Control of neurotransmitter release by an internal gel matrix in synaptic vesicles

Abstract

Neurotransmitters are stored in synaptic vesicles, where they have been assumed to be in free solution. Here we report that in Torpedo synaptic vesicles, only 5% of the total acetylcholine (ACh) or ATP content is free, and that the rest is adsorbed to an intravesicular proteoglycan matrix. This matrix, which controls ACh and ATP release by an ion-exchange mechanism, behaves like a smart gel. That is, it releases neurotransmitter and changes its volume when challenged with small ionic concentration change. Immunodetection analysis revealed that the synaptic vesicle proteoglycan SV2 is the core of the intravesicular matrix and is responsible for immobilization and release of ACh and ATP. We suggest that in the early steps of vesicle fusion, this internal matrix regulates the availability of free diffusible ACh and ATP, and thus serves to modulate the quantity of transmitter released.

Figure 1

ACh and ATP release from permeabilized cholinergic synaptic vesicles. Synaptic vesicle fraction was dialyzed against low ionic strength medium, and ACh and ATP release were continuously monitored by light emission of luminescent enzymatic reactions. (A) In a hemolysis tube containing the recording solution (600 mM sucrose/1 mM Hepes/Tris, pH 5.5) the following reagents were added (see arrowheads): 1, choline oxidase; 2, horseradish peroxidase; 3, luminol; 4, synaptic vesicle fraction; 5, acetylcholinesterase; 6, Triton X-100; 7, 8 mM NaCl; 8, 2 nmol of ACh; and 9, 5 nmol of ACh. (B) In a hemolysis tube containing the recording solution (600 mM sucrose/50 mM Hepes/Tris, pH 5.5), the following reagents were added in the following order: 1, luciferin–luciferase mixture; 2, synaptic vesicle suspension; 3, Triton X-100 detergent; 4, 8 mM NaCl; 5, 100 pmol of ATP; and 6, 500 pmol of ATP. The exogenous doses of ATP (5 and 6) were used to calculate the amount of ATP released. (C) The order of addition of the reagents was changed and the NaCl solution was added before the detergent Triton X-100: 1, luciferin–luciferase mixture; 2, synaptic vesicle suspension; 3, 8 mM NaCl; 4, Triton X-100 detergent; 5, 100 pmol of ATP; and 6, 500 pmol of ATP.

Figure 2

Salt concentration-dependent release of ATP. (A) Superimposed traces of recordings obtained with increasing concentrations of NaCl. The following reagents were added in the following order: 1, luciferin–luciferase mixture; 2, synaptic vesicle suspension; 3, Triton X-100; and 4, NaCl (in increasing concentrations). Traces: black, ultrapure water; red, 2 mM NaCl; green, 4 mM NaCl; magenta, 6 mM NaCl; and blue, 8 mM NaCl. (B) Salt concentration-dependent release of ATP. Traces: red, KCl; black, NaCl; olive green, sodium phosphate; sky blue, sodium sulfate; green, CaCl₂; and dark blue, calcium acetate. (C) In a hemolysis tube containing ultrapure water, the reagents were added in the following order: 1, luciferin–luciferase mixture; 2, synaptic vesicle suspension; 3, 8 mM NaCl; 4, 100 pmol of ATP; and 5, 500 pmol of ATP.

Figure 3

Release of ATP from the matrix of synaptic vesicles. (A) The matrix was refilled according to Materials and Methods. In these conditions, the matrix is able again to release ATP in an ionic force-dependent manner. The reagents were added in the following order: 1, luciferin–luciferase mixture; 2, refilled matrix suspension; 3, 8 mM NaCl; and 4, 100 pmol of ATP. (B) Superimposed traces of recordings obtained with increasing concentrations of NaCl. The reagents were added in the following order: 1, luciferin–luciferase mixture; 2, refilled matrix suspension; and 3, NaCl (in increasing concentrations). Traces: black, ultrapure water; red, 2 mM NaCl; green, 4 mM NaCl; magenta, 6 mM NaCl; and blue, 8 mM NaCl. The matrix was refilled with ACh according to Materials and Methods. In these conditions, the matrix was again able to release ACh when challenged with a NaCl solution. The hemolysis tube contained the refilled matrix and the chemiluminescence reagents (arrowhead 1). (C) To test the presence of free ACh, acetylcholinesterase was added (arrowhead 2). The amount of ACh detected is very small compared with a dose of 5 pmol of ACh (arrowhead 3). (D) NaCl-induced release of ACh. The addition of 20 mM NaCl (arrowhead 2) did not increase the luminescence until acetylcholinesterases (arrowhead 3) were added. The signal is specific to ACh, because a new addition of acetylcholinesterases (arrowhead 4) did not trigger other signal. Finally, the amount of ACh released can be estimated by comparing it to 100 pmol of ACh (arrowhead 5). (E and G) NaCl-dependent release of ATP from immunoprecipitated intravesicular matrix with (E) and without (G) mAb against SV2. The reagents were added in the following order: 1, luciferin–luciferase mixture and, when a baseline was reached, the other reagents were added; 2, immunoprecipitated intravesicular matrix suspension; 3, 8 mM NaCl; and 4, 10 pmol of ATP. (F and H) Western blot detection of SV2 content in immunoprecipitated solution with mAb against SV2 (F) or without the antibody (H).

Figure 4

Synaptic vesicle matrix under AFM. (A) Immunogold detection of synaptic vesicle protein SV2 over the intravesicular matrix by AFM. The white spots correspond to 5-nm gold particles. The image was obtained in air. The remaining images were obtained on matrix immersed in liquid. (B) Imaging by AFM of vesicular matrix in ultrapure water. (C) Effect of 8 mM NaCl on the height of the disks. (D) Histogram of the frequency distribution of the height of matrix disks before and after adding NaCl; white, ultrapure water; black, NaCl. (E) Effect of 0.8 mM CaCl₂ on the height of the matrix disks. (F) Histogram of the distribution of the height of matrix particles before and after adding CaCl₂; white, ultrapure water; black, CaCl₂. (Lower) x, y, and z scales for A–C and E.

Figure 5

Model of ACh and ATP interaction with the synaptic vesicle matrix. Keratan sulfate and sialic acid of the vesicular matrix would support the adsorption of ACh and ATP, respectively. (A) Sulfate groups of galactose (Gal) and N-acetylglucosamine (GlcNAc) residues from the keratan molecule would be found where electrostatic interaction with the quaternary ammonium of ACh takes place (dashed line). S atoms are yellow, O atoms are red, and N, C, and H atoms are blue, black, and white, respectively. (B) For ATP interaction with the matrix, we propose that a calcium intermediate may be formed of the calcium ions, sialic acid, galactose, and ATP. (C) A simplified tube model of the same complex. The calcium ion, in green for clarity, is coordinated with six O atoms, which are red. P atoms are yellow, and N, C, and H atoms are blue, black, and white, respectively.