A polybasic juxtamembrane sequence in Syn1A comprises the lipid-binding domain. (A) Top, sequence alignment of juxtamembrane regions of Syn1A across species. Note the five highly conserved basic residues between positions 260-265 (NCBI accession numbers: HS: AAA53519; MMul: NP_001028037; RN: AAF23017; MMus: NP_058081; LP: AAK70494; LS: AAO83845; DM: A55673; CR: AAY54265). Bottom, sequence alignments of Syn1A juxtamembrane mutants used in this study. (B) Progressive neutralization of Syn1A juxtamembrane basic residues correlates to a progressive reduction in apparent binding affinity for PA. Binding between Syn1A mutants and PA was determined using protein–lipid overlays (n = 4), in which increasing amounts of PA were spotted on nitrocellulose. Soluble Syn1A(1-267) was at a fivefold molar excess to PA. Integrated Syn1A immunoreactivity was measured at each PA spot, normalized for each mutant, and the data were fitted with a Hill equation. (C) The 5RK/A Syn1A mutant exhibits significantly reduced binding to PA-containing liposomes compared with WT Syn1A. Percentage of Syn1A bound to liposomes was defined as the percentage of Syn1A immunoreactivity in the top three gradient fractions relative to the total Syn1A immunoreactivity. (D) Representative protein–lipid overlay demonstrating that Syn1A 5RK/A has reduced binding to all acidic phospholipids tested. (E) Representative protein–lipid overlay demonstrating that a peptide of Syn1A's juxtamembrane region (aa 252-265) closely recapitulates the lipid binding of the full-length soluble Syn1A.

Syn1A 5RK/A interacts normally with Munc18-1 and SNAP25. (A) Analysis of direct CFP-Syn1A-citrine-Munc18-1 interactions by sensitized emission FRET imaging. Graph shows a pixel-by-pixel plot of FRET efficiency versus molar ratio (Syn1A:Munc18-1). Both Syn1A (WT) and Syn1A (5RK/A) bind Munc18-1 with similar FRET efficiency. By comparison, Syn1A I233A demonstrates strongly reduced FRET efficiencies with Munc18-1 compared with WT. Solid horizontal line demonstrates background FRET values determined from coexpression of CFP and citrine fluoroproteins. (B) Average FRET efficiency for each Syn1A construct, at equimolar ratios of Syn1A and Munc18-1 (0.9 < ratio < 1.1; see vertical lines in B). Bars, mean ± SEM. (C) Representative immunoblot demonstrating co-IP of EGFP-SNAP25 with Syn1A from HEK293-S3 cells transiently transfected with Syn1A (WT, L205A/E206A, or 5RK/A), Munc18-1, and EGFP-SNAP25. Note that Syn1A 5RK/A pulls down similar amounts of EGFP-SNAP25 compared with WT Syn1A. In contrast, a mutant of Syn1A (L205A/E206A) previously shown to have reduced binding to SNARE proteins demonstrates a reduced amount of EGFP-SNAP25 pulldown. (D) Integrated densities of Syn1A and EGFP-SNAP25 bands were measured to determine a ratio of EGFP-SNAP25 to Syn1A for each condition. Ratios within a single experiment were then normalized to that of WT Syn1A to allow for comparison across experiments. Bars, means ± SEM, from three independent experiments.

Neutralization of Syn1A's polybasic juxtamembrane region results in a decrease in evoked secretion. (A) Progressive neutralizing mutations of Syn1A's polybasic juxtamembrane domain result in a graded reduction in evoked hGH secretion. Transfected PC-12 cells were stimulated with 70 mM K⁺ for 6 min. Data were normalized to control rescue (Syn1A_K253I) for each experiment and pooled across at least three independent experiments for each condition. Dotted line represents the baseline level of remaining secretion following BoNT-C treatment. Bars, means ± SEM. Asterisks indicate statistical significance (p < 0.001) from control. Graph at right shows the same data as on the left, but with the average residual BoNT-C baseline secretion for each experiment subtracted from each condition before normalization to control rescue. Note that when the data are corrected for residual BoNT-C secretion, the actual effect of the Syn1A neutralization mutants becomes substantially larger. (B) Evoked [Ca²⁺] dynamics reported by changes in Fura2 fluorescence intensity ratio (F340 nm/380 nm) demonstrate that depolarization-induced (100 mM K⁺ for 5 s) calcium fluxes are similar between control and 5RK/A cells. Left, averaged ratio traces for control and 5RK/A cells. Horizontal bar indicates period of stimulus application. Averaged baseline (middle) and evoked change (right) in F340 nm/380 nm ratio for control and 5RK/A cells. (C) Representative amperometric recordings from bovine adrenal chromaffin cells transfected with Syn1A_K253I or Syn1A_K253I (5RK/A) together with BoNT-C. Exocytotic secretion was evoked by a 60-s application of locally perfused 100 mM K⁺. Amperometric spikes were detected and quantified by an automated analysis software program written by Eugene Mosharov (Columbia University). (D) Comparison of averaged number of spikes per cell in control and 5RK/A cells. Bars, mean ± SEM. Only spikes >10 pA in amplitude above RMS baseline were considered for analysis. (E) Cumulative spike frequency distribution, normalized for the total number of spikes for control (Syn1A_K253I) and Syn1A_K253I (5RK/A) cells. The distributions were not significantly different.

Functional phenotypes of Syn1A_K253I control and 5RK/A mutant are differentially regulated by manipulation of phosphatidic acid levels in live PC-12 cells. Graph demonstrates averaged results from hGH secretion assays utilizing the BoNT-C knockdown and rescue. External application of 1 μM LPC has no effect on control secretion, but results in a partial rescue of the 5RK/A phenotype. Overexpression of PLD1 results in near-complete rescue of the 5RK/A phenotype to control secretion levels. On the other hand, knockdown of PLD1 activity via a targeted siRNA construct results in a drastic decrease in control secretion, while having little to no effect on secretion from 5RK/A cells. Results are normalized to the untreated Syn1A_K253I control rescue in each experiment to allow comparison across multiple experiments, and data were pooled from at least three independent experiments. Bars, mean ± SEM. Letters above bars indicate pairs of conditions in which the differences are statistically significant (p < 0.05).

Syn1A directly binds a subset of acidic phospholipids that includes the fusogenic lipid phosphatidic acid. (A) Protein–lipid overlay demonstrating concentration-dependent binding between soluble Syn1A(1-267) and multiple acidic phospholipids. Left, schematic representing the arrangement of lipids spotted on the nitrocellulose. Filled circles indicate those lipids for which an interaction was demonstrated. Right, α-Syn1A immunoreactivity from four representative protein–lipid overlay experiments, in which decreasing concentrations of Syn1A were overlaid onto nitrocellulose membranes spotted with 100 pmol each of indicated phospholipids. (B) Averaged level of interaction of Syn1A for each phospholipid relative to that of PA. Integrated immunoreactive density associated with each lipid on the overlays was averaged from three independent experiments where [Syn1A] = 3 nM and was normalized to the integrated density for PA. Bars, means ± SEM. (C) Representative Western blots of Syn1A immunoreactivity in fractions collected from liposome flotation assays. Bound Syn1A floats with liposomes to the top of the gradient.

Syn1A 5RK/A targets to plasma membrane regions in PC-12 cells. (A) Representative confocal fluorescence and corresponding DIC images of PC-12 cells transiently transfected with eCFP-tagged Syn1A(WT) and Syn1A (5RK/A) together with Munc18-1. (B) Representative epifluorescence images of PC-12 cells transiently transfected with mRFP-Syn1A-pHluorin (WT or 5RK/A) and Munc18. The pHluorin signal (left) reports only on the pool of Syn1A present at the cell surface, whereas the mRFP signal (right) reports on the entire pool of exogenous Syn1A within the cell. (C) Average pHluorin and mRFP fluorescence intensity for PC-12 cells transfected as in B. Bars, means ± SEM. Numbers above each bar represent the number of cells used to calculate the average for each condition. (D) Scatterplot of mean pHluorin intensity versus mean mRFP intensity for each individual cell. Cells demonstrating moderate Syn1A expression levels (defined as a mean RFP signal between 20 and 170, a range within which >85% of the cells analyzed fell) were used to generate linear fits for each data set, as indicated by the solid lines. Dotted lines represent the 95% confidence intervals of the linear fits, which overlap for the WT and 5RK/A data sets.

BoNT-C knockdown allows isolation of functional effects of exogenous Syn1A constructs in live secretory cells. (A) Top, representative epifluorescence images demonstrating that Syn1A (K253I) is resistant to cleavage by BoNT-C. Images compare the subcellular distribution of fluorescent signal in PC-12 cells transfected with CFP-Syn1A (WT) and CFP-Syn1A (K253I) with/without the light chain of BoNT-C. Munc18-1 was coexpressed in each condition. Bottom, normalized fluorescence intensity profiles, as determined by averaging line scans taken from the outside to the middle of each cell, corresponding to the expression conditions shown above. Line scans were normalized, the peaks were aligned, and scans were averaged for each condition. (B) Functional rescue of BoNT-C knockdown in elevated K⁺ evoked (70 mM, 6 min) hGH secretion, by cotransfection with Syn1A (K253I) and Munc18-1. Note that S1A (K253I) or Munc18-1 alone did not rescue secretion, nor did Syn1A (WT) cotransfected with Munc18-1. Bars, means ± SEM.

Neutralization of Syn1A's polybasic juxtamembrane region results in a decrease in fusion pore diameter and a lengthening of fusion pore duration. (A) Representative PSF for control (Syn1A_K253I) or Syn1A_K253I (5RK/A) expression conditions (+ BoNT-C) during stimulation by 60-s application of locally perfused 100 mM K⁺. (B) Left, mean PSF amplitudes for control and 5RK/A cells, from all PSF >1 ms in duration and <30 pA in amplitude (n = 584 control PSF analyzed, n = 576 5RK/A PSF analyzed). Right, averaged median PSF amplitudes (n = 66 control cells analyzed, n = 78 5RK/A cells analyzed). In both cases, the PSF from 5RK/A cells demonstrate statistically significant decreases in amplitude (p < 0.05). Bars, mean ± SEM. (C) Left, mean PSF durations for control and 5RK/A cells, from all PSF >1 ms in duration and <30 pA in amplitude (n = 584 control PSF analyzed, n = 576 5RK/A PSF analyzed). Right, averaged median PSF durations (n = 66 control cells analyzed, n = 78 5RK/A cells analyzed). In both cases, the PSF from 5RK/A cells demonstrate statistically significant lengthening in duration (p < 0.05). Bars, mean ± SEM.