Alternative splicing of SNAP-25 regulates secretion through nonconservative substitutions in the SNARE domain

Abstract

The essential membrane fusion apparatus in mammalian cells, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consists of four alpha-helices formed by three proteins: SNAP-25, syntaxin 1, and synaptobrevin 2. SNAP-25 contributes two helices to the complex and is targeted to the plasma membrane by palmitoylation of four cysteines in the linker region. It is alternatively spliced into two forms, SNAP-25a and SNAP-25b, differing by nine amino acids substitutions. When expressed in chromaffin cells from SNAP-25 null mice, the isoforms support different levels of secretion. Here, we investigated the basis of that different secretory phenotype. We found that two nonconservative substitutions in the N-terminal SNARE domain and not the different localization of one palmitoylated cysteine cause the functional difference between the isoforms. Biochemical and molecular dynamic simulation experiments revealed that the two substitutions do not regulate secretion by affecting the property of SNARE complex itself, but rather make the SNAP-25b-containing SNARE complex more available for the interaction with accessory factor(s).

Figure 1.

Alternative splicing of exon 5 introduces nine amino acid substitutions in SNAP-25. (A) The substitutions are located in the C-terminal end of the first SNARE motif and the first part of the linker and include a relocalization of one of the palmitoylated cysteines. The gray boxes and numbers show residues that are buried in the inside of the complex. (B) Crystal structure of the ternary SNARE complex (Sutton et al., 1998). The membrane anchors of syntaxin and synaptobrevin would attach at the right side. The linker between the two SNAP-25 SNARE domains (SN1 and SN2) was added using a drawing program. The structure was downloaded from PubMed (1SFC) and drawn using Swiss-Pdb viewer (Guex and Peitsch, 1997; http://www.expasy.org/spdbv/).

Figure 2.

SNAP-25 and syntaxin 1 on membrane sheets from embryonic mouse chromaffin cells. Plasma membrane sheets were generated from SNAP-25 +/+ chromaffin cells (A), SNAP-25 null chromaffin cells (B), SNAP-25 null chromaffin cells expressing SNAP-25a (C), and SNAP-25 null chromaffin cells expressing SNAP-25b (D). Sheets were immediately fixed with paraformaldehyde and immunostained for SNAP-25 and syntaxin 1. The samples were imaged in three channels: membranes were identified in the presence of TMA-DPH dye in the blue (not shown), SNAP-25 signal was detected in the red and syntaxin 1 in the long red channel. Overlay from SNAP-25 and syntaxin 1 indicates partial colocalization of two proteins. Note that the SNAP-25 images in A, C, and D were scaled independently of each other to preserve spatial information; however, the absolute immunofluorescence intensities in C and D were much higher than in A (see Figure 3). The colocalization was quantified using correlation analysis (see text).

Figure 3.

Quantification of SNAP-25 isoforms and syntaxin 1 in the plasma membrane of embryonic mouse chromaffin cells. The immunofluorescence of membrane sheets from 12 SNAP-25 null animals, 12 SNAP-25 WT(+/+) animals, 9 SNAP-25 heterozygous (+/-) animals, 9 SNAP-25 null animals expressing SNAP-25a and 7 SNAP-25 null animals expressing SNAP-25b were analyzed and plotted. A minimum of 10 sheets per animal were analyzed. The values indicate relative abundance (normalized to the mean of SNAP-25+/+ animals from the same litter) ± SEM of SNAP-25 (A) and syntaxin 1 (B) protein.

Figure 4.

A group of three amino acids is sufficient to switch SNAP-25a to SNAP-25b phenotype. (A) Mean [Ca²⁺]_i (top, error bars represent SEM), capacitance change (middle), and amperometric current (bottom) were measured simultaneously after a step-like elevation of [Ca²⁺]_i caused by flash photolysis of caged Ca²⁺ (flash at arrow). The traces are averages of many experiments, so the individual fusion events (spikes) are not recognizable in the amperometric signal. Left, secretion after the first stimulation; right, secretion in response to the second stimulation (left). Shown are means of 38 SNAP-25 null cells expressing SNAP25b cells (black) and 38 cells overexpressing SNAP-25a^{N65D/H66Q/Q69K} for 6–8 h (gray). There was no difference in preflash [Ca²⁺]_i between two groups (our unpublished data). The data for SNAP-25a overexpression were taken from another series of experiments and are shown here for comparison. Secretion from cells transfected with SNAP-25a^{N65D/H66Q/Q69K} and SNAP-25b was similar. (B) Amplitudes of exponential fits to individual responses. The amplitudes (mean ± SEM) of the fast and the slow burst component and the rate of sustained component were similar in both stimuli (dark bars, SNAP-25b; gray bars, SNAP-25a^{N65D/H66Q/Q69K}).

Figure 5.

H66Q/Q69K: the minimal mutation in SNAP-25a that gives SNAP-25b phenotype. (A) Response to the first stimulation in SNAP-25 null cells expressing SNAP25a^N65D (red, 23 cells), SNAP25a^Q69K (blue, 30 cells) and SNAP25a^{N65D/H66Q/Q69K} (black, 40 cells). For explanation, see the legend to Figure 4. (B) Size of the burst (fast + slow burst) component and the rate of sustained secretion. The secretory phenotype of SNAP25a^Q69K is intermediate between SNAP25a^N65D and the triple mutation. No difference in the rate of the sustained component was detected. (C) Response to the first flash stimulation in SNAP-25 null cells expressing SNAP25a^H66Q/Q69K (green, 30 cells), SNAP25a^H66Q (gray, 16 cells) and SNAP25b (black, 23 cells). (D) The amplitudes of the burst components and the rate of sustained release in cells expressing SNAP25a^H66Q/Q69K were indistinguishable from cells expressing SNAP-25b.

Figure 6.

Q66H/K69Q mutation in SNAP-25b results in SNAP-25a-like phenotype. (A) The opposite experiment to the one shown in Figure 5. Here, we mutated the positions 66 and 69 in SNAP-25b into the residues found in SNAP-25a. Response to a first flash stimulation in SNAP-25 null cells expressing SNAP25b^Q66H/K69Q (gray, 26 cells) in comparison with SNAP25a (black, 26 cells). The data for SNAP-25b overexpression were taken from another series of experiments and are shown here for comparison. (B) Response to a first flash stimulation in SNAP-25 null cells expressing SNAP25b^K69Q (gray, 26 cells) in comparison with SNAP25a (black, 26 cells). Again, data for SNAP-25b overexpression were taken from another experimental series (C). Size of fast and slow burst and rate of the sustained component. Dark bars, SNAP-25a; gray bars, SNAP-25b Q66H/K69Q; and white bars, SNAP-25b K69Q. No significant changes were found between these three constructs (e.g., for the sustained component; p = 0.13, Kruskal–Wallis test).

Figure 7.

A neighboring aspartate (D70) present in both isoforms is necessary for the SNAP-25b-like phenotype. (A) Response to a first flash stimulation in SNAP-25 null cells expressing SNAP25a^D70A (gray, 22 cells) in comparison with SNAP25a (black, 21 cells). Bottom, size of fast and slow burst and rate of sustained component. (B) Response to a first flash stimulation in SNAP-25 null cells expressing SNAP25b^D70A (gray, 35 cells) in comparison with SNAP25a (black, 41 cells). The data for SNAP-25b overexpression were taken from another series of experiments and are shown here for comparison. Bottom, sizes of fast and slow burst of release, and rate of sustained component. No significant changes were found between SNAP-25a and SNAP-25b^D70A, showing that the aspartate at position 70 is necessary for the SNAP-25b secretory phenotype but not for the SNAP-25a-like secretory phenotype.

Figure 8.

The thermal stability of the ternary SNARE complex is slightly higher in the presence of SNAP-25b. Thermal melts of purified ternary SNARE complexes containing different SNAP-25 variants or point mutations. Thermal stability of ternary SNARE complexes formed overnight was assayed by CD spectroscopy in the presence of 2 M guanidine hydrochloride. Complexes formed with SNAP-25b unfolded at a slightly higher temperature (4–5°C) than SNAP-25a-containing complexes. The double mutation H66Q/Q69K in SNAP-25a was sufficient to get a complex of higher stability, whereas the single mutation Q69K did not change stability. The D70A mutation in SNAP-25b led to a complex with SNAP-25b-like stability, but a SNAP-25a-like secretory phenotype (Figure 7B). This finding shows that the difference in stability is not causing the difference in secretory phenotype.

Figure 9.

Structural arrangement of the SNARE complex around the SNAP-25 residues 66 and 69. A. Structure of the SNAP-25a-containing ternary SNARE complex (Sutton et al., 1998) after MD simulation for 20 ns (as explained in the text). (B) Structure of the ternary SNARE complex after introduction of Q66 and K69 in the SNAP-25a-containing ternary SNARE complex and MD simulation for 20 ns. Note that the structural arrangements shown here are “snapshots” taken from a longer simulation and do not represent a preferred or typical arrangement of the side chains.