Snare Proteins Entropically Expand Membrane Fusion Pores

Abstract

Neurotransmitters and hormones are released through fusion pores whose dynamics are a critical determinant of the release amount and rate. Fusion pores may flicker repeatedly, before resealing (“kiss & run” fusion) or dilating irreversibly (full fusion). These dynamics are physiologically regulated, but the mechanisms are not understood. Prior studies either monitored single exocytotic pores in live cells with uncontrolled biochemistry, or used in vitro reconstitutions lacking single pore sensitivity. The unavailability of key parameters has hampered development of meaningful models to describe pore dynamics.Here we combine mathematical modeling and single pore measurements to investigate regulation of pore dilation. Experimentally, we measured individual pore conductances with controlled SNARE copy number for the first time. Reconstituting v-SNAREs into ∼23 nm diameter nanolipoprotein particles (discs), we fused discs with patch-clamped cells expressing complementary t-SNAREs and measured currents.We found that SNARE numbers dramatically affected pore size. Just ∼2 SNAREs nucleated pores, but with increasing numbers of SNAREs pores became progressively more dilated. We determined pore size distributions and free energy versus pore size.We developed a coarse-grained mathematical model that reproduced the experimental free energy profiles. Free energies were averaged over fusion pore shapes and configurations of SNARE complexes which could zipper at the pore waist or roam unzippered. Protein-free pores resisted expansion with force ∼19 pN due to membrane tension and bending, close to the experimental value. With SNAREs, zippering drove SNARE assembly at the pore, where SNARE crowding generated entropic pore expansion forces, since bigger pores gave zippered SNAREs more entropy. With 15 SNAREs the entropic expansion force of 14 pN was close to the 19 pN resistance.Our results suggest that SNAREs critically regulate pores post-fusion by entropic expansion forces. Several SNAREs are required to fully dilate pores for efficient contents release.