Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Apr 20;120(8):1367-1377.
doi: 10.1016/j.bpj.2021.02.005. Epub 2021 Feb 12.

Cholesterol stabilizes recombinant exocytic fusion pores by altering membrane bending rigidity

Lanxi Wu  1 , Kevin C Courtney  1 , Edwin R Chapman  2
Affiliations

Cholesterol stabilizes recombinant exocytic fusion pores by altering membrane bending rigidity

Lanxi Wu et al. Biophys J. .

Abstract

SNARE-mediated membrane fusion proceeds via the formation of a fusion pore. This intermediate structure is highly dynamic and can flicker between open and closed states. In cells, cholesterol has been reported to affect SNARE-mediated exocytosis and fusion pore dynamics. Here, we address the question of whether cholesterol directly affects the flickering rate of reconstituted fusion pores in vitro. These experiments were enabled by the recent development of a nanodisc⋅black lipid membrane recording system that monitors dynamic transitions between the open and closed states of nascent recombinant pores with submillisecond time resolution. The fusion pores formed between nanodiscs that bore the vesicular SNARE synaptobrevin 2 and black lipid membranes that harbored the target membrane SNAREs syntaxin 1A and SNAP-25B were markedly affected by cholesterol. These effects include strong reductions in flickering out of the open state, resulting in a significant increase in the open dwell-time. We attributed these effects to the known role of cholesterol in altering the elastic properties of lipid bilayers because manipulation of phospholipids to increase membrane stiffness mirrored the effects of cholesterol. In contrast to the observed effects on pore kinetics, cholesterol had no effect on the current that passed through individual pores and, hence, did not affect pore size. In conclusion, our results show that cholesterol dramatically stabilizes fusion pores in the open state by increasing membrane bending rigidity.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Illustration of the experimental setup and a representative recording of an individual fusion pore. (a) Schematic drawing of a fusion pore measured in the nanodisc·black lipid membrane (ND·BLM) assay system. The layout is shown in the left panel; the middle and right panels depict the binding and assembly of trans-SNARE complexes and the opening of a fusion pore. The precise structure of the subsequent reversible, closed state is unknown, but flickers to this state are likely mediated by folding transitions of trans-SNARE complexes (12,15). (b) Representative current histogram showing the open (O) and closed (C) states of a single fusion pore formed by trans-SNARE complexes. (c) Sample trace depicting how the open and closed states were defined. After fusion pore opening, any current values lower than 50% of the open-state current value are defined as closures. The time between two closures is defined as the open time, and the time that the current remains in the closed state is defined as the closed time.
Figure 2
Figure 2
Cholesterol stabilizes the open state of fusion pores without changing current amplitude. (a) Sample traces are given showing the effects of cholesterol on fusion pore dynamics in BLMs comprising PC or PC:PE:PS. (b) Closed and open dwell time distributions from BLMs composed of PC (n = 8 individual fusion pores, formed using three independent preparations of NDs), PC-cholesterol (n = 8 fusion pores from three batches of NDs), PC:PE:PS (n = 11 pores from three batches of NDs), and PC:PE:PS-cholesterol (n = 11 pores from three batches of NDs) are shown. (c) Closures per 100 s for fusion pores formed in the indicated lipid compositions (four to six 100 s epochs were taken from each individual recording; this sampling approach applies to all subsequent analysis of closures per 100 s) are shown. (d) Open probability for fusion pores formed in the indicated lipid compositions is shown. (e) Single pore current values were plotted for each of the indicated BLM lipid compositions. Data are shown as mean ± SEM. Significance was determined using a Kolmogorov-Smirnov test or Student’s t-test as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 3
Figure 3
Real-time incorporation of cholesterol into ND⋅BLM fusion pores using MβCD-cholesterol. (a) Sample traces showing the effects of cholesterol incorporation on fusion pore dynamics are given. Cholesterol was incorporated into the PC:PE:PS BLM, after fusion pore opening by adding MβCD-cholesterol to the cis chamber. (b) Closed and open dwell time distributions of fusion pores before and after the addition of 1 or 5 pM MβCD-cholesterol (n = 5 pores, from three batches of NDs) are shown. (c) Pore closures per 100 s, before and after the addition of MβCD-cholesterol, are shown. (d) Open probability before and after the addition of MβCD-cholesterol is shown. (e) Current amplitude of opened fusion pores before and after the addition of MβCD-cholesterol. Data are shown as mean ± SEM. Significance was determined using a Kolmogorov-Smirnov test or Student’s t-test as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 4
Figure 4
Destabilization of fusion pores upon extraction of cholesterol. (a) Sample traces showing the effects of cholesterol extraction on fusion pore dynamics are given. MβCD was used to extract cholesterol from a PC:PE:PS:cholesterol BLM. (b) Closed and open dwell time distributions of fusion pores before and after the addition of MβCD (n = 5 pores from three batches of NDs) are shown. (c) Pore closures per 100 s before and after the addition of MβCD are shown. (d) Open probability of fusion pores before and after the addition of MβCD is shown. (e) Current amplitude of fusion pores before and after the addition of MβCD is shown. Data are shown as mean ± SEM. Significance was determined using a Kolmogorov-Smirnov test or Student’s t-test as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 5
Figure 5
Membrane rigidity alters fusion pore dynamics. (a) Sample traces showing the effects of phospholipid acyl chain structure on individual pores are given. (b) Closed and open dwell time distributions of pores formed with BLM lipid composed of PC:DOPE:DOPS (n = 11 pores from three batches of NDs) and PC:POPE:POPS (n = 10 pores from three batches of NDs) are shown. (c) Closures per 100 s with the indicated BLM lipid composition are shown. (d) Open probability at the indicated BLM lipid composition is shown. (e) Plot of pore currents obtained using the indicated BLM lipid compositions is shown. Data are shown as mean ± SEM. Significance was determined using a Kolmogorov-Smirnov test or Student’s t-test as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Wickner W., Schekman R. Membrane fusion. Nat. Struct. Mol. Biol. 2008;15:658–664. - PMC - PubMed
    1. Brunger A.T. Structure and function of SNARE and SNARE-interacting proteins. Q. Rev. Biophys. 2005;38:1–47. - PubMed
    1. Weber T., Zemelman B.V., Rothman J.E. SNAREpins: minimal machinery for membrane fusion. Cell. 1998;92:759–772. - PubMed
    1. Jahn R., Fasshauer D. Molecular machines governing exocytosis of synaptic vesicles. Nature. 2012;490:201–207. - PMC - PubMed
    1. Sutton R.B., Fasshauer D., Brunger A.T. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature. 1998;395:347–353. - PubMed

Publication types

LinkOut - more resources

-