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J Physiol 556.2 (2004) pp 347352 347

R A P I D R E P O RT

Hydrogen ions control synaptic vesicle ion channel activityin Torpedo electromotor neurones

Ronit Ahdut-Hacohen 1, Dessislava Duridanova 2, Halina Meiri 1 and Rami Rahamimoff 1

1Department of Physiology and the Bernard Katz Minerva Centre for Cell Biophysics, The Hebrew University- Hadassah Medical School, Jerusalem91120, 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.Whenthe synapticvesicleis inside thepresynapticnerve terminal its internalpHis about5.5andafterfusion,theinsideofthevesiclecomesincontactwiththeextracellularmediumwitha pH of about 7.25. We examined the effect of such pH jumpon the opening of thenon-specicion channel in the synaptic vesicle membrane, in the context of the post-fusion hypothesisof transmitter release control. The vesicles were isolated from Torpedo ocellata electromotorneurones. The pH dependence of the opening of the non-specic ion channel was examinedusingthefusedvesicle-attachedcongurationofthepatchclamptechnique.Therateofopening depends on both pH and voltage. Increasing the pH from 5.5 to 7.25 activated dramatically the non-specic ionchannel of thevesicle membrane. Thesingle channel conductance did notchangesignicantly withthe alteration in thepH, andneitherdidthemeanchannel open time.These results support the hypothesis that during partial fusion of the vesicle with the surfacemembrane, ion channels in the vesicle membrane open, admit ions and thus help in the ionexchange process mechanism, leading to the release of the transmitter from the intravesicularionexchangematrix.Thisprocessmayhavealsoapathophysiologicalsignicanceinconditionsof 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 CellBiophysics, The Hebrew UniversityHadassah Medical School, Jerusalem 91120, Israel. Email: [email protected]

Many intracellular organelles are equipped with ionchannels. These organelles include the endoplasmic andsarcoplasmic reticulum (Szewczyk, 1998; Debska et al.2001), mitochondria (Bernardi, 1999; Debska et al. 2001),nucleus (Mazzanti et al. 2001) and secretory vesicles (Meiret al. 1999). In some of these organelles the physiologicalrole of ion channels has been clearly demonstrated, whilein 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, suchas releaseof transmitters andhormones, and the insertion of ion channels, transportersand receptors into the surface membrane. It has beenrepeatedly shown that many types of ion channels operatein 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, arebound inside the secretory vesicle to a negatively chargedintravesicularmatrix(Stadler & Kiene, 1987; Reigada et al.2003). For these transmitter molecules to be releasedfrom the matrix, a supply of cations is necessary. Several years ago it was proposed that some of the ion channelsin the vesicle membrane serve as a pathway for ions todisplace the transmitter molecule from the ion exchange

matrix (Rahamimoff & Fernandez, 1997). This may beparticularly important during a partial fusion of thevesicle with the surface membrane, the so-called kiss andrun exocytotic event (Ceccarelli & Hurlbut, 1980). Thishypothesis raises the question of the factors governingthe opening of vesicular ion channels. It has already beenshown that the shift in the membrane potential of thevesicle increases the open state probability of the non-specic ion channel (Yakir & Rahamimoff, 1995). Anotherprocess that takes place during the fusion pore formation,

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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 (5.2 5.5) (Michaelson & Angel, 1980;Fuldner & Stadler, 1982), whereas after fusion, the interiorof the vesicle is exposed to the almost neutral pH of theextracellular uid. Thus, our working hypothesis assumes

that the change of H+

ion concentration in the synapticvesicle during formation of the fusion pore may affect theprobability of ion channels opening in the synaptic vesiclemembrane. Hence, we examined the open probability of non-speci c ion channels at different pH values.

The data obtained clearly show that exposure to neutralpH causes a very substantial increase in the open stateprobability of the non-speci c ion channels in synapticvesicles, which could contribute to their emptying.

We speculate that the pH control of the synaptic vesicleion channel activity may have a physiological and apathophysiological importance.

Methods

Electric sh, Torpedo ocellata, were purchased fromshermen at the Mediterranean seaport of Jaffa Tel Aviv.The Ethical Committee for Animal Experimentation of theHebrew UniversityHadassahMedicalSchool approvedthe procedures. The animals were killed humanely andthe 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 smallchamber on the stage of an inverted microscope (ZeissAxiovert 135).Thevesicle-containing medium was furtherdiluted with a bath solution having an ionic compositionidentical to the fusion buffer, thus representing theintravesicular solution.

Patch electrodes were fabricated from borosilicate glasscapillary tubes (Hilgenberg, Malsfeld, Germany), pulledby a puller (P-97 Sutter Instrument, USA), coated withSylgard (184 Silicone, Dow Corning Corporation) andre polished (L/M-CPZ 101, List Medical Instruments)to achieve a nal resistance of 36 M .

The experiments were performed at room temperature.The activity of ion channels was monitored using the cell-attached con guration (in fact, vesicle attached) of thepatch clamp technique (Hamill et al. 1981). The potentialdifference was changed between the pipette and the bathsolution. At each potential the probability of nding thenon-speci c channel in the open state was monitored for

about 1 8 min and the average opening rate and durationof the channel was estimated.

The electrode was sealed to the vesicular membrane.The seal resistance was 3 G . Currents were recordedwith an EPC-7 patch clamp ampli er (List MedicalInstruments, Darmstadt, Germany) at a gain of 50 mV

pA 1

. Measurements were corrected for liquid potentialsby adjusting the offset in the ampli er. In symmetricalsolutions, the membrane potential across the vesiclemembrane is probably close to 0 mV.

pCLAMPsoftware (Axon Instruments,version 5.5) andaninterface(Tl-1,DMAinterface,AxonInstruments)wereused for setting and clamping the membrane potentialat the required value. The information was recordedwith a Neuro-corder (DR-384, Neuro Data Instruments)and stored on a videocassette. The data were digitizedat 20 kHz by using the DigiData 1200 interface and theFetchex software (part of pCLAMP, version6.03) andweresubsequently analysedandplotted usingFetchanand pStat(part of pCLAMP, version 6.03) and SigmaPlot (version 5,Jandel Scientic, San Rafael, CA, USA) programs.

In thevesicle attached con guration of the patch-clamptechnique, there are three compartments: the bath, thepipette and the intravesicular solutions. While the rsttwo can be manipulated at will, the third solution can bedetermined only at the fusion stage, where a large increasein the intravesicular volume occurs see (Rahamimoff et al.1989). Hence, the activity of channels in each patch wasexamined at the pH imposed during the fusion.

All chemicals were obtained from Sigma Chemicals,except forpolyethyleneglycol 1500(PEG1500,BoehringerMennheim, GmbH Germany).

Thecomposition of solutionsusedin most experimentswas as follows (mm ). Pipette solution: mannitol 500,potassium glutamate 70, KCl 10, Na-Hepes 10, CaCl 22. Bath solution (extra- and intravesicular): potassiumglutamate 350, KCl 50, Na-Hepes 10, CaCl 2 1. Mannitolwas used to adjust the osmolarity of the pipette and thebath solutions. The pH was adjusted by adding 10 m mNa-Hepes and either HCl or KOH when necessary. Theexperiments 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 jumpaccompanying the fusion of the vesicle with the surfacemembrane causes a change in the ion channel activity, weexamined theopening rate of thenon-speci c ion channelat the pH range of 5.5 7.25.

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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-speci c channel openingincreaseddramaticallywiththe increase in pH.Ifonetakesthe mean open channel frequency at pH 5.5 as a unit, thenthefrequencyatpH6.0is2.82-,atpH6.5is23.9-andatpH7.25 is 261-fold greater. A complementary representation

ofthesame datais shown inFig. 1 B , where a huge decreasein mean channel close time was observed with increase inpH.

It was shown previously, that theprobability of openingof the non-speci c channel is highly dependent on thepotential difference across the fused vesicle membrane(Yakir & Rahamimoff, 1995). Therefore we examinedthe combined effect of pH and voltage. The potentialdifference across the membrane was changed. At eachpotential the probability of nding the non-speci cchannel in the open con guration was examined.

Figure 2A shows that at pH of 5.5 the channel was inthe closed con guration at most of the voltages and only when the membrane was totally depolarized there was asmall number of openings of the ion channels.

The situation changed signi cantly when the pH wasincreased to 6.0, 6.5 and 7.25. At pH 7.25, the probability ofthe channel tobe in the opencon gurationat 0 mVwashigher than 90%.

Figure 2E shows the three-dimensional representationof the probability of opening as a function of voltage andof 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 analysiswas performed in a smaller number of experiments.

These results clearly indicate that the probability of opening changes with voltage andpH. Similar dependenceof opening on voltage was observed also with otherchannels (Pohlmeyer et al. 1997; Valiunas & Weingart,2000). From a physiological point of view, the number of ions owingthrough thechannel is the relevantparameter,since these ions serve as transmitter substitutes for theion exchange matrix. To estimate the number of ionsowing through the non-speci c channels, we need toknow whether the channel conductance and the meanchannel open-time change with pH. Figure 3 deals withthese control considerations. Figure 3 A D shows thecurrent voltage relations at different pH values. One canclearly see that the single channel conductance does notchange signicantlyasa functionofthe pHof the solution.The last parameter that is necessary to evaluate is themean channel open time (MCOT). We found that thedifferences in the MCOT at the various pH values wereless than 25% compared to the average MCOT (Fig. 3 E ).

Since the increase in the number of openings with pHelevation was higher than 1000% (the value depends onthe voltage applied) and the increase in conductance wassmaller than 10%, the dominantfactor causing elevationof ions current through the vesicle membrane with increaseinpH is the dramatic increase in the probability ofopening

of the non-speci c ion channel.

Discussion

This article addresses the question of how the non-speci cion channel in the vesicle membrane could be involved inthe regulation of transmitter release.

Figure 1. The pH dependence of the opening of the non-specicvesicle channel A, the mean frequency of channel opening (mean S.D.): 2.9 1.4Hz, 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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Our working hypothesis was that inside the restingpresynaptic nerve terminal these ion channels are in theclosed state. Following an action potential, when fusion of the vesicles with the presynaptic surface membrane takesplace, these channels are activated. Two major factors cancontribute to this opening of thenon-speci c ion channel.

The rst is the voltage jump, which occurs during thefusion of vesicles with the surface membrane (DeRiemeret al. 1988; Yakir & Rahamimoff, 1995; Meir et al. 1999),and the second is the pH jump, which also occursduring the fusion process. While stored inside the nerveterminal, the intravesicular solution has a pH of about 5.5.Under these conditions the ion channels of the vesicularmembrane are not active. After formation of the fusion

Figure 2. Open probability of thenon-speci c ion channel as a function ofvoltage at four different pH valuesThe open probability ( NP o ) decreases withacidication. In each pH it reaches the highestvalue at 0 mV and decreases fast uponimposing voltage on the membrane. A, pH5.5; B, pH 6.0; C , pH 6.5; D, pH 7.25. E , 3Dpresentation of the probability of opening asa function of both voltage and pH. The errorbars represent standard errors of the mean.

pore, H + ions leave the vesicle along their concentrationgradient, until the new equilibrium is reached with thepH of the extracellular medium. The data presented heredemonstrate that the change in pH inside the synapticvesicles does affect theopen probability of thenon-speci cion channel with high conductance. Hence, the fusion of

the vesicle withthe surface membrane results inopeningof non-speci c ion channels, allowing a massive ow of ionsfrom the cytosol into the synaptic vesicle. It is expectedthat prolongation of the action potential may furtherincrease theprobabilityof opening of thesechannels.Fromelectrochemical considerations the main cation chargecarriers are the potassium ions. These ions could in turnreplace 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 synapticcleft.

Before accepting this hypothesis one should examinewhen 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, suggesteda complete fusion of the vesicle membrane with thesurface membrane (Heuser & Reese, 1973). If this occurs,then the synaptic vesicle should face the bulk of theextracellular uid containing an abundance of positively charged ions. Under such conditions, the contribution of the non-speci c ion channel in the control of transmitterliberation would be minimal. The second model, the kissand 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 , pH6.5, slope conductance = 165.80 pS,intercept = 16.4 mV; r 2 = 0.97; n = 5. D, pH7.25, slope conductance = 162.78 pS,intercept = 17.0 mV; r 2 = 0.99; n = 3. Theerror bars represent standard deviations. E ,mean channel open time does not dependsignicantly on pH. Student s t test yieldedP -values greater than 0.05 for most of theslopes indicating that there is no signi cantdifference in the slope conductance at theexamined pH values.

experimental data accumulated over the past decades (forreview see (Schneider, 2001). According to this model,a fusion pore forms between the vesicle and the surfacemembrane following the action potential, but it does notbecome immediately part of the cell membrane. Undersuch circumstances the role of the vesicular ion channels

in providing cations necessary for transmitter liberationcould be very substantial. The entry of ions from eitherthe synaptic cleft or from the axoplasm into the vesiclecandisplace the transmitter fromthe ion-exchangematrix,thusfacilitating its discharge (Marszalek et al. 1996).Thereis a growing body of evidence that this mechanism isnot an exception, but occurs quite frequently during thesynaptic release process (Reigada et al. 2003). Hence, thepH jump can be one of the factors controlling transmitterrelease from synaptic vesicles. The pH dependence of theopening of the non-speci c ion channel in the vesicle


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membrane may have not only physiological signi cancebut also pathophysiological relevance. Any in uence,which could affect the generation of the pH gradientin the synaptic vesicle membrane, would also alter theproperties of vesicular ion channels, thus interfering withthe release process. Similarly, conditions of severe acidosis

caninterfereandaffectindirectlytheprocessoftransmitterrelease and synaptic transmission. In this context, it is of interest to note that a reduction of the extracellular pHto about 6.4 causes a decrease in hormone release fromneurohypophysial nerve terminals (Cazalis et al. 1987).

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Acknowledgements

We thank Mrs Laura Brendel for her unfailing administrativeassistance during the preparation of this work and Ms AnnaFendyur for her help with the gures. This work was supportedby grants from the Israel Science Foundation (ISF), US-IsraelBinational Science Foundation (BSF) and the Bernard KatzMinerva Centre for Cell Biophysics.


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