In 55th Biophysics Meeting (2011 Baltimore), there was a interesting post about the SNAP-25 conformational change during the fusion pore formation and expansion, from Manfred Lindau Group.
The title is: The conformational change of SNAP25 during the exocytosis
Ying Zhao, Qinghua Fang, Adam Drew Herbst, Khajak Berberian, Kassandra Kisler, Wolfhard Almers and Manfred Lindau
Ying Zhao, Qinghua Fang, Adam Drew Herbst, Khajak Berberian, Kassandra Kisler, Wolfhard Almers and Manfred Lindau
It is widely assumed that a conformational change in the SNARE complex induces fusion pore opening and transmitter release. The SNAP-25 FRET construct SCORE (SNARE complex reporter) has CFP as FRET donor inserted at the N-terminal end of SN1 and the fluorescent protein Venus (YFP) as FRET acceptor inserted at the N-terminus of SN2. It was previously shown that SCORE that reports a FRET change in response to stimulation that is calcium dependent but is not abolished by tetanus toxin treatment, which abolishes fusion (An and Almers 2004 Science 306:1042). To determine if the conformational change reported by SCORE is directly associated with fusion we monitored SCORE fluorescence in chromaffin cells in TIRF mode while exocytotic events at the cell bottom were spatially and temporally monitored by a microfabricated electrochemical detector (ECD) array with four Pt electrodes patterned on a glass coverslip (Hafez et al. PNAS 2005 102:13879). After the SCORE-expressing cells were placed on the top of the ECD array, the amperometric currents and fluorescence images were simultaneously recorded. Based on the charge ratios of oxidation currents recorded by the four electrodes, the localization of single exocytotic events was determined. The time course of CFP and YFP fluorescence in the 4 camera pixels at the release site was extracted for 10 s time intervals before and after the amperometric spike. Averaging >300 events revealed a transient increase of YFP and a transient decrease of CFP fluorescence by ~2.5% at the time of amperometric events, The increased FRET level returned to the pre-exocytotic level within ~5s. This result directly indicates that the SNAP25 conformational change is directly associated with fusion. Supported by NIH grant R01GM085808.
