Subnanometer fusion pores in spontaneous exocytosis of peptidergic vesicles.
Kiss-and-run exocytosis, consisting of reversible fusion between the vesicle membrane and the plasma membrane, is considered to lead to full fusion after stimulation of vesicles containing classical transmitters. However, whether this is also the case in the fusion of peptidergic vesicles is unknown. Previously, we have observed that spontaneous neuropeptide discharge from a single vesicle is slower than stimulated release, because of the kinetic constraints of fusion pore opening. To explore whether slow spontaneous release also reflects a relatively narrow fusion pore, we analyzed the permeation of FM 4-64 dye and HEPES molecules through spontaneously forming fusion pores in lactotroph vesicles expressing synaptopHluorin, a pH-dependent fluorescent fusion marker. Confocal imaging showed that half of the spontaneous exocytotic events exhibited fusion pore openings associated with a change in synaptopHluorin fluorescence but were impermeable to FM 4-64 and HEPES. Together with membrane capacitance measurements, these findings indicate an open fusion pore diameter <0.5 nm, much smaller than the neuropeptides. In stimulated cells, >70% of exocytotic events exhibited a larger, FM 4-64-permeable pore (>1 nm). Interestingly, capacitance measurements showed that the majority of exocytotic events in spontaneous and stimulated conditions were transient. Stimulation increased the frequency of transient events and the fusion pore dwell time but decreased the fraction of events with lowest measurable fusion pore. Kiss-and-run is the predominant mode of exocytosis in resting and in stimulated peptidergic vesicles. Stimulation prolongs the effective opening of the fusion pore and expands its primary subnanometer diameter to enable hormone secretion without full fusion.
Vardjan
Nina
N
Celica Biomedical Center, 1000 Ljubljana, Slovenia.
Stenovec
Matjaz
M
Jorgacevski
Jernej
J
Kreft
Marko
M
Zorec
Robert
R
D000143:Acids / Q000378:metabolism; D000818:Animals; D002021:Buffers; D002462:Cell Membrane / Q000502:physiology*; D022162:Cytoplasmic Vesicles / Q000502:physiology*; D005089:Exocytosis / Q000502:physiology*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006863:Hydrogen-Ion Concentration; D052682:Lactotrophs / Q000502:physiology; D008297:Male; D008561:Membrane Fusion / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D010903:Pituitary Gland, Anterior / Q000166:cytology; D011388:Prolactin / Q000378:metabolism*; D011726:Pyridinium Compounds / Q000493:pharmacokinetics; D000644:Quaternary Ammonium Compounds / Q000493:pharmacokinetics; D051381:Rats; D017208:Rats, Wistar
D000143:Acids; D002021:Buffers; C092350:FM 4-64; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D011388:Prolactin