Review
The Rockefeller University Press $30.00
J. Gen. Physiol. 2017 Vol. 149 No. 3 301–322
https://doi.org/10.1085/jgp.201611724
The Journal of General Physiology
301
Introduction
The packaging of signaling molecules into vesicles
gives exocytosis an intrinsically quantal character. In
its simplest form, the fusion of a vesicle with the plasma
membrane can be viewed as complete and instanta-
neous. Very rapid release can raise the concentration
of a signaling molecule to high levels around the re-
lease site at the cell surface, creating an extremely
steep gradient that collapses rapidly as signaling mole-
cules spread out by diffusion. However, release is often
graded and slow; a vesicle can release a variable por-
tion of its content over an extended period of time.
Partial release makes exocytosis subquantal, and slow
release keeps the extracellular concentration from
getting very high. Furthermore, most secretory vesicles
contain multiple signaling molecules that may be re-
leased in different proportions and at different rates
depending on the triggering signal.
The speed with which signaling molecules are se-
creted is determined by the fusion pore, which pro-
vides a narrow aqueous connection from the vesicle
lumen to the extracellular space. As essential kinetic
intermediates of membrane fusion, fusion pores are
the natural object of studies of molecular mecha-
nisms. For these reasons, fusion pores have been the
subject of intense interest for many years (Monck and
Fernandez, 1994; Lindau and Almers, 1995; Lindau
and Alvarez de Toledo, 2003; Harata et al., 2006; Jack-
son and Chapman, 2008; Sørensen, 2009; Vardjan et
al., 2009). A fusion pore must be quite narrow when
it rst forms, but it can expand to allow the vesicle
membrane to merge with the plasma membrane. Tun-
ing exocytosis can be achieved by stabilizing the pore
in an intermediate state, by grading the speed of fu-
sion pore expansion, by shrinking the fusion pore, or
by closing the fusion pore to recover and recycle the
vesicle. Synapses can achieve submillisecond speed
and precise timing by opening and expanding fusion
pores rapidly. In contrast, endocrine functions rarely
depend on rapid release. Hormone diffusion through
tissue, organ, or organism can take seconds or lon-
ger, so nothing is lost by spreading exocytosis out over
time scales of hundreds of milliseconds or seconds.
Endocrine release is tailored for exibility and con-
trol; synaptic release is tailored for speed. Both neu-
rons and endocrine cells use a canonical molecular
complex (Walch-Solimena et al., 1993; Martin, 1994),
and it is fascinating that this basic complex can be
adapted to such a wide range of functions.
Distinct functional needs translate into require-
ments for fusion pores with specic structures, perme-
abilities, and dynamic behavior. This article presents
current knowledge about fusion pores with an empha-
sis on their molecular composition, dynamic transi-
tions, and functional roles. Clues about fusion pores
come from many sources, and a wide range of exper-
imental approaches have provided glimpses into dif-
ferent aspects and properties. We will survey fusion
pore structure, composition, and dynamics and em-
phasize the diversity of forms and behavior. Wher-
ever possible, we will discuss the molecular basis and
functional implications of fusion pore properties and
diversity. Finally, we present a detailed analysis of syn-
aptic release to illustrate the impact of pore state on
biological function.
Ca
2+
-triggered exocytosis functions broadly in the secretion of chemical signals, enabling neurons to release
neurotransmitters and endocrine cells to release hormones. The biological demands on this process can vary
enormously. Although synapses often release neurotransmitter in a small fraction of a millisecond, hormone re-
lease can be orders of magnitude slower. Vesicles usually contain multiple signaling molecules that can be re-
leased selectively and conditionally. Cells are able to control the speed, concentration profile, and content
selectivity of release by tuning and tailoring exocytosis to meet different biological demands. Much of this regu-
lation depends on the fusion pore—the aqueous pathway by which molecules leave a vesicle and move out into
the surrounding extracellular space. Studies of fusion pores have illuminated how cells regulate secretion. Fur-
thermore, the formation and growth of fusion pores serve as a readout for the progress of exocytosis, thus reveal-
ing key kinetic stages that provide clues about the underlying mechanisms. Herein, we review the structure,
composition, and dynamics of fusion pores and discuss the implications for molecular mechanisms as well as for
the cellular regulation of neurotransmitter and hormone release.
Fusion pores and their control of neurotransmitter and hormone release
Che-WeiChang, Chung-WeiChiang, and MeyerB.Jackson
Department of Neuroscience, University of Wisconsin–Madison, Madison, WI 53705
© 2017 Chang et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the
publication date (see http ://www .rupress .org /terms /). After six months it is available under
a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International
license, as described at https ://creativecommons .org /licenses /by -nc -sa /4 .0 /).
Correspondence to Meyer B. Jackson: mbjackso@wisc.edu
Abbreviation used: TMD, transmembrane domain.
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Fusion pores and their control of exocytosis | Chang et al.302
The fusion landscape
Fusion pores should be viewed within the broader
framework of mechanisms of exocytosis. To fuse, a ves-
icle must contact the plasma membrane, and this con-
tact must undergo structural transitions. Fig. 1
illustrates the key intermediates that have been in-
voked, starting with a vesicle touching the plasma
membrane (state 1). A contact becomes more intimate
by establishment of a protein connection in which the
proteins could be separate (state 2a) or associated
(state 2b). State 2b could be viewed as a closed fusion
pore, which could open to a proteinaceous pore (state
3). These structures require transmembrane domains
(TMDs) of proteins in the vesicle and plasma mem-
brane. A connection represented in state 2 could also
evolve to a lipid stalk (state 4a), which can expand to a
hemifusion diaphragm (state 4b). A lipidic fusion
pore (state 5) is an essential intermediate in any model
of membrane fusion, and its immediate precursor
could be either state 3 or state 4b, or possibly even
state 2, although this last route is more speculative. In
a lipidic pore, the two membranes have fused, but the
process is actually not complete at this point. Full
merger to a single at membrane requires that the li-
pidic pore expand (state 6). Possible transitions be-
tween these various states are indicated by arrows in
Fig.1, and most of these transitions are thought to be
reversible. Expansion of state 5 to state 6 can also be
reversed by a budding process performed by mem-
brane curvature–inducing proteins, but reversal at this
stage is irrelevant to the present focus. States 3 and 5
are fusion pores, and states 2 and 4 can become fusion
pores. So fusion pores are where the action is with re-
gard to mechanistic studies. The landscape illustrated
in Fig. 1 serves as a useful road map to the present
discussion, and experimental studies of fusion can be
interpreted in terms of these hypothesized structures.
Structure
There have been many attempts to use electron mi-
croscopy to observe the initial steps of fusion during
exocytosis. The smallest pores captured between fusing
vesicles and the plasma membrane have diameters of
8–20 nm (Chandler, 1991). Freeze fracture images show
pores with a range of shapes and possibly without par-
ticles (no protein). There can be little doubt that even
the smallest of these pores seen in the electron micro-
scope consist of a contiguous lipid bilayer that curves
smoothly to join the two fusing membranes (Fig. 1,
state 5). However, the initial fusion pores inferred from
conductance measurements must be smaller, with di-
ameters <1 nm (discussed below in Transport proper-
ties–Fusion pore conductance). Whether these pores
are composed of lipid bilayer (Fig.1, state 5) or protein
(Fig.1, state 3) remains unresolved.
Stimulation of synapses or endocrine cells generally
induces fusion of only a small fraction of the available
vesicles, so fusion pores are rare kinetic intermediates.
This together with generally short lifetimes makes it
especially difcult to capture fusion pores for struc-
Figure 1. Putative intermediates of membrane fusion and their transitions. Transitions that are less relevant or speculative are in-
dicated by dashed arrows. Protein elements are colored green. (1) A vesicle approaches the plasma membrane. (2) Proteins hold the
vesicle and plasma membrane together, either through separate contacts (a) or through one central contact (b). (3) A proteinaceous
fusion pore could form from a central contact as in 2b. (4) Lipid mixing of the outer (proximal) leaflets can begin, first through the
formation of a stalk (4a) and then through the formation of an extended hemifusion diaphragm (4b) in which the fused proximal leaf-
lets retract and leave a bilayer formed by the two distal leaflets. (5) A fusion pore formed by a contiguous lipid bilayer curved into an
hourglass-like shape. (6) A greatly expanded lipid fusion pore on the way to complete merger of the plasma and vesicle membranes.
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303JGP Vol. 149, No. 3
tural work, and there are no structures of initial fusion
pores from biological samples. So far, what little struc-
tural insight we have of early-stage pores was derived
from studies of model systems with dened chemical
compositions. The vesicle proteins synaptophysin and
synaptobrevin associate into hetero-oligomers, and
these complexes have been puried and studied with
negative stain electron microscopy. Because one of the
TMDs of synaptophysin is homologous to a connexin
TMD, the gap junction Cx26 crystal structure was used
to build a model in which 6 synaptophysin molecules
and 12 synaptobrevin molecules form a ring-like ar-
rangement (Fig.2A). The relevance of this structure
to exocytosis remains unclear, but it has remarkable
parallels with a model based on amperometric mea-
surements of fusion pore ux in cells expressing syn-
aptobrevin TMD mutations (Fig.4B). The model of
the synaptobrevin–synaptophysin complex places syn-
aptobrevin in two different congurations, with TMDs
oriented differently relative to the pore axis. Of the
four residues of the synaptobrevin TMD implicated as
pore liners by amperometry (Chang et al., 2015), two
face into the pore lumen for one of the synaptobre-
vins in the structure, and the other two residues face
into the lumen in the other synaptobrevin in the struc-
ture (Fig.2B).
The model in Fig.2 suggests that synaptophysin forms
part of the fusion pore. Synaptophysin forms channels
in lipid bilayers (Thomas et al., 1988), but a function in
fusion pores was thought to be unlikely when a synapto-
physin knockout was shown to have normal synaptic re-
lease (McMahon et al., 1996). Without synaptophysin,
synapses have slower endocytosis (Kwon and Chapman,
2011), and in endocrine cells, synaptophysin–dynamin
interactions modulate release at a step downstream
from the initial fusion pore (González-Jamett et al.,
2010). However, genetic studies with synaptophysin are
complicated by the presence of three homologous pro-
teins that could substitute for synaptophysin (Arthur
and Stowell, 2007). Recent amperometry experiments
in chromafn cells have demonstrated that molecular
manipulations of synaptophysin produce signicant
changes in fusion pore properties (unpublished data).
Although the synaptophysin–synaptobrevin model in
Fig.2 is intriguing, it implies a pore that is too large. A
total of 24 TMDs line the putative pore, including 12
molecules of synaptobrevin. Estimates of the number of
SNA RE molecules required for fusion vary widely but
are generally much lower (discussed below in Composi-
tion–SNA RE number). Fusion pore conductance mea-
surements (also discussed below in the same section)
indicate that the pore could be lined by as few as ve to
eight TMDs. This makes it unlikely that the structure in
Fig.2 forms the initial fusion pore of exocytosis. How-
ever, the parallel with amperometry results are intrigu-
ing, and the capacity of synaptobrevin and synaptophysin
TMDs to come together is probably relevant. If the
TMD–TMD interfaces between these two proteins are
Figure 2. A structural model of a synaptophysin–synaptobrevin complex. The model was based on a study by Adams et al.
(2015) and generated with PyMOL using a pdb file provided by M. Stowell. (A) The complete complex viewed from the vesicle lumen
shows 6 synaptophysins (green) and 12 synaptobrevins (red) in a hexagonal formation. 12 synaptophysin TMDs and 12 synaptobre-
vin TMDs face inward and could line a fusion pore. (B) The TMDs of two synaptophysin and two synaptobrevin molecules are viewed
from within the plane of the membrane. Residues highlighted in yellow were implicated as pore liners by amperometry experiments
with synaptobrevin TMD mutants (Chang et al., 2015); synaptobrevin residues 99 and 103 are highlighted on one chain, and residues
101 and 105 are highlighted on the other (compare with Fig.4B).
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Fusion pores and their control of exocytosis | Chang et al.304
exible, then smaller complexes and pores with fewer
TMDs may be possible.
Transport properties
A fusion pore provides a conduit for transport, and
most of what we know about fusion pores derives from
measurements of some form of ux. Ions and signal-
ing molecules can ow through the aqueous pore
lumen, and membrane can ow along the pore walls.
The transport of ions, signaling molecules, and mem-
brane can be measured and used to gain insight into
fusion pore structure and composition. Fig. 3 illus-
trates how patch clamp measurements of conductance
and amperometry measurements of content ux sig-
nal the evolution of the fusion pore. These two tech-
niques both report a nascent pore that supports low
but detectable transport. Each measurement reveals a
sequence of steps beginning with pore opening and
continuing with pore expansion.
Fusion pore conductance. Fusion pore conductance
can be determined with various electrical recording
congurations (Breckenridge and Almers, 1987; Zim-
merberg et al., 1987; Lindau and Neher, 1988; Lindau
and Alvarez de Toledo, 2003). One must measure the
complex impedance of a membrane and interpret the
results with the aid of an equivalent circuit consisting
of a fusing vesicle and its pore in parallel with a patch
of membrane. An initial increase in conductance con-
comitant with an increase in capacitance provides in-
formation about the nascent fusion pore, and ensuing
increases provide a readout of the progress of fusion
over time (Fig.3).
Fusion pore conductance is related to pore geometry,
and the simplest way to envisage this relationship is to
hypothesize that a fusion pore behaves like the solution
that lls it. This involves using the macroscopic expres-
sion for the conductance, γ, of an element of solution.
A pore with length,
l
, and constant cross-sectional area,
A
, has a conductance of
γ =
A
__
ρl
. (1)
ρ is the resistivity of the aqueous solution lling the
pore (ρ ∼100 Ω cm for standard physiological saline). It
is common to take
l
as roughly twice the thickness of a
lipid bilayer (∼10 nm). If the pore is cylindrical with
radius
r
, then
A
is π
r
2
, and we can use the conductance
to estimate
r
. This expression has been tested with ion
channels, and with γ ∼1 nS, calculations with Eq. 1 using
structural estimates of
A
and
l
give conductance values
roughly three times higher than experimental measure-
ments (Jackson, 2006). The error is much larger for
smaller channels. The main reason for this discrepancy
is that Eq. 1 neglects interactions between the ions and
the walls of the pore and surrounding membrane. Ac-
tual fusion pores are likely to be wider than estimates
based on conductance, so Eq. 1 provides a lower limit
for
r
. One might hope to have more condence in Eq.
1 for very high conductances, but then another prob-
Figure 3. Impedance and amperometry
measurements of fusion pores can be inter-
preted in terms of three successive stages
of membrane fusion. (1) Contact (top left),
(2) fusion pore opening (top middle), and (3)
fusion pore expansion (top right). (left) Imped-
ance recording reveals fusion pore openings
as a change in the complex impedance of a
patch of membrane to which a vesicle fuses.
The imaginary component of the impedance
(blue) and real component (red) are used
to calculate the fusion pore conductance, γ
(green; Lollike et al., 1995). The opening of
a fusion pore produces an initial conductance
increase, and fusion pore expansion increases
the conductance further. (right) Amperometry
recording reveals a fusion pore opening as a
pre-spike foot (shaded), which represents the
flux of catecholamine out of the vesicle at a
limited rate. Fusion pore expansion allows
content release much more rapidly to pro-
duce an amperometric spike.
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305JGP Vol. 149, No. 3
lem arises: the conductance of the approach to the pore
must be included. Eq. 1 then must incorporate an addi-
tive term that is proportional to
r
rather than
A
(Hille,
1992; Nanavati et al., 1992; Jackson, 2006).
Despite the reservations just discussed regarding the
quantitative dependence of conductance on radius, the
wide range of values for fusion pore conductance in-
dicates that fusion pore sizes also vary. Conductances
vary by over two orders of magnitude, ranging from
∼20 pS (Klyachko and Jackson, 2002) to several nanos-
iemens (Monck et al., 1990; Spruce et al., 1990; Nana-
vati et al., 1992; Curran et al., 1993). Thus, pore radii
may vary by roughly one order of magnitude. In the
same patch of membrane of nerve terminals, fusion of
small synaptic-like vesicles and large dense-core vesicles
have fusion pore conductances differing by a factor
of ∼10 (Klyachko and Jackson, 2002). The large con-
ductances of endocrine fusion pores have been inter-
preted in terms of a lipidic pore (Fig.1, state 5) using
a continuum elasticity model based on the mechanical
properties of lipid bilayers (Nanavati et al., 1992). Vesi-
cles fusing with membranes in the absence of proteins
produce pores with large conductances that uctuate
very widely (Chanturiya et al., 1997). Theoretical work
on the shape of fusion pores formed by an elastic lipid
bilayer suggests that their radii range from 1.1 to 4.2
nm (Jackson, 2009). Pores of this size should have con-
ductances in the nanosiemens range. So larger fusion
pore conductances are consistent with the elastic prop-
erties of a lipid bilayer, but smaller fusion pore conduc-
tances are not. Smaller pores are therefore less likely
to be purely lipidic. Conductances overlapping with
the range of ion channels was one of the earliest argu-
ments for a proteinaceous pore (Almers, 1990; Lindau
and Almers, 1995; Lollike et al., 1995). Furthermore,
ion channel conductances generally do not uctuate
much, so the greater stability of small conductance fu-
sion pores points to a proteinaceous structure.
Content flux. Amperometry provides the most sensitive
measurement currently available for ux of vesicle con-
tent through a fusion pore. This electrochemical tech-
nique detects readily oxidized molecules contained in
many vesicles such as catecholamines, histamine, dopa-
mine, and serotonin. The loss of content from a single
vesicle is readily observed (Wightman et al., 1991), and
a single-vesicle release event progresses through distinct
stages. First comes the “pre-spike foot” that reports ux
through an initial fusion pore (shaded region of the
amperometry trace in Fig.3; Chow et al., 1992; Jankow-
ski et al., 1993). The spike comes next when the pore
starts to expand. Kiss-and-run exocytosis also is accom-
panied by ux through a fusion pore (Alvarez de To-
ledo et al., 1993; Wang et al., 2003, 2006), and the
implications for pore closure are discussed below (Dy-
namic properties–Closure).
Because vesicles often contain multiple signaling
molecules with different sizes, fusion pores can act as
lters to select which molecules will pass. The mole-
cules detected by amperometry are generally small
with molecular masses <200 D. Permeation of the ini-
tial pore by molecules of this size ts with the ∼1-nm
dimensions of pores inferred from conductance mea-
surement. The vesicles of pancreatic β cells contain
ATP and GABA and can be loaded with exogenous se-
rotonin. By expressing GABA
A
receptors and puriner-
gic P
2
X
2
receptors, the release of ATP and GABA can
be monitored as an ion current through the plasma
membrane (Braun et al., 2007). ATP, GABA, and sero-
tonin all were found to pass through the initial fusion
pore during β cell exocytosis, but at different rates. The
ux was greatest for the smallest molecule, GABA, and
lowest for the largest molecule, ATP, consistent with a
ltering action by an ∼1.4-nm pore.
N
-methyl--glu-
camine is also small and should pass through fusion
pores easily, but passage of this molecule could not be
seen in pores formed during SNA RE-mediated fusion
of nanodiscs with cells (Wu et al., 2016). These fusion
pores may be different from dense-core vesicle fusion
pores, but it is also possible that factors other than size
have an impact on permeability.
The most pronounced ltering appears with pep-
tides, both between different sized peptides and be-
tween peptides versus smaller molecules. As soon as a
fusion pore opens it allows pH to equilibrate, but vesi-
cles begin to lose their peptides with a delay of about a
second after pH equilibration (Barg et al., 2002; Tsuboi
and Rutter, 2003). The smaller peptide neuropeptide Y
is released very rapidly, and the larger peptide tissue
plasminogen activator is released very slowly, even when
the two are contained in the same vesicle (Perrais et al.,
2004). The vesicles of chromafn cells contain both cat-
echolamine and large peptide hormones. Chromafn
cells release norepinephrine in response to weak stimu-
lation, but stronger stimulation elicits release of both
catecholamine and the large peptide chromogranin
(Fulop et al., 2005). Thus, with weak stimulation the fu-
sion pore remains in its initial narrow state, and strong
stimulation drives its expansion to a wider state. Like-
wise, stronger stimulation of β cells triggers fusion pore
expansion to allow insulin release (MacDonald et al.,
2006). In lactotrophs, weak stimulation opens a small
pore that only allows pH to equilibrate, whereas stron-
ger stimulation allows larger uorescent tracers to pass
(Vardjan et al., 2007). These various experiments sug-
gest that pore size is a critical determinant in selecting
which signaling molecules are secreted and that the
mode of release depends on the form of stimulation.
However, as a cautionary note, it was found that subtle
variations in the structures of uorescently tagged pep-
tides had a profound impact on release kinetics (Mi-
chael et al., 2004). These results cannot be explained by
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