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J Physiol Volume 556, Number 2, 347-352, April 15, 2004 DOI: 10.1113/jphysiol.2003.058818
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RAPID REPORT

Hydrogen ions control synaptic vesicle ion channel activity in Torpedo electromotor neurones

Ronit Ahdut-Hacohen1, Dessislava Duridanova2, Halina Meiri1 and Rami Rahamimoff1

1 Department of Physiology and the Bernard Katz Minerva Centre for Cell Biophysics, The Hebrew University- Hadassah Medical School, Jerusalem 91120, Israel2 Institute for Biophysics, The Bulgarian Academy of Sciences, Sofia, Bulgaria


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
During exocytosis the synaptic vesicle fuses with the surface membrane and undergoes a pH jump. When the synaptic vesicle is inside the presynaptic nerve terminal its internal pH is about 5.5 and after fusion, the inside of the vesicle comes in contact with the extracellular medium with a pH of about 7.25. We examined the effect of such pH jump on the opening of the non-specific 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-specific ion channel was examined using the fused vesicle-attached configuration of the patch clamp technique. The rate of opening depends on both pH and voltage. Increasing the pH from 5.5 to 7.25 activated dramatically the non-specific ion channel of the vesicle membrane. The single channel conductance did not change significantly with the alteration in the pH, and neither did the mean channel open time. 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 from the intravesicular ion exchange matrix. This process may have also a pathophysiological significance in conditions of altered pH.

(Received 27 November 2003; accepted after revision 13 February 2004; first published online 20 February 2004)
Corresponding author R. Rahamimoff: Department of Physiology and the Bernard Katz Minerva Centre for Cell Biophysics, The Hebrew University–Hadassah Medical School, Jerusalem 91120, Israel. Email: ramir{at}cc.huji.ac.il


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
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 significance 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-specific ion channel (Yakir & Rahamimoff, 1995). Another process that takes place during the fusion pore formation, between the vesicle and the extracellular medium, is a pH jump. When the secretory vesicle is inside the terminal, it has an acid pH (5.2–5.5) (Michaelson & Angel, 1980; Fuldner & Stadler, 1982), whereas after fusion, the interior of the vesicle is exposed to the almost neutral pH of the extracellular fluid. 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-specific ion channels at different pH values.

The data obtained clearly show that exposure to neutral pH causes a very substantial increase in the open state probability of the non-specific 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
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Electric fish, Torpedo ocellata, were purchased from fishermen at the Mediterranean seaport of Jaffa–Tel Aviv. The Ethical Committee for Animal Experimentation of the Hebrew University Hadassah Medical 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 from electric 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 medium was 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 fire polished (L/M-CPZ 101, List Medical Instruments) to achieve a final resistance of 3–6 M{Omega}.

The experiments were performed at room temperature. The activity of ion channels was monitored using the cell-attached configuration (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 finding the non-specific channel in the open state was monitored for about 1–8 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{Omega}. Currents were recorded with an EPC-7 patch clamp amplifier (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 amplifier. In symmetrical solutions, the membrane potential across the vesicle membrane is probably close to 0 mV.

pCLAMP software (Axon Instruments, version 5.5) and an interface (Tl-1, DMA interface, Axon Instruments) 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 analysed and plotted using Fetchan and pStat (part of pCLAMP, version 6.03) and SigmaPlot (version 5, Jandel Scientific, San Rafael, CA, USA) programs.

In the vesicle attached configuration of the patch-clamp technique, there are three compartments: the bath, the pipette and the intravesicular solutions. While the first 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 (PEG 1500, 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, CaCl2 2. Bath solution (extra- and intravesicular): potassium glutamate 350, KCl 50, Na-Hepes 10, CaCl2 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
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
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-specific ion channel at the pH range of 5.5–7.25.

Figure 1A shows the main experimental observation. The frequency of the non-specific channel opening increased dramatically with the increase in pH. If one takes the mean open channel frequency at pH 5.5 as a unit, then the frequency at pH 6.0 is 2.82-, at pH 6.5 is 23.9- and at 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.



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  		Figure 1.  The pH dependence of the opening of the non-specific 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.

 
It was shown previously, that the probability of opening of the non-specific 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 finding the non-specific channel in the open configuration was examined.

Figure 2A shows that at pH of 5.5 the channel was in the closed configuration at most of the voltages and only when the membrane was totally depolarized there was a small number of openings of the ion channels.



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  		Figure 2.  Open probability of the non-specific ion channel as a function of voltage at four different pH values
The open probability (NPo) decreases with acidification. 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.

 
The situation changed significantly 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 configuration at 0 mV was 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 with voltage 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 flowing through the channel is the relevant parameter, since these ions serve as transmitter substitutes for the ion exchange matrix. To estimate the number of ions flowing through the non-specific 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 3A–D shows the current voltage relations at different pH values. One can clearly see that the single channel conductance does not change significantly as a function of the pH of 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 than 10%, the dominant factor causing elevation of ions current through the vesicle membrane with increase in pH is the dramatic increase in the probability of opening of the non-specific ion channel.



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  		Figure 3.  Control experiments
A–D, i–V relation at different pH values. A, pH 5.5, slope conductance = 173.36 pS, intercept = 15.0 mV; r2= 0.99; n= 7. B, pH 6.0, slope conductance = 181.77 pS, intercept = 17.7 mV; r2= 0.97; n= 8. C, pH 6.5, slope conductance = 165.80 pS, intercept = 16.4 mV; r2= 0.97; n= 5. D, pH 7.25, slope conductance = 162.78 pS, intercept = 17.0 mV; r2= 0.99; n= 3. The error bars represent standard deviations. E, mean channel open time does not depend significantly on pH. Student's t test yielded P-values greater than 0.05 for most of the slopes indicating that there is no significant difference in the slope conductance at the examined pH values.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
This article addresses the question of how the non-specific ion channel in the vesicle membrane could be involved in the regulation of transmitter release.

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-specific ion channel. The first 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 pH of about 5.5. Under these conditions the ion channels of the vesicular membrane are not active. After formation of the fusion 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 open probability of the non-specific ion channel with high conductance. Hence, the fusion of the vesicle with the surface membrane results in opening of non-specific ion channels, allowing a massive flow 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 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 first 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 fluid containing an abundance of positively charged ions. Under such conditions, the contribution of the non-specific 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 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 can displace the transmitter from the 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-specific ion channel in the vesicle membrane may have not only physiological significance but also pathophysiological relevance. Any influence, 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 can interfere and affect indirectly the 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).


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Bernardi P (1999). Mitochondrial transport of cations: Channels, exchangers, and permeability transition. Physiol Rev 79, 1127–1155.[Abstract/Free Full Text]

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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 figures. 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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This Article
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