Small molecules demonstrate the role of dynamin as a bi-directional regulator of the exocytosis fusion pore and vesicle release.
Hormones and neurotransmitters are stored in specialised vesicles and released from excitable cells through exocytosis. During vesicle fusion with the plasma membrane, a transient fusion pore is created that enables transmitter release. The protein dynamin is known to regulate fusion pore expansion (FPE). The mechanism is unknown, but requires its oligomerisation-stimulated GTPase activity. We used a palette of small molecule dynamin modulators to reveal bi-directional regulation of FPE by dynamin and vesicle release in chromaffin cells. The dynamin inhibitors Dynole 34-2 and Dyngo 4a and the dynamin activator Ryngo 1-23 reduced or increased catecholamine released from single vesicles, respectively. Total internal reflection fluorescence (TIRF) microscopy demonstrated that dynamin stimulation with Ryngo 1-23 reduced the number of neuropeptide Y (NPY) kiss-and-run events, but not full fusion events, and slowed full fusion release kinetics. Amperometric stand-alone foot signals, representing transient kiss-and-run events, were less frequent but were of longer duration, similarly to full amperometric spikes and pre-spike foot signals. These effects are not due to alterations in vesicle size. Ryngo 1-23 action was blocked by inhibitors of actin polymerisation or myosin II. Therefore, we demonstrate using a novel pharmacological approach that dynamin not only controls FPE during exocytosis, but is a bi-directional modulator of the fusion pore that increases or decreases the amount released from a vesicle during exocytosis if it is activated or inhibited, respectively. As such, dynamin has the ability to exquisitely fine-tune transmitter release.
Discipline of Human Physiology, Centre for Neuroscience, Flinders University, Adelaide, Australia.
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J
Jackson
Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, Queensland, Australia.
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A
Papadopulos
Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, Queensland, Australia.
F A
FA
Meunier
Centre for Chemical Biology and Chemistry, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, New South Wales, Australia.
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A
McCluskey
Children's Medical Research Institute, University of Sydney, Westmead, New South Wales, Australia.
P J
PJ
Robinson
1] Discipline of Human Physiology, Centre for Neuroscience, Flinders University, Adelaide, Australia [2] South Australian Health and Medical Research Institute (SAHMRI), Adelaide, Australia.
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DJ
Keating
D000818:Animals; D002395:Catecholamines / Q000378:metabolism; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism; D003487:Cyanoacrylates / Q000494:pharmacology; D034281:Dynamins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D006835:Hydrazones / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D009284:Naphthols / Q000494:pharmacology; D009478:Neuropeptide Y / Q000378:metabolism; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism*; D020032:Tyrphostins / Q000494:pharmacology
D002395:Catecholamines; D003487:Cyanoacrylates; D006835:Hydrazones; D007211:Indoles; D009284:Naphthols; D009478:Neuropeptide Y; C000601112:Ryngo 1-23; D020032:Tyrphostins; C568360:dyngo-4a; C000597838:dynole 34-2; D034281:Dynamins