The Dual Function of the Polybasic Juxtamembrane Region of Syntaxin 1A in Clamping Spontaneous Release and Stimulating Ca2+-Triggered Release in Neuroendocrine Cells

Abstract

The exact function of the polybasic juxtamembrane region (5RK) of the plasma membrane neuronal SNARE, syntaxin 1A (Syx), in vesicle exocytosis, although widely studied, is currently not clear. Here, we addressed the role of 5RK in Ca²⁺-triggered release, using our Syx-based intramolecular fluorescence resonance energy transfer (FRET) probe, which previously allowed us to resolve a depolarization-induced Ca²⁺-dependent close-to-open transition (CDO) of Syx that occurs concomitant with evoked release, both in PC12 cells and hippocampal neurons and was abolished upon charge neutralization of 5RK. First, using dynamic FRET analysis in PC12 cells, we show that CDO occurs following assembly of SNARE complexes that include the vesicular SNARE, synaptobrevin 2, and that the participation of 5RK in CDO goes beyond its participation in the final zippering of the complex, because mutations of residues adjacent to 5RK, believed to be crucial for final zippering, do not abolish this transition. In addition, we show that CDO is contingent on membrane phosphatidylinositol 4,5-bisphosphate (PIP2), which is fundamental for maintaining regulated exocytosis, as depletion of membranal PIP2 abolishes CDO. Prompted by these results, which underscore a potentially significant role of 5RK in exocytosis, we next amperometrically analyzed catecholamine release from PC12 cells, revealing that charge neutralization of 5RK promotes spontaneous and inhibits Ca²⁺-triggered release events. Namely, 5RK acts as a fusion clamp, making release dependent on stimulation by Ca²⁺SIGNIFICANCE STATEMENT Syntaxin 1A (Syx) is a central protein component of the SNARE complex, which underlies neurotransmitter release. Although widely studied in relation to its participation in SNARE complex formation and its interaction with phosphoinositides, the function of Syx's polybasic juxtamembrane region (5RK) remains unclear. Previously, we showed that a conformational transition of Syx, related to calcium-triggered release, reported by a Syx-based FRET probe, is abolished upon charge neutralization of 5RK (5RK/A). Here we show that this conformational transition is dependent on phosphatidylinositol 4,5-bisphosphate (PIP2) and is related to SNARE complex formation. Subsequently, we show that the 5RK/A mutation enhances spontaneous release and inhibits calcium-triggered release in neuroendocrine cells, indicating a previously unrecognized role of 5RK in neurotransmitter release.

Keywords: FRET; amperometry; exocytosis; fusion clamp; neuroendocrine cells; syntaxin-1A.

Figure 1.

5RK is crucial for the CDO of CSYS. A, Domain structure of CSYS (Greitzer-Antes et al., 2013), and its mutants, CSYS-5RK/A and CSYS-YQ/AA. Two fluorescent molecules, CFP and YFP, are fused to Syx via flexible linkers (L). B, Changes in the average normalized FRET ratio in PC12 cells expressing CSYS in response to high K⁺ depolarization. Addition of 200 μm Cd to both physiological and high K⁺ solutions results in a smaller decrease in the FRET ratio compared with its absence (white diamonds, n = 109, and black diamonds, n = 122, respectively; 9 experiments; F_(79,15800) = 22.68, p < 0.0001 ANOVA with repeated measures). C, No significant difference in the FRET ratio reduction between cells expressing CSYS-5RK/A in the presence and absence of Cd (white squares, n = 67, and gray squares, n = 51, respectively; 5 experiments). D, The initial FRET efficiencies (E^app) of PC12 cells expressing CSYS and CSYS-5RK/A are similar (black bar, n = 31, and gray bar, n = 25, respectively; 2 experiments). E, CDO involves ternary SNARE complex assembly Ea, The reduction in the FRET ratio of CSYS (black diamond, n = 47; 4 experiments) was statistically smaller (F_(79,6636) = 5.232, p < 0.0001, ANOVA with repeated measures) upon Syb2 cleavage by 30 nm TeTx-LC (gray diamond, n = 39; 4 experiments). Eb, On the contrary, the FRET ratio of CSYS-5RK/A (gray square, n = 31; 4 experiments) is not affected by the Syb2 cleavage (black square, n = 35; 4 experiments). Dashed lines indicate a similar FRET ratio reduction of CSYS-5RK/A compared with CSYS+TeTx-LC. F, Addition of Cd to CSYS-YQ/AA-expressing cells results in a smaller decrease in the FRET ratio compared with its absence (white triangles, n = 33 and black triangles, n = 41, respectively; 3 experiments; F_(79,3239) = 5.621, p < 0.0001 ANOVA with repeated measures).

Figure 2.

PIP2 depletion abolishes CDO of CSYS. A–C, PIP2 hydrolysis by PLCη2 (tagged with m-Kate fluorophore) in PC12 cells. A, PLCη2 coexpression with CSYS resulted in a significantly smaller decrease in the FRET ratio in response to high K⁺ depolarization compared with no treatment (black diamonds, n = 49, and gray diamonds, n = 50, respectively; 3 experiments; F_(79,7110) = 7.192, p < 0.0001, ANOVA with repeated measures). B, Addition of Cd had no effect on the FRET ratio reduction of CSYS coexpressed with PLCη2 (black circles, n = 9; gray circles, n = 8), which was similar to that of CSYS alone but in the presence of Cd (white diamonds, n = 11); all cells were assayed in a single experiment. C, Hydrolysis of PIP2 by PLCη2 did not have any effect on the FRET reduction of CSYS-5RK/A (gray squares, n = 17; black squares, n = 17; 2 experiments). D, E, PIP2 depletion induced by the 5-phosphatase Inp54p system in PC12 cells. Da, Rapamycin addition induced heterodimerization between FRB and mRFP FKBP Inp54p, resulting in mRFP tagging of PM (top) and a decrease in the PM tagging of GFP-PH PLCδ, which binds PIP2 (bottom). Scale bar, 5 μm. Db, Normalized fluorescence intensity profiles of the above cells, indicating PM expression, determined from line scans (Da, white lines) taken from the outside to the middle of each cell. E, PIP2 depletion by the 5-phosphatase Inp54p system resulted in a significantly smaller decrease in the FRET ratio of CSYS in response to high K⁺ depolarization (white triangles, n = 16; black diamonds, n = 20; 2 experiments; F_(79,2686) = 7.632, p < 0.0001, ANOVA with repeated measures).

Figure 3.

Inhibition of PA production in the PM has no effect on CSYS opening. A, Left, Confocal images, taken with a 488 excitation laser, demonstrating the cellular distribution of a wild-type PA-binding probe coupled to EGFP (wt-PABD) in PC12 cells. The cells were incubated for 1 h with (Ac, Ad) or without (Aa, Ab) 6 μm PLD-inhibitor, VU0155056. In both groups the cells were maintained under resting conditions (Aa, Ac) and stimulated for 5 min with high K⁺ solution (Ab, Ad). Middle, Normalized fluorescence intensity profiles, as determined by line scans (5 μm; white line) taken from the outside to the middle of each cell, corresponding to PA distribution on PM shown on the left. Line scans were normalized and the peaks were aligned, for each condition. Right, Changes in membrane fluorescence before and after high K⁺ depolarization from cells expressing wt-PABD, in the presence (right) or absence (left) of the PLD-inhibitor (**p < 0.001). B, No significant difference between the changes of the FRET ratio of CSYS in response to high K⁺ depolarization in the absence (n = 22) and presence (n = 21) of the PLD-inhibitor.

Figure 4.

Neutralization of 5RK increases the frequency of spontaneous events and diminishes evoked events. A, CSYS-5RK/A-R is resistant to cleavage by BoNT-C1 and targets to PM regions in PC12 cells. Top, Confocal images of PC12 cells demonstrating that CSYS-5RK/A-R expression with (Aiv) or without (Aiii) BoNT-C1 is distributed to the PM, in contrast with the cytosolic expression of CSYS-5RK/A in the presence of BoNT-C1 (compare Aii with Ai). Scale bar, 5 mm. Bottom, Normalized fluorescence intensity profiles of the above cells indicating PM or cytosolic expression. The fluorescence profiles were determined from line scans (red lines, top) taken from the outside to the middle of each cell. B–E, Representative amperometric recordings of catecholamine release (left), total charge release (middle), and average number of events (right) for spontaneous and evoked release from either native PC12 cells (control, B; t₍₁₉₎ = 2.72, *p = 0.012 and t₍₁₉₎ = 3.98, **p = 0.001, for total charge release and average number of events, respectively; paired t test) or those transfected with BoNT-C1 and cotransfected with either BoNT-C1-resistant CSYS (C; t₍₁₉₎ = 2.22, *p = 0.038 and: t₍₁₉₎ = 2.1, *p = 0.05, for total charge release and average number of events, respectively; paired t test) or BoNT-C1-resistant CSYS-5RK/A (D), before and after application of high K⁺ depolarization by a 10 s pressure pulse (horizontal line) through a micropipette positioned close to the cell. Ea, An amperometric spike example. Eb–Ed, Single-spike analysis of the cells in A–C: mean half-width (Eb), mean peak amplitude (Ec), and mean quantal size (Ed) of individual events.

Figure 5.

5RK neutralization mutation diminishes evoked release. Cumulative distribution of spike frequency (A) or quantal release (B) normalized to the total number of spikes or to the total quantal release, respectively, in all cells in Figure 4B and C. Each point was normalized to the total number of spikes or to the total quantal release in each group before and after application of high K⁺ solution (horizontal line). The cumulative distribution of each plot was fitted with a linear function; r² (A) = 0.98 and 0.99, r² (B) = 0.97 and 0.98 for CSYS and CSYS-5RKA, respectively.

Figure 6.

YQ neutralization mutant, CSYS-YQ/AA, does not affect the secretion phenotype. A, Average number of events for spontaneous and evoked release per cells transfected with BoNT-C and either CSYS or CSYS-YQ/AA, both of which are resistant to BoNT-C cleavage (CSYS t₍₁₆₎ = 2.89, *p = 0.011; CSYS-YQ/AA t₍₁₃₎ = 2.17, *p = 0.048; paired t test:). B, Cumulative distribution of spike frequency in the cells of A, normalized to the total number of spikes in each group after 120 s of recording; 60 s in low K⁺ (5 mm K⁺) and 60 s after 10 s application of high K⁺ (100 mm) solutions (indicated by a line).

Figure 7.

3D-alignment-based reconstitution of Syt1-SNARE model. A, SNARE complex protein alignment based on magic fit (Swiss-prot viewer) derived from the pdb files 5ccg.pdb (containing the Syt-SNARE complex without the linker and transmembrane regions of Syx and Syb2) and 3ipd.pdb (containing the cis-SNARE complex with linker and transmembrane regions of Syx and Syb2). Aa, Superimposition of SNARE complex proteins (red/purple, Syx; gray/yellow, SNAP25; dark/light blue, Syb2) together with the C2B domain of Syt1 (green). Ab, Underlined alignment of Syx helices from 5ccg.pdb (purple) and 3ipd.pdb (red) files. Amino acid stretches of Syx used in 5ccg.pdb (purple) and 3ipd.pdb (red) files are denoted. The root mean square deviation of the alignment was 0.95Å. B, A reconstituted Syt1-SNARE complex with an electrostatic surface for the Syx linker region, alone (Ba) and together with the Syb2 linker region (Bb). Stick model representing amino acids of the linker regions of Syx and Syb2 (red and blue, respectively). C, Close-up views of the electrostatic surface of the linker region (the electrostatic potential was visualized within a −5 to 5 kT/e range) composed of Syx, alone (Ca) and together with Syb2 (Cb), in longitudinal projection along the helices (left) and projection through a cross-section (black bars) at the Syx-K264 level, looking toward the N-termini of Syx and Syb2 (right). D, Electrostatic surface relevant for synaptotagmin-SNARE complex (reconstituted based on the cis-SNARE complex model; A) interaction with presynaptic PM and a vesicular membrane, in longitudinal (Da) and cross sectional (Da, black bar; Db) projections. The linker region of Syx and the polybasic region of Syt1 are denoted to emphasize the similar orientations of their positive charges with respect to the PM.