interaction between ca2+ release from inositol trisphosphate sensitive stores and ca2+ entry through...

cell calan (lw4) 15, 411422 QLongmul~Lm1984

Interaction between Ca*+ release from inositol trisphosphate sensitive stores and Ca*+ entrv through neuronal Ca”+ channels expressed Xenopus oocyte

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F. FOURNIER’, P. NAVARRE’, F. MATIFAT’, C. VILBERT’, T. COLIN2, P. GUlLBAULT2, G. BRULE’ and D. MARLOT’

’ Laboratoire de Neurobiologie Cellulaire, UFR des Sciences exactes et fondamentales, Universit6 de Picardie Jules Verne, Amiens and 2Laboratoire de Physiologie Cellulaire, UFR Biologic, Univetsit6 des Sciences et Techniques de Lille, Villeneuve tlAscq, France

Abstract - Rat cerebeiiar RNA injected into Xenopus oocytes leads to the expression of putative P-type vottagedependent Ca2+ channels (VDCCS). The monitoring of intraceiiuiar Ca2’ variations by recording the Ca2’ dsip”dent chloride current in voltage clamped oocytes indicates that activation of these Ca + channels by depoiarization gives rise to two distinct components of cytosoiic Ca2+ elevation. if the eariy component (Tr) can be di aMbuted to the Ca2+ entry through VDCCs, the second one4(T2)ls~toa + 3

reiease from insP3 sensitive stores activated following entry. Modlficaflons of cyt* soiic Ca2+ by direct ln)ection of c$+ into oocytes or by increasing the Q?+ influx through VDCCs suggest that the Ca2’ release from intraceliuiar InsPs sensitive stores can be modulated in a dffferentiai manner. Nameiy, discrete elevations of cytosoiic Ca2+ switch on the Ca2’ release whereas higher Ca2+ concentrations dampen the release.

These results suggest a functional coupling beWeen P-type VDCCs and InsPs receptors.

The spatio-temporal characteristics of the release of Ca2+ from InsP3 sensitive stores are complex, dis-

be modulated by both cytosolic and/or luminal calcium ions, (for review see 181). The regulation of

playing either an all-or-none mechanism [l-4] the InsP3 response by cytosolic Ca2+ appeared to be which could account for the recently observed quan- tal mobilization of Ca2+ [5,6], or a steady release

a complex mechanism since Ca2+ elevation near the external side of the 11@3 qtor can exert opposite

where the libemtion is a czontinuous pmcess toauumdativeinuease in cytosolic Ca2+

leading effects, namely facilitation or inhibition [9.10]. [7] . Hence, moderate cytosolic Ca2’ concentrations are

Inmanycelltypes,thereleaseofsequ~ abletosensitizetheInsp3receptorandtriggerfur- Ca2’ from InsP3 sensitive stores has been shown to ther Ca2’ liberation fkom In* stores [ll], whms

411

412 CEU. CALCIUM

higher Ca2’ co~~tions switch off the InsPs-induced Ca2+ release [12,13]. In this context, any Ca’” entry through the plasma membrane can poten- tially regulate the Ca2’ release from Ins& sensitive stores by increasing the cytosolic Ca2+ concentration near the InsP3 receptors.

The Xen0pu.s oocyte represents the most suitable convenient model to study the intrinsic properties of Ca2+ signalling linked to InsPa, since the release of in~ce~ul~ stores was shown to be mediated from only one kind of store which is mobile by InsPs and regulated by cytoplasmic C&r’+ f14]. Further- mom, the oocyte system can be used to express ion channels such as voltage dependent Ca2’ channels (VDCCs). Therefore, cembellar mRNA injected oocytes preferentially expressed one kind of VDCCs which is pharmacologically related to the neuronal P-type [15]. These P-type Ca2+ channels am found at high density in mammalian cerebellar Purkinje cells, throughout the central nervous system and at the motor nerve terminal [16-201.

Our study shows that the Cazt entry which is primarily induced by activation of the putative P- type VDCCs expressed in oocytes is able to modu- late the Ca2+ release from InsP3 sensitive stores. Such an interaction appears to be mediated by the cytosolic Ca2’ elevation which is directly derived from the Ca2’ entry through P-type VDCCs. These observations demonstrate a functional coupling between VDCCs and InsPa receptors.

Materinls and methods

Cerebellar RNA from 15-17-day old Wistar rats was extracted using a phenoV&lorofomr procedure [21]. Total RNA was dissolved in sterile water at about 5 mg/ml concentration.

Pieces of adult Xenopus luevis ovary were surgi- cally removed under general anesthesia and dis- sected away in ND96 solution (mM): NaCl96, KCl 2, MgCla 2, CaCls 1.8, HEPES 5, pH 7.4. To dis- card follicular cells, oocytes were treated for 2-3 h with coB~ena~ (type IA) at 2 mg/ml in a calcium free medium 50-80 nl of cerebellar RNA were pressure injected per oocyte (stages V, VI). Oocytes were kept for 2-6 days at 20°C in ND96 medium supplemented with gentamycin (50 pg/ml).

~~physiolo~~ ~~ ts were performed using the standard two microelectrode voltage clamp tecbuique. Oocytes were placed in a ret- ording chamber (100 pI) and impaled with eleo tmdes filled with 3 M KCl. Stimulation of tbe preparation and data acquisition were conducted using a personal computer interfaced with a Labmaster (Axon Instruxrn%t, Burlingatrsz, CA, USA). Off-line analysis was performed using the pCLAMP soft- ware (~5.5, Axon ~s~nt). The ND96 soluti~ was mu~ly used to monitor the [Ca2+]i variations and occasional rn~~~ons of Exodus Ca2+ were compensated by adequate modifications of cat- ion concentrations (Mg2+). To directly record the VDCC activity, mRNA injected oocytes were bathed in BaMS medium (mM): Ba(OH)s 40, NaOH 50, CsOH 2, HEPES 5, pH 7.4. Therefore, Ba2+ was used as charge carrier and the activity of VDCCs was recorded as an inward Ba2+ current (IF&. Drugs were applied by addition to the superfusate. For intracellular injection, an additional micropipette (3-10 pm tip diameter) was used and all pressure injected co~unds (InsP3, heparin, EGTA, CaCl2) were dissolved in HEPlWKOH 5 mM, pH 7.

The optical arrangement for the light-flash experiments was essentially the same as previously described [22]. Flashes were trigged at the begin- ning of the depolarization step from a xenon short arc flash tube which delivered a total output energy of 200 W in less than 1 ms. The light was filtered to mmove h-1 wavelengths (< 300 nm) and fo- cused onto the cell with a spot diameter of about 6 mm. The caged-InsPs was pressure injected 1 h before ~~~n~. Final intraoocyte connation wasestimatedtobe5pM.

Results

Macroscopic currents were recorded in voltage- clamped Xenopus oocytes 2-6 days after injection with cerebellar RNA. Electrophysiological recording of the native ~ci~-~~~nt chloride current (IClc$ was used as an ~~~~r of the intracellular Ca2+ variations 1231.

In this study, an appm~~ depolarization (-430 /+20 mV) of an oocyte batbed in a $~ysiological medium (ND96 containing 1.8 mM Ca ) elicited a

targe transient outward chIoride current with two distinct c~~n~~ (Fig. lA, upper pa&1. This complex chloride current (TI and Tz ~~~n~~ is triggered by the cytosolic Ca2* elevation immedi- ately induced following Ca2’ entry thmugh newly expressed VDCCs and is mainIy supported by en- dogenous Cl- channels as reported in other studies [X4]. In order to directly investigate the Ca2” channel activity, Cl- ions were suppressed fmm the external medium and 13a2’ ions (40 mM), which do not activate the Ca2’ dependent chlotidc cnrren% were used as the charge carriers (BaMS ~~~~ Under these co~~~~ a ~~l~~g voltage step ~~~~20 mV) evoked the classical inward mono- p&sic Ba2+ current (I&), reGX%iIlg the exogenous VDCC activity directed by ce~bellar RNA pig. lA, bottom panel [15,25]). Therefore, as described earlier for brain mRNA injected oocytes [%,Wl, the outward chloride cnncnt elicited by depolarization presents more commonly the additional delayed co~oent named T2. This cornet can be spo~~~usly elicited a&z the fmt deviation or cau develop pr~sively with an iterative stimn- lation (Fig. 1% left and right panels).

Since the T2 co~nent appeared when depolarization steps were applied to the oocyte membrane, one m&y propose that Ca2’ entry tbmugh expressed VDCCs can play a pivotal role in tbc occurrence of T2. Abolition of the Ca2’ entry by suppressing the external Ca2’ (n = 4) or by applying the inorganic 02’ channel blocker Cd2’ (300 pM, n = 3) inhibited both TI and T2 components (Fig. 1C). Con- versely, an increase of external Ca’+ con~n~tion is able to amplify T2 and T1 fn = 3) or to trigger this cornpot%& when it was pm~ionsly absent in the in- itiaI current (n = 2). Tbesc results indicate tbat Ca2* influx is obviously a necessary condition for the oc- curmnce of the T2 component in mlWA injected oocytes.

In order to clearly determine the link between the 112 component and the expressed VDCC activity, the properties of voltage dependence of this component were correlated to those of Tl and IB*, respec- tively. As descrii in Fzgnxe 2A, T2, TI and 1Ba are all activated at the same voltage threshold and the maxim& current ~~~ for all these cnmznts was recoded at the same voltage value. Beyond this value, the current amplitude of Tr, T2 and 1Ba

decreased when the membrane potentiaI was made mom positive. However, if 1~~ and T2 cao@ot be ~~~ in a simple way because of the &imic voltage dtyendtmx properties of Cl- flux through

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l%g. 2 Dependence of tk ‘I’2 component upon VDCC activity expressed b RNA injected occytes. (A) W relatboships of TI and T2 armposlen$~m~dtothatofe~~IBa~indu~eocn, bcmmentsbthe memhmne voBag~ wem performed from a

hoklbg potential of-80 mV. ?%a insets mpresented heside the I/V curves are, mqectively, the chbkle cunents (upper traces) and Be

~WRWS (lower traces) elicited at the different munbmnc vokagca indicated at ead~ trace. @) Phammcobgkal bhibition of the

outward chloride current0 (-Ii and Tz, left panel) induced by the crude Ag&nops~ uptia venom contabbg the two specific toxins of P-type channels: E;Tx and o_Aga-IV A. ‘I% right pa1~1 reprments tk blockade of the comqonding Be cucmnt induced on the same

cell by Aga V. Membrane voltage was stepped tiom -80 to +lO mV. (C) Left panel: spontaneous occmmnm of the T2 component in

an oocyte injected with cbmd P-type cabium channel. Right panel: trig@ng of the T2 compormt fbllowiag btrace~lular application

of InsPj into cRNA injected occytcs. Membrane voltage was stepped from -80 to +I0 mV.

416 CELL CALCXUM

evolution of Tl and T2 ampliMe versus time presented in Figun! 3B shows that the IMP3 effect induced by an unique injectiou of Ins& into oocytes principally consists in a progressive boost of T2

while the Ca” entry represented by Tl stays con- stant Following InsP3 injection, the T2 amplitude

grows up to reach a maximal value and then slowly decreases to return back towaxls its iuitial value.

When oocytes were injected with the (Ztr5) isomer of II&?, which is not a substrate of the InsI+-3&i- nase, the amplitude of T2 was also po~tia~ but the effect was quite obviously per&e.@ (n = 3, not shown) suggesting that the slow decrease of T2 during the In& rasponse (Fig. 3B) was mainly due to the metabolic degradation of hsP3 over the time,

In ad~tion, caffeine (10 mhQ, which is a puta-

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Time (dn)

Fig. 3 Involvement of the IusR seusitive stores hn the emergence of Tz. (A) T&e curmnt (TFMJ evoked by the release of Cal4 after flash photolysis of wed-lnsF% (Ins%FL) dunhg a &polarizatkm step to +20 mV from -SO mV. The flash was deiivemd 350 ms a&r the b&m&g of the dcp&rimtion. (-SW20 mV). The ioset mpmsents TM curcent e&&d wbnt the ceE was contiouously maiu- tained at a l&t@ potential of -SO mV. Tbe flash was dclkred m dcsc&ed in Materials and methods and caged-Ios& was injected 1 h prior to the experiment. (B) Evolution of TI and Ta components vetxus time following iutaacclhdar injection of ha+& (2 pmok). Thetimcof~e~queI~inpaionwasindiartedbythesmucK. Th:~tnet~~ofT1andT2waslncrarundasindicattdinthe inset. Far all cum&s pmsantcd, the holding potential was -SO mV aad the membmoc voltage was transiently stepped to +20 mV. Examples of currents fec&ed at the iudicated times are alno preseuted.

P-TYPE VDCC~ & lNSp3 MEEPTO~ FlJNCl’lONAL CGUPLlNG 417

A

B

tive inhibitor of the InsP3 response in oocytes arxi

without emptying the intracellular pools 1313, typi- cally inhibited Tz when used in ex&ac&&u appli- cations (Hg. 4A, n = 4). The injection of low mole- cular weight heparin (50 @ml. final concentration in oocytes), a competitive antagonist of the IusP3 re-

ceptor in numerous systems including oocytes

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Time (ah)

[ 14,321, completely abolished the T2 component

while no effect was ever detected on the Tl comp0

nent (Fig. 4B, n = 4).

-fore, the second ant T2 induced by Ca2’ entry through putative P-type VDCCs appears to be linked, in addition, to a Ca2’ release from InsP3-sensitive Ca2+ stores. Acconiing to our R.- suits, one may propose that the Ca2’ transient T2

was mediated by a Ca2’ release fkom the InsP3-sen-

sitive stores enslaved to the rapid modification of Ca2’ entry directed by ~tivation of VDCCs.

In order to determine more precisely the fimc- tional link between the early Ca2+ entry through VDCCs and the Ca2+ release from. the InsF%ensi- tive stores, different amounts of Ca2+ were directly injected into oocytes. The aim of these ex

Eri=nb was to establish the dependence of the Ca release upon different cytosolic Ca2+ inactions. Injec- tion of small amounts of Ca2+ into oocytes (0.1-0.3 pmoles) ki to a potent&ion of T2 amplitude (Fig. 5A, n = 3) or even mom could trigger this component when it was p~vio~ly absent in the initial outward chloride cunent (Fig. 5B, n = 2). Converse1

& direct iutraoocyte injection of higher doses of Ca (10 pmoies) markedly reduced the amplitude of T2

(Fig. 5C, n = 3). These results suggest that the cytosolic Ca2+ can exert differential effects depending on its concentration.

These ~sul~~int out that the transient z&ease which is represented by the T2 current arises because the en@ of Ca2’ into the oocyte through the VDCCs may lead to a moderate increase in cyto- s0I.k Ca2+ concentration which, in turn, triggers Ca2+ release from the InsP3 stem.

Lastly, we have compred the InsP3-evoked re-

sponses obtained with two difkrent levels of Ca2’ entry into oocytes. The goal of these experiments was to deduce the influence of the depolarkation-induced Ca2’ influx on the qualitative properties of the InsP3/Ca2+ Greg. For this issue, we have compared the properties of the specific InsP3 currents from native oocytes (unin’ with cerebellar mRNA), where the inward Fted Ca + current is weak, to those of RNA-injected oocytes expressing a large density of Ca2+ channels in their plasma membrane. In native control oocytea bathed in a nominal Ca2+ medium, the specific net In@3 qmse (the control cumznt was subtracted fium the maximal current

418

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Fig. 5 DiKemntial modulation of Tz by direct injection of Ca” into oocyte. Modulation of the Ca2’ release by the cytosolic csz’. The tmces marked 1 nprescnt cunents before the Ca2’ injection (control currents). The traces madced 2 represent the cmrents recotded at the maximal effect observed after Ca2’ injec-

tion. (A) Potentiation of Tz amplitude induced by an unique injection of a small amount of Ca2’ (0.3 pmoles) into oocyte. (B) Triggering of T2 induced by an unique injection of a small amount of Ca2’ (0.3 pmoles) into oocyte. (C) Reduction of Tz amplitude when a high amount of Ca2+ (10 pmoles) was injected into oocyte. For all currents presented, the holding potential was -80 mv and the membrane voltage was transiently stepped

t20 mV.

&Cited itY the presence Of III&) Con&S in

passive variation of the chloride current arising at both the holding and the depolarixed levels (Pig. 6A, upper panel, n = 7). No T24ike component was ever discernible during the depolarixation-evoked current after the injection of InsP3 in native oocytes. The typical I/V curves of the maximal InsP3 current

CELL CALCIUM

recorded in a control oocyte presented in Figure 6A (lower panel) shows that the InsP3-induced current grows up rather passively with the increments of depolarization. Only an outward rectification could be detected when the membrane voltage was modi- tied indicating that Ca2+ entry through native VDCCs has no signitlcant intluence on the InsP3 response. Conversely, the increase of the rate of Ca2+ entry through the plasma membrane by expressing additional VDCCs in RNA-injected oocytes dra- matically modify the nature of the InsP3 response. Indeed, as described in Figure 6B (upper panel) and as already noted earlier (see Fig. 3), the net InsP3 response in RNA-injected cells exclusively consists of the appearance of the smooth hump T2. Further- more, the I/V curve of the maximal InsP34nduced current demonstrates that the InsP3 response gains a new shape of voltage dependence which is close to that of the expressed VDCCs (Fig. 6B, lower panel). It may be concluded that the VDCCs impart their excitable features to the Ca2’ release pmss. This transfer of properties seems to be specifically mediated by the expected Ca2’ elevation in cytosol following VDCC activation. Indeed, InsP3 responses obtained in RNA injected oocytes bathed in Ca2’ free medium become again quite similar to the typical responses obtained in uninjected oocytes (results not illustrated).

Discussion

The involvement of the InsP3-sensitive stores in T2 occurrence was defined in our study by using InsP3 receptor antagonists (heparin and caffeine), by InsP3 flash photolysis during the depolarization step, and finally by the effect of direct injection of InsP3 into oocytes. In addition, ruthenium red, dantmlene and ryanodine did not affect the transient Ca2’ &ease (data not illustrated). This indicates that Ca2+-induced Ca2’ release operating via the ryanodine-sensitive channels is not involved in the T2 response. Testing the activity of ryanodine receptor inhibitors appeared to be warranted in our investigation since the theory predicts that the ryanodine-gated channels would be expressed in cerebellar RNA injected oocytes too. Immunological and biochemical experiments have recently revealed the complete ab-

P-TYPE VDCCs & INSP3 RECJZITORS FUNCITONAL CGUPLING 419

sent.% of tbe lyanodine receptor in oocyres, confer- cations of a?+ rele in xt?nf3ps octqtes by a2’ ring to the InsP3 receptor a pivotal role in tbe b&c- tion of both Ca2+ osci&tions and waves in this ceil

iofhuxes evoked either by ~~ or by h~~~~ have been recently provided

r141* [2?33,34f. T2 amplitude evolves in a voltige-dependeut

manner and the pharmsbcological sensitivity of the T2 component to AgaV indicates that tbe channels responsible for the inward Ca2’ current underlying Tz were actily the expressed P-type VDCCs. This point was confii by using the clone encoding for motive P-type ca2’ channels.

Therefore, the expe&kents presented here sbow that the release of Ca2+ fkom ~~~~~ stores, visualized as the membrane etnrent 1‘2, was closely de~n~ent upon the variation of the P-type induced Ca &~EL Similar observations showing modifi-

Several grwps have pointed out tbe role of Ca2+ as a coagonist of the InsP3 xeceptor [8,11,35]. This positive effect on JBsP3-sensitive stoze axon induced by ca2’ itself bas been demon&a&d in Xeqnus oocytes 114,341. In our study, we have shown that direct intmoocyte injection of small amounts of Ca2+ could amplify or trigger tbe T2 comJlolle3l~ These d&a showing It faciBt&on of the Ca refease from ~-~ti~ stozes argue for a direct sensitiz.&ioo of the Ins& receptor by cyt~sok fke ca2’ and were relevant enouq to pmpose tbe cellular me&a&m by which Ca + entry tbrougb

420 CELL CALCIUM

VDCCs acts on the Ca2+ release from InsPs-sensitive stores. One may put forward that Ca2+ influx induced following depolarization leads to a transient increase of cytosolic free Ca2’ (visualized by the Tt component) near the external side of hSP3 receptors which are connected to the stores just beneath the plasma membrane. This local Ca2+ elevation is able, in turn, to sensitize the InsPa receptor to the ambient htsPs concentration, and to switch on the Ca2+ release process.

Local eon of Ca2+ is edify condi- tioned by the rate of Ca2+ entering through the plasma membrane but is also dependent upon the veloc- ity of Ca2’ entry and the ypcity of Ca2’ bu&ring systems in the cytosol (Ca -ATPases, Ca’+-binding proteins). Such a Ca2’ confin~t can be obtained by transiently overflowing the Ca2+ bufferin capac- ity. Therefore, following a swift flowing Ca + entry through expressed VDCCs, the Ca” concentration probably becomes high enough to produce the effect of facilitation on the InsPs receptors. The fact that Tz was mainly observed in RNA injected oocytes, where the density of VDCCs was obvious1 en-

It hanced, supports the hypothesis that the Ca release sensitization would occur only when the Ca” influx through plasma membrane was sufficiently extensive.

An alternative to the proposed model is that calcium entry stimulates PLC to promote small amounts of InsPs which can account for Tz. How- ever, recent studies have already discussed this possibility and the conclusions, based on the shape of the Tz curmt (smooth graded) and the propaga- tion of calcium into the oocyte, was that a direct activation of the PLC enzyme by cytosolic calcium seems to be unlikely 1271. ‘ibus, the proposed mechanism specifying a direct activation of the calcium release process by calcium itself constitutes, in the oocyte, the most convincing model.

We have observed that injection of high amounts of Ca2’ into oocytes leads to inhibition of the T2

current. This argues for the existence of a dual modulation of the Ix@3 receptor depending on the cytosolic Ca2’ inanition. However, in our experiments and as previously described [12], the in- bibitory effect of Ca’+ takes $ace only for supra- maximal doses of injected Ca . Such amounts of Ca’” lead to submicromolar concentrations of Ca2’

in the cytosol, which are bamly reached under nominal physiological conditions. This indicates that, under physiological conditions, the

Y tentiation of

InsP3 receptor activity induced by Ca ’ is predomi- nant. This dual regulation has been already described in different structures including Xerwpus oocytes, smooth muscle cells, synaptosomederived microsomal vesicles aud cerebellar microsomes [9- 11,141. In our hands, the optimal positive effect was obtained for 0.2-0.4 pmoles of injected Cazt giving the maximal auction at a fiual in- &aooqte coucentration after the Ca2+ injection of about 0.3-0.5 pM, assuming the basal concentration of ca2’ is 0.1 pM [36]. This optimal concentration is close to tbat revealed by Parys and coworkers concerning tbe maximal rate of the InsP3-evoked 45Ca2” efflux from a crude preparation of oocyte microsomes [14].

Even though most of the ItuP3 is confined to re- stricted regions of cytosol, other compartments of the cell would not be absolutely free of IosPs. In these particular regions of we& InsP3 concentrations, the Ca2’ release could be efflciendy activated by ambient InsPs, in so far as the InsPs reayptors have been 2freviously sensitized by Ca (for example, Ca coming from activation of VDCC).

One interesting point emphasized in this work is represented by the typical shape of the T2 response which very much looks like the current evoked by the flash photolysis of pminjected caged-InsPs ho- mogeneously distributed throughout the cell. Since in flash experiments the entire cell was briefly il- lets, tbe TFM current would result from a s~c~no~ liberation of Ca2+ tiom ~ltiple stores which have been described in oocytes as tide- pendent units with differential sensitivity and lib- erating their Cazt in a near all-or-none process 1271. Then, the smooth graded shape of the Tnaah curmnt probably represents an integrative signal derived from a simultaneous activation of numerous stores. As the T2 component presents the same characteristics as mash, we can speculate that tbe cytosolic Ca2+ eIevat.ion (derived here from VDCC activation) would level the differences of sensitivity of Ins-~nsitive stores to give an homog~~us ca2’ release. Therefore, the cytosol could be considered in this way as an excitable synchronizer-medium operating though its basal level of Ca2’.

P-TYPE VDCCs & INSP3 RECEPTORS FWNCK’KGNAL COUPLING 4221

As previously noted, the VIXCs expressed in oocytes from cerebelIar or whole brain mRNA are related to P-type &ham&S [l&25,28]. Immnnolocal- ization experiments showed that these ca2” chau- nels, preferentially expressed in Pu&inje cells of cerebellar cortex, were also encountered throughout the central nervous system (CNS) and particularly at the presynaptic terminals [M-20,37]. Mintx et al. have pointed out that o_Aga-IVA-sensitive P-type channels were responsible for the “‘sCa2+ fluxes monitomd in mammalian brain synaptosomes 1171. Pierre, a recent study ~~ns~~ that glutamate release from these synap~so~s was effec- tively sensitive to the P-type autagonist o_Aga-IVA, suggesting that P charmels are involved in the sy- naptic transmission at the

2t lutamate@c synapse

level of CNS [19]. The Ca entry thmugh pmsy- naptic Ca2’ channels is susceptible to rapidly stimu- lating neurotrausmitter liberation by means of cytosolic Ca2+ elevation. The local concentrations iu the immediate vicinity of Ca2’ channels can culminate in several hundred micromolar valnes (local microdomains of Ca’+ $381) but become rapidly less wheu the Ca2+ diffuses through the ~i~~~g cytosol regions. According to our results, one may propose tbat the InsP3-sensitive stores could enlarge the local microdomains of Ca2+ . This would occur by a sensitization of Ca2’ release through InsPs receptors induced by the Ca2+ entry. In other words, the InsPs- sensitive stores could be consider& as a natural am- plifier relay of the presynaptic Ca’” influx leading to the prolongation of the Ca2+ effects on the neuro- transmitter release.

This work was supperred by grants fmm 1e Minist& de Ia Rwherche et de 1’Enseigncment Sup&iwr and from la R&ion de F’icardie. Fmancial support to PN was f&m le Conscil Regional de P&die, Fman&l support to TC WBS &om Association Fraqaise con&v lera Myopathics. I-ho authors are indebted to Drs P, C%rnet and N. Mori for the generous gift of cRNA encoded for the cakkm cfrannel. We also thank f)rs 3. Nargwt and E. Bourinct for their help with ~gat-MIJa experiments aML I% S. O’Reagan for helpftd diswions. We wish to thank JJ? Pozzo di Borgo and C. Chat&in for technicsf assistance,

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Please send reprint requests to : Dr Franch Foumier, Labor&& de Neurobiologie Celhtkire, UFR des Sciences exactes et fondamentales, Universite de Picardie Jules Verne, 33 Rue Saint Leu, F-80039 Amiens C&x 1, France.

Received : 20 October 1993 Revised : 11 Januaty 1994 Accepted : I4 January 1994

interaction between ca2+ release from inositol trisphosphate sensitive stores and ca2+ entry through...

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