J Physiol 556.

2 (2004) pp 347352 347

RAPI D REPORT
Hydrogen ions control synaptic vesicle ion channel activity
in Torpedo electromotor neurones
Ronit Ahdut-Hacohen
1
, Dessislava Duridanova
2
, Halina Meiri
1
and Rami Rahamimoff
1
1
Department of Physiology and the Bernard Katz Minerva Centre for Cell Biophysics, The Hebrew University- Hadassah Medical School, Jerusalem
91120, Israel
2
Institute for Biophysics, The Bulgarian Academy of Sciences, Soa, Bulgaria
During exocytosis the synaptic vesicle fuses with the surface membrane and undergoes a pH
jump. Whenthesynapticvesicleis insidethepresynapticnerveterminal its internal pHis about
5.5andafterfusion, theinsideof thevesiclecomesincontact withtheextracellularmediumwith
a pHof about 7.25. We examined the effect of such pHjump on the opening of the non-specic
ion channel in the synaptic vesicle membrane, in the context of the post-fusion hypothesis
of transmitter release control. The vesicles were isolated from Torpedo ocellata electromotor
neurones. The pH dependence of the opening of the non-specic ion channel was examined
usingthefusedvesicle-attachedcongurationof thepatchclamptechnique. Therateof opening
depends on both pH and voltage. Increasing the pH from 5.5 to 7.25 activated dramatically
the non-specic ion channel of the vesicle membrane. The single channel conductance did not
change signicantly withthe alterationinthe pH, andneither didthe meanchannel opentime.
These results support the hypothesis that during partial fusion of the vesicle with the surface
membrane, ion channels in the vesicle membrane open, admit ions and thus help in the ion
exchange process mechanism, leading to the release of the transmitter fromthe intravesicular
ionexchangematrix. Thisprocessmayhavealsoapathophysiological signicanceinconditions
of altered pH.
(Received 27 November 2003; accepted after revision 13 February 2004; rst published online 20 February 2004)
Corresponding author R. Rahamimoff: Department of Physiology and the Bernard Katz Minerva Centre for Cell
Biophysics, The Hebrew UniversityHadassah Medical School, Jerusalem 91120, Israel. Email: ramir@cc.huji.ac.il
Many intracellular organelles are equipped with ion
channels. These organelles include the endoplasmic and
sarcoplasmic reticulum (Szewczyk, 1998; Debska et al.
2001), mitochondria (Bernardi, 1999; Debska et al. 2001),
nucleus (Mazzanti et al. 2001) and secretory vesicles (Meir
et al. 1999). In some of these organelles the physiological
role of ion channels has been clearly demonstrated, while
in other organelles, such as the secretory vesicles, the
functional signicance of the channels is still obscure.
Secretory vesicles are very important in a number of
physiological functions, such as release of transmitters and
hormones, and the insertion of ion channels, transporters
and receptors into the surface membrane. It has been
repeatedly shown that many types of ion channels operate
in the membrane of secretory vesicles (Rahamimoff
et al. 1988, 1989; Stanley et al. 1988; Lee et al. 1992;
Woodbury, 1995; Yakir & Rahamimoff, 1995; Thevenod,
2002).
Many transmitter molecules, such as acetylcholine, are
bound inside the secretory vesicle to a negatively charged
intravesicular matrix (Stadler &Kiene, 1987; Reigada et al.
2003). For these transmitter molecules to be released
from the matrix, a supply of cations is necessary. Several
years ago it was proposed that some of the ion channels
in the vesicle membrane serve as a pathway for ions to
displace the transmitter molecule from the ion exchange
matrix (Rahamimoff & Fernandez, 1997). This may be
particularly important during a partial fusion of the
vesicle with the surface membrane, the so-called kiss and
run exocytotic event (Ceccarelli & Hurlbut, 1980). This
hypothesis raises the question of the factors governing
the opening of vesicular ion channels. It has already been
shown that the shift in the membrane potential of the
vesicle increases the open state probability of the non-
specic ion channel (Yakir &Rahamimoff, 1995). Another
process that takes place during the fusion pore formation,
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The Physiological Society 2004 DOI: 10.1113/jphysiol.2003.058818
348 R. Ahdut-Hacohen and others J Physiol 556.2
between the vesicle and the extracellular medium, is a pH
jump. When the secretory vesicle is inside the terminal,
it has an acid pH (5.25.5) (Michaelson & Angel, 1980;
Fuldner &Stadler, 1982), whereas after fusion, the interior
of the vesicle is exposed to the almost neutral pH of the
extracellular uid. Thus, our working hypothesis assumes
that the change of H
+
ion concentration in the synaptic
vesicle during formation of the fusion pore may affect the
probability of ion channels opening in the synaptic vesicle
membrane. Hence, we examined the open probability of
non-specic ion channels at different pH values.
The data obtained clearly showthat exposure to neutral
pH causes a very substantial increase in the open state
probability of the non-specic ion channels in synaptic
vesicles, which could contribute to their emptying.
We speculate that the pH control of the synaptic vesicle
ion channel activity may have a physiological and a
pathophysiological importance.
Methods
Electric sh, Torpedo ocellata, were purchased from
shermen at the Mediterranean seaport of JaffaTel Aviv.
The Ethical Committee for Animal Experimentation of
the HebrewUniversity HadassahMedical School approved
the procedures. The animals were killed humanely and
the electric organs were removed and frozen at 70

C.
Isolation (Tashiro & Stadler, 1978; Yakir & Rahamimoff,
1995) and fusion (Rahamimoff et al. 1988) of the synaptic
vesicles fromelectric organs were performed as previously
described.
Twenty microlitres of the diluted fusion medium,
containing fused synaptic vesicles was placed in a small
chamber on the stage of an inverted microscope (Zeiss
Axiovert 135). The vesicle-containing mediumwas further
diluted with a bath solution having an ionic composition
identical to the fusion buffer, thus representing the
intravesicular solution.
Patch electrodes were fabricated from borosilicate glass
capillary tubes (Hilgenberg, Malsfeld, Germany), pulled
by a puller (P-97 Sutter Instrument, USA), coated with
Sylgard (184 Silicone, Dow Corning Corporation) and
re polished (L/M-CPZ 101, List Medical Instruments)
to achieve a nal resistance of 36 M.
The experiments were performed at roomtemperature.
The activity of ion channels was monitored using the cell-
attached conguration (in fact, vesicle attached) of the
patch clamp technique (Hamill et al. 1981). The potential
difference was changed between the pipette and the bath
solution. At each potential the probability of nding the
non-specic channel in the open state was monitored for
about 18 min and the average opening rate and duration
of the channel was estimated.
The electrode was sealed to the vesicular membrane.
The seal resistance was 3 G. Currents were recorded
with an EPC-7 patch clamp amplier (List Medical
Instruments, Darmstadt, Germany) at a gain of 50 mV
pA
1
. Measurements were corrected for liquid potentials
by adjusting the offset in the amplier. In symmetrical
solutions, the membrane potential across the vesicle
membrane is probably close to 0 mV.
pCLAMP software (Axon Instruments, version 5.5) and
aninterface(Tl-1, DMAinterface, AxonInstruments) were
used for setting and clamping the membrane potential
at the required value. The information was recorded
with a Neuro-corder (DR-384, Neuro Data Instruments)
and stored on a videocassette. The data were digitized
at 20 kHz by using the DigiData 1200 interface and the
Fetchex software (part of pCLAMP, version 6.03) and were
subsequently analysedandplottedusing FetchanandpStat
(part of pCLAMP, version 6.03) and SigmaPlot (version 5,
Jandel Scientic, San Rafael, CA, USA) programs.
In the vesicle attached conguration of the patch-clamp
technique, there are three compartments: the bath, the
pipette and the intravesicular solutions. While the rst
two can be manipulated at will, the third solution can be
determined only at the fusion stage, where a large increase
in the intravesicular volume occurs see (Rahamimoff et al.
1989). Hence, the activity of channels in each patch was
examined at the pH imposed during the fusion.
All chemicals were obtained from Sigma Chemicals,
except for polyethylene glycol 1500 (PEG1500, Boehringer
Mennheim, GmbH Germany).
The composition of solutions used in most experiments
was as follows (mm). Pipette solution: mannitol 500,
potassium glutamate 70, KCl 10, Na-Hepes 10, CaCl
2
2. Bath solution (extra- and intravesicular): potassium
glutamate 350, KCl 50, Na-Hepes 10, CaCl
2
1. Mannitol
was used to adjust the osmolarity of the pipette and the
bath solutions. The pH was adjusted by adding 10 mm
Na-Hepes and either HCl or KOH when necessary. The
experiments were performed at four different pH values:
5.5, 6.0, 6.5 and 7.25.
Results
To test the working hypothesis that the pH jump
accompanying the fusion of the vesicle with the surface
membrane causes a change in the ion channel activity, we
examined the opening rate of the non-specic ion channel
at the pH range of 5.57.25.
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J Physiol 556.2 H
+
dependence of synaptic vesicle ion channels 349
Figure 1A shows the main experimental observation.
The frequency of the non-specic channel opening
increaseddramatically withthe increase inpH. If one takes
the mean open channel frequency at pH5.5 as a unit, then
the frequency at pH6.0 is 2.82-, at pH6.5 is 23.9- andat pH
7.25 is 261-fold greater. A complementary representation
of the same data is shown in Fig. 1B, where a huge decrease
in mean channel close time was observed with increase in
pH.
It was shown previously, that the probability of opening
of the non-specic channel is highly dependent on the
potential difference across the fused vesicle membrane
(Yakir & Rahamimoff, 1995). Therefore we examined
the combined effect of pH and voltage. The potential
difference across the membrane was changed. At each
potential the probability of nding the non-specic
channel in the open conguration was examined.
Figure 2A shows that at pH of 5.5 the channel was in
the closed conguration at most of the voltages and only
when the membrane was totally depolarized there was a
small number of openings of the ion channels.
The situation changed signicantly when the pH was
increased to 6.0, 6.5 and 7.25. At pH 7.25, the probability
of the channel to be in the open conguration at 0 mVwas
higher than 90%.
Figure 2E shows the three-dimensional representation
of the probability of opening as a function of voltage and
of the pH. The number of experiments performed at every
pH was as follows: pH 5.5, 17; pH 6.0. 13; pH 6.5, 18;
and pH 7.25, 14. The effect of pH on the probability of
opening was observed in all experiments. A full analysis
was performed in a smaller number of experiments.
These results clearly indicate that the probability of
opening changes withvoltage and pH. Similar dependence
of opening on voltage was observed also with other
channels (Pohlmeyer et al. 1997; Valiunas & Weingart,
2000). From a physiological point of view, the number of
ions owing throughthe channel is the relevant parameter,
since these ions serve as transmitter substitutes for the
ion exchange matrix. To estimate the number of ions
owing through the non-specic channels, we need to
know whether the channel conductance and the mean
channel open-time change with pH. Figure 3 deals with
these control considerations. Figure 3AD shows the
current voltage relations at different pH values. One can
clearly see that the single channel conductance does not
change signicantly as a functionof the pHof the solution.
The last parameter that is necessary to evaluate is the
mean channel open time (MCOT). We found that the
differences in the MCOT at the various pH values were
less than 25% compared to the average MCOT (Fig. 3E).
Since the increase in the number of openings with pH
elevation was higher than 1000% (the value depends on
the voltage applied) and the increase in conductance was
smaller than10%, the dominant factor causingelevationof
ions current through the vesicle membrane with increase
inpHis the dramatic increase inthe probability of opening
of the non-specic ion channel.
Discussion
This article addresses the question of howthe non-specic
ion channel in the vesicle membrane could be involved in
the regulation of transmitter release.
Figure 1. The pH dependence of the opening of the non-specic
vesicle channel
A, the mean frequency of channel opening (mean S.D.): 2.9 1.4
Hz, pH 5.5; 8.19 2.6 Hz, pH 6.0; 69.5 9.7 Hz, pH 6.5; 757.9
69.5 Hz, pH 7.25; 1 s bins. B, the mean channel closed time (ms)
decreases with the elevation of pH (mean S.E.M.): 1456.87 95.8,
pH 5.5; 691.66 130.76, pH 6.0; 62.07 9.06, pH 6.5; 0.476
0.12, pH 7.25.
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350 R. Ahdut-Hacohen and others J Physiol 556.2
Our working hypothesis was that inside the resting
presynaptic nerve terminal these ion channels are in the
closed state. Following an action potential, when fusion of
the vesicles with the presynaptic surface membrane takes
place, these channels are activated. Two major factors can
contribute to this opening of the non-specic ion channel.
The rst is the voltage jump, which occurs during the
fusion of vesicles with the surface membrane (DeRiemer
et al. 1988; Yakir & Rahamimoff, 1995; Meir et al. 1999),
and the second is the pH jump, which also occurs
during the fusion process. While stored inside the nerve
terminal, the intravesicular solution has a pHof about 5.5.
Under these conditions the ion channels of the vesicular
membrane are not active. After formation of the fusion
Figure 2. Open probability of the
non-specic ion channel as a function of
voltage at four different pH values
The open probability (NP
o
) decreases with
acidication. In each pH it reaches the highest
value at 0 mV and decreases fast upon
imposing voltage on the membrane. A, pH
5.5; B, pH 6.0; C, pH 6.5; D, pH 7.25. E, 3D
presentation of the probability of opening as
a function of both voltage and pH. The error
bars represent standard errors of the mean.
pore, H
+
ions leave the vesicle along their concentration
gradient, until the new equilibrium is reached with the
pH of the extracellular medium. The data presented here
demonstrate that the change in pH inside the synaptic
vesicles does affect the openprobability of the non-specic
ion channel with high conductance. Hence, the fusion of
the vesicle withthe surface membrane results inopening of
non-specic ion channels, allowing a massive ow of ions
from the cytosol into the synaptic vesicle. It is expected
that prolongation of the action potential may further
increase the probability of opening of these channels. From
electrochemical considerations the main cation charge
carriers are the potassium ions. These ions could in turn
replace the positively charged transmitter molecules from
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J Physiol 556.2 H
+
dependence of synaptic vesicle ion channels 351
the ion exchange matrix inside the vesicle (Rahamimoff &
Fernandez, 1997) facilitating their delivery to the synaptic
cleft.
Before accepting this hypothesis one should examine
when it might occur. Two models of the fusion of
the secretory vesicles have been proposed to explain
the mechanism of transmitter release. The rst one,
which has been recognized a long time ago, suggested
a complete fusion of the vesicle membrane with the
surface membrane (Heuser & Reese, 1973). If this occurs,
then the synaptic vesicle should face the bulk of the
extracellular uid containing an abundance of positively
charged ions. Under such conditions, the contribution of
the non-specic ion channel in the control of transmitter
liberation would be minimal. The second model, the kiss
and run suggested about a quarter of a century ago
(Ceccarelli & Hurlbut, 1980), has been substantiated by
Figure 3. Control experiments
AD, iV relation at different pH values. A,
pH 5.5, slope conductance = 173.36 pS,
intercept = 15.0 mV; r
2
= 0.99; n = 7. B,
pH 6.0, slope conductance = 181.77 pS,
intercept = 17.7 mV; r
2
= 0.97; n = 8. C, pH
6.5, slope conductance = 165.80 pS,
intercept = 16.4 mV; r
2
= 0.97; n = 5. D, pH
7.25, slope conductance = 162.78 pS,
intercept = 17.0 mV; r
2
= 0.99; n = 3. The
error bars represent standard deviations. E,
mean channel open time does not depend
signicantly on pH. Students t test yielded
P-values greater than 0.05 for most of the
slopes indicating that there is no signicant
difference in the slope conductance at the
examined pH values.
experimental data accumulated over the past decades (for
review see (Schneider, 2001). According to this model,
a fusion pore forms between the vesicle and the surface
membrane following the action potential, but it does not
become immediately part of the cell membrane. Under
such circumstances the role of the vesicular ion channels
in providing cations necessary for transmitter liberation
could be very substantial. The entry of ions from either
the synaptic cleft or from the axoplasm into the vesicle
candisplace the transmitter fromthe ion-exchange matrix,
thus facilitating its discharge (Marszalek et al. 1996). There
is a growing body of evidence that this mechanism is
not an exception, but occurs quite frequently during the
synaptic release process (Reigada et al. 2003). Hence, the
pH jump can be one of the factors controlling transmitter
release from synaptic vesicles. The pH dependence of the
opening of the non-specic ion channel in the vesicle
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352 R. Ahdut-Hacohen and others J Physiol 556.2
membrane may have not only physiological signicance
but also pathophysiological relevance. Any inuence,
which could affect the generation of the pH gradient
in the synaptic vesicle membrane, would also alter the
properties of vesicular ion channels, thus interfering with
the release process. Similarly, conditions of severe acidosis
caninterfere andaffect indirectlythe process of transmitter
release and synaptic transmission. In this context, it is of
interest to note that a reduction of the extracellular pH
to about 6.4 causes a decrease in hormone release from
neurohypophysial nerve terminals (Cazalis et al. 1987).
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Acknowledgements
We thank Mrs Laura Brendel for her unfailing administrative
assistance during the preparation of this work and Ms Anna
Fendyur for her help with the gures. This work was supported
by grants from the Israel Science Foundation (ISF), US-Israel
Binational Science Foundation (BSF) and the Bernard Katz
Minerva Centre for Cell Biophysics.
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