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Exocytosis from chromaffin cells:
hydrostatic pressure slows vesicle fusion
rstb.royalsocietypublishing.org
Walter Stühmer
Department of Molecular Biology of Neuronal Signals, Max Planck Institute of Experimental Medicine,
Hermann-Rein-Strasse 3, Göttingen 37075, Germany
Review
Cite this article: Stühmer W. 2015 Exocytosis
from chromaffin cells: hydrostatic pressure
slows vesicle fusion. Phil. Trans. R. Soc. B 370:
20140192.
http://dx.doi.org/10.1098/rstb.2014.0192
Accepted: 26 March 2015
One contribution of 16 to a discussion meeting
issue ‘Release of chemical transmitters from
cell bodies and dendrites of nerve cells’.
Subject Areas:
physiology
Keywords:
secretion, hydrostatic pressure, patch clamp,
kinetics, capacitance measurements
Author for correspondence:
Walter Stühmer
e-mail: [email protected]
Pressure affects reaction kinetics because chemical transitions involve changes
in volume, and therefore pressure is a standard thermodynamic parameter
to measure these volume changes. Many organisms live in environments at
external pressures other than one atmosphere (0.1 MPa). Marine animals
have adapted to live at depths of over 7000 m (at pressures over 70 MPa),
and microorganisms living in trenches at over 110 MPa have been retrieved.
Here, kinetic changes in secretion from chromaffin cells, measured as capacitance changes using the patch-clamp technique at pressures of up to 20 MPa
are presented. It is known that these high pressures drastically slow down
physiological functions. High hydrostatic pressure also affects the kinetics
of ion channel gating and the amount of current carried by them, and it
drastically slows down synaptic transmission. The results presented here
indicate a similar change in volume (activation volume) of 390 + 57 Å3 for
large dense-core vesicles undergoing fusion in chromaffin cells and for degranulation of mast cells. It is significantly larger than activation volumes of
voltage-gated ion channels in chromaffin cells. This information will be
useful in finding possible protein conformational changes during the reactions
involved in vesicle fusion and in testing possible molecular dynamic models of
secretory processes.
1. Hydrostatic pressure allows measurements of activation
volumes
Stepwise changes in thermodynamic variables can yield information helpful to
gain insight into an unknown mechanism. For example, changes in temperature
allow conclusions about the energetics of chemical reactions. Another global
fundamental thermodynamic variable, albeit often neglected, is pressure. The
dependence of kinetics on pressure of a simple chemical reaction allows a direct
measurement of the volume change associated with the formation of the transient
activated state of the reaction (DV*) [1]. In other words, it is possible to measure
changes in volume as cubic Ångström per molecule (a microscopic quantity)
during transitions from one state to another simply by measuring changes in reaction rates induced by changes in external pressure (both are macroscopic
quantities). As the activated transition states have generally a larger volume
than the initial or final state, an increase in pressure leads to a slowing-down of
reaction kinetics, given that an increase in volume has to work against pressure
(which tends to decrease volume). Physiological chemical processes, such as the
gating of voltage-dependent ion channels or the fusion of vesicles necessary for
exocytosis, are affected by pressure and therefore it is possible to estimate activation volumes, and also to discriminate between possible complex molecular
reactions pathways leading to ion flow through channels or vesicle fusion.
2. Effect of hydrostatic pressure on ion channels
(a) Extrasynaptic acetylcholine receptors
As an example, figure 1 compares single-channel records obtained from extrasynaptic acetylcholine receptors recorded at atmospheric pressure (0.1 MPa)
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
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500 ms
Figure 1. Comparison of single-channel currents through extrasynaptic
acetylcholine receptors in cultured embryonic rat muscle cells at atmospheric
((a), 0.1 MPa) and 30 MPa (b), corresponding to the pressure found close to
3000 m below the water surface. Currents were recorded at a membrane
potential of 280 mV in the presence of saturating acetylcholine (20 mM).
Note the change in lifetimes of open and shut states, whereas the channel
conductance is unchanged. Adapted from Heinemann et al. [2].
and 30 MPa [2]. A significant slowing-down of gating kinetics
can be seen. However, the conductance of the channel did not
significantly change. After a temperature correction to 13.38C
for an increase of less than 28C due to compression, using the
appropriately scaled amount of change known in reaction
rate produced by a change in temperature of 108C (Q10) of
1.34 for single-channel currents, the conductance was 29.6 +
1.2 pS at 0.1 MPa, and 28.4 pS + 1.7 pS at 40 MPa. Analysing
close-times and open-times histograms at various pressures,
and correcting for temperature effects, activation volumes for
the closed and open state associated with closing (DV*c) and
opening (DV*o) the acetylcholine channel of 139 + 11 Å3 and
59 + 4 Å3, respectively, were obtained. This gives a difference
in volume between the closed and open states, DV, of 80 +
12 Å3. As closing the pore involves a larger volume than opening it, increased pressure favours the closed state. It should be
noted that these results include the activation volumes of binding and unbinding of acetylcholine. Also, activation volume in
molecular terms is rather abstract, as it includes, for example,
changes in packing of water associated with the changes in
exposure of charges to an aqueous environment.
(b) Effects of pressure on other ion channels
Many types of excitable cells are sensitive to pressure [3].
Hydrostatic pressure has profound effects on drug action,
particularly general anaesthetics. Organisms living in
oceans as well as divers are subject to large changes and/or
high hydrostatic pressures. So in addition to being a useful
tool, pressure is a parameter that needs to be considered in
physiology. Most types of voltage-gated ion channels have
pressure-dependent properties, mostly a slowing-down of
2
Phil. Trans. R. Soc. B 370: 20140192
(b)
gating kinetics. The ionic currents [4–6], and gating currents
[7] of voltage-gated sodium channels have been extensively
studied. Voltage-gated sodium channels from squid giant
axons slow their activation kinetics by a factor of about 1.6
at 62 MPa, giving a DV*o of about 58 Å3, and a DV of 26 Å3
between the closed and the open state [6]. Marine animals
are exposed to such pressures at depths of 7000 m, at which
squids, among other living organisms, have been reported.
Seven thousand metres is about one-half of the maximal
ocean depth. Comparing the slowing-down of kinetics for
the gating currents and the corresponding sodium currents,
it becomes clear that higher pressures slow down the ionic
currents more than the corresponding gating currents. This
means that in addition to an increase in volume during
gating, the opening of the ion conduction pathway requires
additional conformational rearrangements that significantly
contribute to the total activation volume, as gating currents
precede the opening of the ion channel pore. The measured
DV*g was 28 Å3. The unavoidable conclusion is that gating
and the channel opening transition are separate phenomena,
with the latter involving larger molecular rearrangements
that carry little or no charge rearrangements [7].
Voltage-gated potassium channels in neurons have a
similar slowing-down of kinetics as voltage-gated sodium currents [4,5,8,9]. Potassium channels from squid giant axons slow
their activation kinetics by a factor of about 1.9 at 41 MPa,
giving a DV*o of about 60 Å3 [9]. The voltages of activation
and inactivation of most ion channels slightly shift by a few
tens of millivolts to the right or left with pressure, and the activation volumes are slightly different at various test potentials.
In addition, the conductance for most channels studied so far
(except for the voltage-gated calcium channel CaV1.2, see
below) does not significantly change. For example, alamethicin
pores in artificial bilayers do not significantly alter their conductance at pressures of up to 100 MPa, while kinetic
analyses reveal an activation volume in the order of 100 Å3
[10]. These results indicate that ions do not change their molecular volume while transiting through the conduction pore,
which is consistent with current theories of ion permeation in
which the hydration shell of permeant ions is replaced by
fixed charges within the permeation pore. This exchange is
not expected to cause significant conformational changes,
and hence will not lead to changes in activation volumes.
However, the closed–open conformational transition of an
ion channel involves large molecular rearrangements.
Voltage-gated Ca2þ channels are relevant for secretion as
they trigger exocytosis. Heinemann et al. [11] measured
effects of pressure on voltage-gated sodium and calcium
channels in bovine chromaffin cells. The activation volumes
obtained for sodium currents (DV ¼ 33 Å3) were similar to
those reported by Conti et al. [6] for sodium channels in
squid giant axons. For Ca2þ currents, an upper limit for the
activation volume of 20 Å3 was obtained, with hardly any
effect on current amplitude. However, Aviner et al. [12] did
see a drastic slowing-down of activation kinetics with
pressure of recombinant CaV1.2 and CaV3.2 channels in
Xenopus oocytes, with concomitant strong effects on current
amplitudes. They also carefully studied inactivation kinetics
at various pressures as well as the dependence on voltage
of activation, inactivation and conductance. Most interesting
is the observation that CaV1.2 currents increased by 70 +
32% at 5 MPa, while currents through CaV3.2 channels
decreased by 27 + 5% at the same pressure. For CaV1.2, a
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2 pA
(a)
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(a)
(b)
BNC
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holder
I
pipette
reservoir
20 mm
10 mm
pressure transducer
thermistor
Figure 2. (a) Cross section of the flying-patch holder. The reservoir and external assembly can be extended and turned while establishing the whole-cell configuration in a standard patch-clamp set-up. Once the cell is patched and detached from the culture dish, the reservoir assembly is placed carefully under the pipette
and cell being patched. The flying-patch holder can then be removed from the head stage and carefully inserted into the pressure chamber (b), which is filled with
paraffin oil (via I, oil inlet). Adapted from Heinemann et al. [2].
DV of 756 Å3 was obtained, meaning that although activation of
this current will be slowed down dramatically with pressure,
the increased Ca2þ influx could well account for effects of
the High-Pressure Nervous Syndrome observed in deep-sea
divers. High-Pressure Nervous Syndrome is observed at pressures larger than about 1 MPa and is characterized by severe
impairment of basic physiological functions, including motor
and cognitive impairments [13–17].
3. Effect of hydrostatic pressure on secretion
Several reports [18,19] have pointed out that severe pressuredependent effects are detectable in synaptic transmission. A
reduction in the frequency of the spontaneous release of transmitter in the frog neuromuscular junction by a factor of two
occurred at pressures as low as 5 MPa. In addition, the amplitude of the excitatory postsynaptic potential in ganglia of
Aplysia californica was reduced to one-half at 10 MPa. These
results clearly point to a very pressure-sensitive presynaptic
event involved in vesicle fusion.
The strong dependence of CaV1.2 currents on pressure,
together with the effects described above for other ion currents,
cannot explain the various severe physiological effects
observed in whole organisms under pressure. One possible
explanation is that other voltage-gated Ca2þ channels in the
presynaptic terminal, particularly those directly involved in
presynaptic Ca2þ entry (P/Q, N or R type), are affected differently. In fact, the opposite behaviour with pressure observed in
CaV1.2 and CaV3.2 indicate that different calcium channel
types react very differently to pressure. Therefore, the release
processes of extra-somatic and somatic vesicle fusion are different in terms of activation volumes. Additionally, binding of
Ca2þ to molecules regulating the vesicle-release and the
reuptake mechanisms in extra-somatic and somatic secretion
might have very different activation volumes. See Daniels &
Grossman [20] for a comprehensive review.
There are many studies reporting how osmotic pressure
affects vesicle fusion and retrieval kinetics, but these forms of
pressures mainly induce lateral stress on the membrane, similar
to stretching the membrane in two dimensions. The magnitude
of this pressure is very limited, as otherwise the membrane
would rupture. A pressure as low as 0.3 KPa is able to split
endocytic events into two pathways, one pressure-insensitive
and another strongly pressure sensitive [21], probably because
endocytosis involves a decrease in membrane surface, which
will be contrasted by the forces stretching the membrane. By
contrast, hydrostatic pressure, which acts in three dimensions,
compresses all components isotropically, and therefore can be
studied at several tens of megapascal in living matter. Only
this kind of pressure application allows a calculation of activation volumes, although these volumes are apparent and
have to be considered as molecular rearrangements.
The patch-clamp technique [22] makes it possible to
record capacitance changes accompanying fusion of single
vesicles with the cell membrane. This is attained by measuring the time course of the total impedance of the cell, which
includes the imaginary part. Towards this aim, the complex
impedance is measured by imposing a small sinusoidal voltage to the cell while measuring the elicited current. Using
a lock-in amplifier, very small changes in phase and amplitude between the voltage excitation and the current
response can be detected at low noise levels [11]. Changes
in the real part of the impedance correspond to changes in
the equivalent resistance and shifts in phase correspond to
changes in capacitance. By designing a special high-pressure
recording chamber (figure 2), patch-clamp experiments have
Phil. Trans. R. Soc. B 370: 20140192
internal
electrode
external
electrode
3
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(a)
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6
pF
0
(b)
6
pF
0
Phil. Trans. R. Soc. B 370: 20140192
been performed at pressures up to 60 MPa, which corresponds to 592 times normal atmospheric pressure or to the
pressure experienced at 5900 m under the surface of the
ocean. The high-pressure chamber consists of a patch-pipette
holder connected to a pressure feed-through that can be
placed on the manipulator of a conventional patch-clamp
set-up. Attached to the holder is a reservoir with a ground
electrode, which will replace the external recording chamber
while transiting the patch to the high-pressure chamber and
performing recordings. The procedure has a surprisingly
high rate of successful transfers of patches or cells attached
to the pipette into the high-pressure chamber. Once
immersed into paraffin oil and after closure of the chamber,
patches were very stable and lasted for up to 1 h. Vibrations
do not affect the assembly, the electrical shielding is extremely good given the thick conducting surrounding of the
pressure chamber, and low noise recordings are possible
due to the reduction of capacitance as the oil excludes extracellular solution from creeping along the external portions of
the pipette tip.
Paraffin oil was chosen as the medium transmitting
pressure to the patch-clamp assembly, as besides being a
very good insulator, it is practically incompressible. Therefore, changes in pressure will lead to only small changes in
volume, thereby causing only small changes in temperature,
and also reducing the energy required and stored to reach
and keep high pressures. Pressure was applied by operating
a manually driven high-pressure pump. A conventional automotive diesel-injection pump, which is able to deliver
pressures of up to 100 MPa, can also be used.
Due to the complication of having to measure changes in
capacitance from a cell under high pressure, vesicle fusion had
to be induced after the patch-clamp assembly was inside the
high-pressure chamber. This was achieved by first establishing
the whole-cell configuration in chromaffin cells already in the
tight-seal cell-attached configuration, and then being immersed
in oil and positioned in the sealed high-pressure chamber. As
under these conditions suction could not be applied to rupture
the membrane patch, brief high-voltage pulses were applied to
the pipette electrode. The pipette solution contained 1 mM free
Ca2þ, thus directly inducing vesicle fusion. Note that this procedure excludes any pressure effect due to changes in kinetics
of calcium channels, and therefore gives a direct readout of
pressure effects on vesicle fusion per se. For details please refer
to Heinemann et al. [11].
Figure 3 shows measurements during secretion in chromaffin cells at various pressures. Figure 3a depicts the time
course of total capacitance at atmospheric pressure, starting
just before obtaining the whole-cell configuration at a holding
potential of 270 mV. Just after rupture of the patch, an artefact due to the high-voltage pulse can be seen (seen also in
figure 3c), thereafter the step in capacitance corresponding
to the addition of the cell capacitance can be seen in the
record. Immediately after obtaining the whole-cell configuration, the steady increase in capacitance due to vesicle
fusion induced by 1 mM free Ca2þ in the pipette solution
equilibrating with the internal solution of the cell can be
seen. Figure 3b shows a similar experiment in which a step
in pressure to 10 MPa was applied (middle trace, dotted
lines) while vesicles were being fused. The concomitant
pressure and change in temperature (lower traces) were
recorded. Note the significant decrease in vesicle-fusion rate
during the pressure step. The same procedure is seen in
10 MPa
34
°C
28
(c)
6
pF
0
10 MPa
34
°C
28
60 s
Figure 3. (a) Time course of chromaffin cell capacitance increase at atmospheric pressure after attaining the whole-cell configuration. The initial peak
to the left is due to a high-voltage pulse used to obtain this configuration.
The initial step corresponds to the capacitance of the cell, the gradual increase
thereafter to vesicle fusion induced by Ca2þ in the pipette solution. (b) Another
experiment as in (a), but inducing a brief increase in pressure to 10 MPa. The
slowing-down of capacitance increase is fully reversible. The other two traces
report pressure and temperature in the pressure chamber by the corresponding
sensors seen in figure 2. (c) Yet another experiment but with a pressure step to
20 MPa. Adapted from Heinemann et al. [11].
figure 3c, but with a pressure step to 20 MPa. This pressure
is sufficient to practically completely inhibit vesicle fusion.
The unavoidable increase in temperature will tend to underestimate the direct pressure effect as the rate of vesicle fusion
is increased at higher temperatures. Heinemann et al. [11] estimated the activation volume of vesicle fusion in bovine
chromaffin cells to be 400 Å3, and therefore roughly an
order of magnitude larger than the effect on voltage-gated
ion channels, except for CaV1.2 channels.
From the above, it can be concluded that, at least in chromaffin cells, the Ca2þ-dependent vesicle-fusion process is
strongly pressure dependent by itself. The data obtained
from the experiments mentioned will be helpful in testing
parameters defined in molecular dynamic models regarding
this particular process for compatibility and in comparing
these to vesicle-fusion models of other cells.
Ethics statement. For this review, no experiments were performed. The
use and care of animals in the experiments from which the data
was taken for this review followed the guidelines of the German
and Italian laws on animal protection, and appropriate ethical
approval and licences had been obtained.
Funding statement. This work was funded by the Max Planck Society.
Conflict of interests. I have no competing interests.
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