SNARE proteins: one to fuse and three to keep the nascent fusion pore open

Abstract

Neurotransmitters are released through nascent fusion pores, which ordinarily dilate after bilayer fusion, preventing consistent biochemical studies. We used lipid bilayer nanodiscs as fusion partners; their rigid protein framework prevents dilation and reveals properties of the fusion pore induced by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor). We found that although only one SNARE per nanodisc is required for maximum rates of bilayer fusion, efficient release of content on the physiologically relevant time scale of synaptic transmission apparently requires three or more SNARE complexes (SNAREpins) and the native transmembrane domain of vesicle-associated membrane protein 2 (VAMP2). We suggest that several SNAREpins simultaneously zippering their SNARE transmembrane helices within the freshly fused bilayers provide a radial force that prevents the nascent pore from resealing during synchronous neurotransmitter release.

Fig 1

(A) Cartoon showing the v-nanodisc model. The nanodisc is a small piece of lipid bilayer wrapped by two MSP (blue). VAMP2 (green) can insert into nanodisc to form v-disc (11). The lipid head groups are shown as grey spheres. (B) Elution profile of nanodisc or v-disc on Superdex 200 10/300 GL column. Embedment of VAMP2 results in the earlier elute volume of v-disc (red major peak) as compared to that of VAMP2 free nanodisc (black peak). By gel filtration, the 6XHis-SUMO tag (cleaved from VAMP2 by SUMO-protease, the red minor peak) can also be removed. (C) SDS-PAGE gel stained with Coomassie Brilliant Blue showing the input and final nanodisc products after gel filtration. (D) V-disc samples were analyzed in an FEI Tecnai-12 electron microscope. V-discs showed regular “disc” shapes (Left panel), VAMP2 protein can hardly be seen because of small protein size and flexible structure. When soluble Syntaxin1A H3 domain and SNAP-25N/C domain were co-incubated with v-disc they form SNARE complexes that can be seen as rod-like structures (red, right panel) protruding from two sides of the v-disc (green, right panel).

Fig 2

(A) Schematics showing how the fusion pore can be envisioned. The diameter of the nanodisc is 16 nm. Lipids that naturally form flat surfaces will favor structures that have a zero net curvature (when neglecting the Gaussian curvature). Hence, in saddle-like (neck-like) structures, the positive (pore) and negative (perpendicular to the pore, seen in this panel) curvatures are of the same order. A 4 nm pore would correspond to a 6 nm curvature for the exterior of the bilayer. This is approximately what is represented here. With no stress a 1 nm pore can form. With a reasonable amount of stress (θ = 20 degrees) a 4 nm pore diameter would result. (B) Lipid mixing is SNARE specific. v-discs exchanged lipids with t-liposome (blue). Discs without VAMP2 do not fuse with t-liposomes (red), and CDV which titrates the free t-SNARE also blocked the fusion (green). (C) Calcium release is SNARE specific. 50 mM calcium is encapsulated into t-liposome. During the liposome-nanodisc fusion, the pore opens and calcium is released from the liposome to the exterior buffer, mag-fluo-4 signal is enhanced. Increasing mag-fluo-4 signal indicates calcium is continuously released during the fusion (blue), no significant calcium release is observed under non-fusogenic conditions (red and black). (D) Dithionite assay showed some NBD protection after 40 minutes fusion (blue). To completely quench all NBD signal, detergent (De) was first added to disrupt the liposomes followed by adding dithionite (Di) to get 100% quench (brown). With CDV to block the fusion, no NBD protection was observed after dithionite treatment (green).

Fig 3

(A) Elution profile of v-discs with different VAMP2 copies per disc. With more VAMP2 incorporation, v-discs eluted in smaller elution volumes on Superdex200 column. (B) Determination of the number of VAMP2 per nanodisc. The v disc samples obtained by gel filtration were analyzed by SDS-PAGE gel staining with Coomassie Brilliant Blue. The copy of VAMP2 per disc was determined by the ratio of VAMP2: MSP according to the quantification of the corresponding protein bands. Each v-disc populations have about 1.2 (ND1), 2.2 (ND2), 3.15 (ND3), 4.3 (ND4), 5.5 (ND5), 7.4 (ND7) and 9.3 (ND9) copies of VAMP2 per disc on average. (C) Lipid mixing assays demonstrate that discs with varying VAMP2 copy numbers are equally efficient in fusing with calcium encapsulated t-liposomes. (D) Calcium release assay showed different kinetics when v discs with different copy numbers of VAMP2 fuse with t calcium liposome. Calcium release is gradually increased with higher copy numbers of VAMP2 inserted into the nanodiscs. The error bars are SEM. (E) The end point values after 40′ fusion reaction are presented for both the lipid mixing (blue) and the calcium release (green). Lipid mixing, normalized by the average end point value, does not vary significantly with the number of VAMP2. Calcium release, normalized by the value for ND9, varies as a sigmoid with an inflection point at ~5 VAMP2 per nanodisc.

Fig 4

(A) Schematics showing wild type and the various chimeric VAMP2. The structure of C18, C45 (solanesyl ester) and protein sequence of VAMP2 or PDGFR TMD. C18 spans only one leaflet of the lipid bilayer, while C45 and protein TMDs cross the lipid bilayer. (B) Lipid mixing assay of t liposome with v disc prepared with wt VAMP2 or the CD VAMP2 with C18 or C45. The lipid anchor, either C18 or C45 showed compromised fusion efficiency comparing with wild type VAMP2 with native TMD. (C) Lipid mixing assay showed the TMD of VAMP2 and PDGFR have similar fusion kinetics, which suggested that these two TMD are equivalent in lipid mixing. (D) Calcium release assay demonstrated substantially reduced release when protein TMD is replaced by lipid anchor, either C18 or C45. (E) Calcium release assay reveals VAMP2 TMD is more efficient than PDGFR TMD for content release. The error bars are SEM.