated by a 300 ms interval, which was used to calculate the IRP. The figure represents (i) the Ca²⁺ currents and (ii) the membrane capacitance changes provoked by application of this protocol during one typical experiment. The two pulses (represented in (i), from −80 to +10 mV) induced identical calcium currents, while the C_m response exhibited a clear depression between the first (ΔC_m1) and the second pulse (ΔC_m2). The values of B_min and B_max for this particular experiment were 42 and 48 fF, respectively. C. (i) The bar diagram summarizes the B_min and B_max values for the IRP obtained in presence of BAPTA (n = 14) or EGTA (n = 13) in the internal solution. BAPTA reduced significantly the exocytosis of IRP (p<0.02) respect to EGTA. (ii) On the other hand, the exocytosis obtained in response to a stronger stimulus (two 100 ms pulses separated by 300 ms), aimed to estimate the whole RRP , , was not affected by the type of calcium buffer added to the internal solution (EGTA, n = 11; BAPTA, n = 12). D. (i) Averaged exocytic response of chromaffin cells loaded with EGTA (n = 10, black) or BAPTA (n = 9, gray) in response to a train of ten depolarizing pulses of 50 ms (2 Hz). Note that BAPTA induced a clear reduction of exocytosis during the first pulses. (ii) The bar diagram represents the averaged Ca²⁺ currents during train stimulation. (iii) Synchronous capacitance changes along the train. Synchronous exocytosis is defined as the change in capacitance during each stimulus, and measured in a 50 ms window starting 50 ms after each depolarization minus the mean pre-stimulus capacitance also measured in a 50 ms window. *p<0.05; **p<0.001.

Figure 2. IRP exocytosis is coupled to P/Q calcium channels. A.

Representative example of recorded Ca²⁺ currents (top) and membrane capacitance changes (bottom) induced by application of the dual 10 ms pulse protocol in presence of AGA (200 nM) in the external solution. B. The bar diagram represents the averaged Ca²⁺ currents in control conditions and in the presence of AGA (n = 8). C. The bar diagram summarizes the averaged capacitance changes obtained in response to the application of the dual 10 ms pulse protocol in control conditions and in the presence of AGA (n = 8). The toxin reduced dramatically both B_max and B_min parameters associated with the exocytosis of the IRP. D. Exocytosis, measured as the change in membrane capacitance, in response to short depolarizations (between 5 and 50 ms) was plotted against the Ca²⁺ entry (calculated as the time integral of I_Ca2+) for cells in control conditions (n = 130), and in presence of NITRE (10 µM) (n = 45) or AGA (n = 45).

Figure 3. Synprint mediates the coupling of IRP with P/Q-type calcium channels. A:

Examples of original records of Ca²⁺ currents (top) and membrane capacitance changes (bottom) in response to the application of the dual 10 ms pulse protocol, in Syn⁻ (i) or Syn⁺ (ii) cells. B. (i) Calcium current densities obtained in Syn⁻ (n = 30), EGFP (n = 7), Syn⁺ (n = 14), and Syn⁺ cells treated with 200 nM ω-agatoxin-IVA (Syn⁺+AGA) (n = 10). (ii) Averaged estimations of B_min and B_max for the IRP, obtained in response to the application of the dual 10 ms pulse protocol under the same conditions mentioned in (i). Please note that while Syn⁻, EGFP and Syn⁺ have almost identical I_Ca2+ values, a highly significant decrease (p<0.005) in the IRP exocytosis was found between Syn⁺ and the other two groups of experiments. C. Confocal images of two chromaffin cells that were positive for EGFP+synprint transfection. D. Exocytosis, measured as the change in membrane capacitance in response to short depolarizations (between 5 and 50 ms), was plotted against the Ca²⁺ entry (calculated as the time integral of I_Ca2+) for Syn⁻ (n = 40) and Syn⁺ cells (n = 30). The plot also represents the data of cells in control conditions (continuous line) or treated with AGA (dashed line) from Fig. 2D. Note that while Syn⁻ followed a similar behavior than control cells, Syn⁺ is superimposed almost perfectly with AGA.

Figure 4. Synprint transfection does not modify the relative contributions of calcium current subtypes.

The figures on the top show the calcium current vs. voltage relationships for (A) Syn⁺ cells in control conditions (Syn⁺) (n = 9), Syn⁺ cells treated with 200 nM ω-agatoxin-IVA (Syn⁺+AGA) (n = 9), and Syn⁺ cells treated with 10 µM nitrendipine (Syn⁺+NITRE) (n = 7); and for (B) nontransfected cells in control conditions (Control) (n = 11), nontransfected cells treated with 200 nM ω-agatoxin-IVA (Control+AGA) (n = 9), and nontransfected cells treated with 10 µM nitrendipine (Control+NITRE) (n = 9). The cells were stimulated with 50 ms square voltage pulses, from a holding potential of −80 mV to the potentials indicated in the abscissas of panels A and B. C. Original records of Ca²⁺ currents obtained in response to square depolarizations to +10 mV for the same three conditions detailed in panel A. D. Original records of Ca²⁺ currents obtained in response to a square depolarization to +10 mV for the same three conditions detailed in panel B.

Figure 5. Effects of synprint on exocytosis provoked by strong stimulation. A.

(i) Averaged exocytic response of Syn⁻ cells (n = 7, black line), Syn⁺ (n = 6, dark gray line) and normal cells treated with AGA (n = 13, light gray line), in response to trains of ten depolarizing pulses of 50 ms (2 Hz). The bar diagrams in (ii) and (iii) represent the averaged I_Ca2+ peaks and the synchronous exocytosis elicited at each depolarization in the three conditions described in (i). B. The exocytosis was measured as the increase in fluorescence due to incorporation of the fluorophore FM4-64 into the membrane of newly fusing vesicles. (i) Typical experiments performed on a Syn⁻ (n = 10) and a Syn⁺ (n = 9) chromaffin cells. The spatially averaged fluorescence (F.A.U.: fluorescence arbitrary units) of the whole cell was measured at the equatorial cell section, previous subtraction of the background fluorescence (see methods), and normalized (in %) with respect to the value obtained at the end of the FM4-64 incubation period. The cell was stimulated for 3 min. with 50 mM K⁺ solution in presence of the fluorophore. Exocytosis was quantified by the increase in fluorescence above the plateau value established previously the stimulus (dotted line). The stimulation period was terminated by changing the extracellular high K⁺ solution to standard solution without FM4-64 (see methods). (ii) Bar diagram showing the results obtained from the type of experiments represented in (i). The results are expressed as the percentage increase in fluorescence induced by depolarization with respect to previous fluorescence values (after background subtraction). No significant differences were found between these two groups.