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Matching kinetics of synaptic vesicle recycling and enhanced neurotransmitter influx by Ca2 in brain plasma membrane vesicles
NEUROCHEMISTRY International Neurochemistry International 22 "0887# 288Ð394
Matching kinetics of synaptic vesicle recycling and enhanced neurotransmitter in~ux by Ca1¦ in brain plasma membrane vesicles va Szarics\ Gabriella Nyitrai\ Tamas Blandl\ Julianna Kardos Ilona Kovacs\ E Group of Neurochemistry\ Institute of Chemistry\ Chemical Research Center\ Hungarian Academy of Sciences\ 0414 POB 06\ Pusztaszeri u t 48Ð56\ 0914 Budapest\ Hungary Received 07 March 0887^ accepted 01 June 0887
Abstract Using native plasma membrane vesicle suspensions from the rat cerebral cortex under conditions designed to alter intravesicular ðCa1¦Ł\ we found that Ca1¦ induced 3624) more in~ux of ð2HŁGABA\ ð2HŁD!aspartate and ð2HŁglycine at 26>C with half!times 0[629[4\ 0[229[3 and 0[229[3 min\ respectively[ We labelled GABA transporter sites with the uptake inhibitor\ ð2HŁ!"R\S#!N!ð3\3! bis"2!methyl!1!thienyl#but!2!en!0!ylŁnipecotic acid and found that Ca1¦ induced a partial dissociation of the bound inhibitor from GABA transporter sites with a similar half!time[ By means of rapid kinetic techniques applied to native plasma membrane vesicle suspensions\ containing synaptic vesicles stained with the amphipathic ~uorescent styryl membrane probe N!"2!triethylammonium! propyl#!3!ð3!"dibutylamino#styrylŁpyridinium dibromide\ we have measured the progress of the release and reuptake of synaptic vesicles in response to Ca1¦ and high!ðK ¦Ł depolarization in the 9[9993Ð099 s range of time[ Synaptic vesicle exocytosis\ strongly in~uenced by external ðCa1¦Ł\ appeared with the kinetics accelerated by depolarization[ These results are consistent with the potential involvement of Ca1¦ in taking low!a.nity transporters to the plasma membrane surface via exocytosis[ Þ 0887 Elsevier Science Ltd[ All rights reserved[
0[ Introduction It has been observed previously that Ca1¦ induced the enhancement of GABA ~ux across rat brain plasma membranes "Kardos et al[\ 0883#[ In the present study the e}ect of Ca1¦ on the transmembrane ð2HŁGABA\ ð2HŁD!aspartate and ð2HŁglycine ~ux was assessed at 26>C in native plasma membrane vesicle suspensions\ quickly prepared in physiologic salt solution containing protease inhibitors and antioxidant with minor preparative per! turbation[ By taking as an experimental model the native plasma membrane vesicle suspensions give results on GABAA receptor and GABA transport kinetics similar to the gradient!puri_ed synaptosomal fractions "Cash and Subbarao\ 0876a\ 0876b^ Kardos\ 0883# and neu! ronal cell cultures "Kardos\ 0882#[ Characterized at the EM level "Kardos et al[\ 0880#\ these native plasma mem! brane vesicle suspensions of resealed plasmalemma frag! ments and synaptosomes contains numerous synaptic vesicles[ Corresponding author[ Tel[] ¦25!0!214!8090^ Fax] ¦25!0!214!6443^ E!mail] jkardosÝcric[chemres[hu
Also\ we described the e}ect of Ca1¦ ion on the kinetics and equilibrium binding of the lypophilic derivative of the inhibitory nipecotic acid\ ð2HŁ!"R\S#!N!ð3\3!bis"2! methyl!1!thienyl#but!2!en!0!ylŁnipecotic acid "ð2HŁNNC! 217# to the GABA transporter sites[ Using the ~uorescent N!"2!triethylammoniumpropyl#!3!ð3! "dibutylamino#styrylŁ!pyridinium dibromide "FM0!32#\ whose quantum yield increases greatly upon insertion into membranes\ we have measured the exocytosis of synaptic vesicles in response to Ca1¦ addition under simi! lar conditions[
1[ Materials and methods 1[0[ Preparation of plasma membrane vesicle suspensions Native plasma membrane vesicle suspensions from the cerebral cortex of 3Ð5 weeks old male Wistar rat were prepared as described previously "Serfo¼ zo¼ and Cash\ 0881^ Kardos and Blandl\ 0883^ Kardos et al[\ 0883#[ The rat was killed by decapitation with a guillotine[ The
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cerebral cortex was homogenized by Ultraturrax "Kardos and Blandl\ 0883# in 29 ml of 09 mM HEPESÐNaOH bu}ered\ pH 6[4 9[21 M sucrose solution containing pro! tease inhibitors "phenyl!methanesulfonyl ~uoride\ 0 mM^ aprotinin\ 9[90 mg:ml^ antipain\ leupeptin\ pepstatin A\ 9[994 mg:ml each^ Serfo¼ zo¼ and Cash\ 0881#\ antioxidant "butylated hydroxy!toluene\ 9[91 mM^ Serfo¼ zo¼ and Cash\ 0881# and 0 mM ATP "Kardos et al[\ 0883# "all from Sigma#[ Thereafter 29 ml of bu}er A "in mM] NaCl\ 034^ KCl\ 4^ MgCl1\ 0^ NaHCO2\ 1^ D!glucose\ 09^ ami! nooxyacetate\ 9[0^ ATP\ 0^ HEPES\ 09^ EGTAÐTris\ 0^ pH 6[4^ ðCa1¦Ł 09 nM#^ Turner and Goldin\ 0878^ Kardos et al[\ 0883# or C "in mM] NaCl\ 034^ KCl\ 4^ MgCl1\ 0^ CaCl1\ 0[2^ D!glucose\ 09^ HEPES\ 09^ pH 6[4# was added and the suspension was centrifuged for 3 min\ 169g "Serfo¼ zo¼ and Cash\ 0881#[ The supernatant was centrifuged for 19 min\ 5499g followed by resuspension of the pellet in a glassÐTe~on homogenizer "19 up and down strokes by hand#[ Thereafter the suspension was centrifuged for 04 min\ 3499g "Serfo¼ zo¼ and Cash\ 0881#[ The _nal pellet resuspended as above in either the bu}er A or in the depolarizing\ bu}er B "in mM] NaCl\ 009^ KCl\ 39^ MgCl1\ 0^ NaHCO2\ 1^ D!glucose\ 09^ ami! nooxyacetate\ 9[0^ ATP\ 0^ HEPES\ 09^ EGTAÐTris\ 0^ pH 6[4^ ðCa1¦Ł 09 nM# was adjusted to 9[79 mg:ml of protein concentration assayed by the Folin reagent method "Lowry et al[\ 0840#[ Native plasma membrane vesicle suspension kept on ice for 2 h have retained the initial transport activity[
are presented as mean2S[E[M[ from two experiments using triplicate incubations[ The progress with time of 5[9 nM ð2HŁNNC!217 binding in native plasma membrane vesicle suspensions in bu}er A and B was followed up to the equilibrium at 26>C[ After 09 min\ 9[23 M CaCl1 solution was added "ðCa1¦Ł 1[3 mM after mixing# and the Ca1¦ ion!induced dissociation was measured until the new equilibrium was attained "09 min#[
1[1[ Neurotransmitter in~ux
In~ux data were analysed "Scientist\ MicroMath Version 1[92# according to a _rst!order kinetics^ the _rst!order overall rate constant "k\ mean2standard deviation of least squares _tting# and _nal in~ux "INFf# for ð2HŁGABA\ ð2HŁD!aspartate and ð2HŁglycine in~ux in low! ðCa1¦Ł "INFf−Ca# and high!ðCa1¦Ł "INFf¦Ca# conditions were determined and the corresponding half!times "ln 1:k# were derived[ Activation energy of transport was obtained from the slope "tg a# of the linearized log"k# f"T−0# Arrhenius equation in kcal:M] DE −1[292R tg a[ ð2HŁNCC!217 dissociation curves were calculated according to a _rst!order kinetics as above^ the _nal bound values "Bf−Ca and Bf¦Ca# and the overall rate constant for dissociation "kd\ mean2standard deviation of least squares _tting# in the virtual absence "ðCa1¦Ł 09 nM# and presence "ðCa1¦Ł 1[3 mM# of Ca1¦ ion in low! and high!ðK ¦Ł bu}er were determined and the corresponding half!times derived[ By taking traces of percent ~uorescence decrease mea! sured in the absence of Ca1¦ ion "ðCa1¦Ł 09 nM# as a background\ the di}erence of traces measured in the presence "ðCa1¦Ł 1[3 mM# and absence of Ca1¦ was calculated "percent change of FM0!32 ~uorescence inten! sity# and the on and off rates of the percent change of ~uorescence intensity oberved so far were analysed
The progress with time of 099 nM ð2HŁGABA "50 Ci: mmol\ 1[15 TBq:mmol^ Amersham#\ 099 nM ð2HŁD!aspar! tate "10 Ci:mmol\ 9[66 TBq:mmol^ Amersham# and 0 mM ð2HŁglycine "29 Ci:mmol\ 0[0 TBq:mmol\ Amersham# in~ux in native plasma membrane vesicle suspension in bu}er A was followed up to steady!state at di}erent temperatures "3\ 19\ 29 and 26>C# as described previously "Kardos et al[\ 0883\ 0886#[ Thereafter\ 9[23 M CaCl1 solution was added "1[3 mM _nal# and the time!course of Ca1¦!induced neurotransmitter in~ux was measured up to steady!state and compared[ 1[2[ ð2HŁNNC!217 binding and dissociation Native plasma membrane vesicle suspensions prepared in bu}er A in the presence "1[3 mM# or absence "09 nM# of Ca1¦ ion were incubated with increasing concentration of ð2HŁNNC!217 "34 Ci:mmol\ 0[54 TBq:mmol^ Amer! sham#[ At the end of incubation\ 0 ml suspension was _ltered "Whatman GF:B _lters#\ washed with 1×2 ml ice!cold bu}er and the radioactivity on the _lter was counted by standard liquid scintillation techniques[ Non! speci_c binding was determined in the presence of 099 mM GABA "Braestrup et al[\ 0889#[ Binding parameters
1[3[ FM0!32 ~uorescence Native plasma membrane vesicle suspensions prepared in bu}er C were stained by exposure for 4 min to 04 mM FM0!32 "Molecular Probes# dissolved in high!ðK ¦Ł "Ryan et al[\ 0882^ Betz et al[\ 0881# bu}er D "in mM] NaCl\ 019^ KCl\ 14^ MgCl1\ 0^ CaCl1\ 0[2^ D!glucose\ 09^ HEPES\ 09^ pH 6[4# at 29>C[ Thereafter the suspension was centrifuged for 04 min\ 2499g "Serfo¼ zo¼ and Cash\ 0881#[ The _nal pellet was washed gently three times and was resuspended in bu}er A[ With the application of rapid kinetic techniques using the SF!50DX1 Stopped! Flow UV:Fluorescence System "Hi!Tech# and varied mixing protocols\ traces of percent ~uorescence intensity as a function of time\ each comprising 539 data acquired automatically\ were recorded on a log time base[ Di}erent mixing protocols were characterized by di}erent traces representing running averages from three recordings[ 1[4[ Data analysis
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according to _rst!order kinetics[ First!order overall rate constants "kon and ko}# in low! and high!ðK ¦Ł conditions were determined and the corresponding half!times "ln 1:kon and ln 1:ko}# were derived[
2[ Results 2[0[ Neurotransmitter in~ux E}ects of Ca1¦ ions on transmembrane ð2HŁGABA\ ð2HŁD!aspartate and ð2HŁglycine in~ux in native plasma membrane vesicle suspensions prepared in low!ðCa1¦Ł condition "bu}er A\ ðCa1¦Ł 09 nM# were measured at 26>C "Table 0#[ By the addition of Ca1¦ ions to these plasma membrane vesicles\ enhancements of trans! membrane ð2HŁGABA\ ð2HŁD!aspartate and ð2HŁglycine ~uxes were observed and characterized by the average increase of _nal in~ux values\ 3624) "Table 0[#[ Onset kinetics for these Ca1¦!induced enhancements were alike\ within the precision of determination\ giving rise to the mean half!time of 0[329[1 min "Table 0[#[ The e}ect of the temperature "3Ð26>C# on the rate constants of Ca1¦! induced transmembrane ~uxes of ð2HŁGABA\ ð2HŁD! aspartate and ð2HŁglycine was characterized by a single activation energy of DE 3[929[4 kcal:M "given as mean2S[E[M[ of DE values\ obtained for GABA\ D! aspartate and glycine#[ 2[1[ ð2HŁNCC!217 binding and dissociation Speci_c binding of ð2HŁNCC!217 in native plasma mem! brane vesicle suspensions in low!ðCa1¦Ł condition "bu}er
A\ ðCa1¦Ł 09 nM# at 29>C was saturable\ data analysis showed no indication of multiple binding sites[ Equi! librium parameters for ð2HŁNCC!217 binding were Kd 4324 nM and Bmax 4[120[0 pmol:mg protein at 29>C[ By comparison\ in Tris!citrate bu}er\ containing 0 M NaCl\ a Kd value of 07 nM was obtained at 29>C with the use of the higher a.nity ð2HŁNO!217 enantiomer in rat forebrain membrane suspensions "Braestrup et al[\ 0889#[ The equilibrium binding of ð2HŁNCC!217 was alt! ered by the addition of Ca1¦ "external ðCa1¦Ł 1[3 mM# and characterized by a 0[6!fold increase of Kd "Kd 7426 nM# with Bmax value unchanged within the precision of determination "Bmax 4[520[0 pmol:mg protein#[ The addition of CaCl1 solution "1[3 mM after mixing# to native plasma membrane vesicle suspensions prepared in low!ðCa1¦Ł condition "bu}er A\ ðCa1¦Ł 09 nM# initiated a partial "1725)# dissociation of ð2HŁNCC!217 speci_cally bound to GABA transporter sites in these suspensions with a half!time of 0[729[3 min at 26>C[ Under high!ðK ¦Ł condition\ Ca1¦ induced a more com! plete dissociation "64203)# of the speci_c ð2HŁNNC! 217 binding in native plasma membrane vesicle sus! pensions with a similar half!time of 0[829[3 min at 26>C Table 0[ 2[2[ FM0!32 ~uorescence The e}ects of mixing suspensions of native plasma mem! brane vesicles containing FM0!32 stained synaptic ves! icles with bu}ers\ designed to alter intravesicular ðCa1¦Ł "external ðCa1¦Ł 09 nM and 1[3 mM after mixing# under low! and high!ðK ¦Ł "4 mM and 39 mM after mixing# conditions on the time dependence of percent FM0!32
Table 0 Comparison of the rate parametersa characteristic for Ca1¦!induced enhancements of ð2HŁGABA\ ð2HŁD!aspartate and ð2HŁglycine in~ux and dissociation of ð2HŁNCC!217 binding in native plasma membrane vesicles from the rat cerebral cortex at 26>C Dissociation of ð2HŁNCC!217
In~ux of
Half!time "min# INFf−Ca "pmol:mg# INFf¦Ca "pmol:mg# D"INFf¦Ca−INFf−Ca# ")# Bf−Ca "pmol:mg# Bf¦Ca "pmol:mg# D"Bf−Ca−Bf¦Ca# ")#
ð2HŁGABA
ð2HŁD!ASPb
ð2HŁGlycine
4 mM KCl
39 mM KCl
0[629[4 09621 05223c 4125 Ð Ð Ð
0[229[3 12323 225200c 3325 Ð Ð Ð
0[229[3 820 0220c 33211 Ð Ð Ð
0[729[3 Ð Ð Ð 0[0029[91 9[7929[94c 1725
0[829[3 Ð Ð Ð 9[1729[90 9[9629[92c 64203
a Data were analysed "Scientist\ MicroMath Version 1[92# according to a _rst!order kinetics^ the _rst!order overall rate constant "k#\ _nal in~ux "INFf# for ð2HŁGABA\ ð2HŁD!aspartate and ð2HŁglycine in~ux and _nal bound values "Bf# for ð2HŁNCC! 217 binding in native neuronal plasma membrane vesicle suspensions in the absence "ðCa1¦Ł 09 nM] INFf−Ca or Bf−Ca# and presence "ðCa1¦Ł 1[3 mM] INFf¦Ca or Bf¦Ca# of Ca1¦ ion were determined and the corresponding half!times "ln 1:k or ln 1:kd# were derived[ Data are given as mean2S[E[M[ from 2Ð4 kinetic experiments[ b 2 ð HŁD!ASP] ð2HŁD!aspartate[ c P³9[990\ when steady!state in~ux in low!ðCa1¦Ł bu}er "ðCa1¦Ł 09 nM# is compared "Student|s t!test#[
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Fig[ 0[ The e}ects of mixing suspensions of native plasma membrane vesicle suspensions from the rat cerebral cortex\ containing FM0!32 stained synaptic vesicles with bu}ers\ designed to alter intravesicular ðCa1¦Ł under low! and high!ðK ¦Ł conditions\ on the time dependence of percent FM0! 32 ~uorescence intensity at 29>C[ Traces with di}erent colours refer to bu}ers with di}erent ðCa1¦Ł and ðK ¦Ł] green "background#\ ðCa1¦Ł 09 nM\ ðK ¦Ł 4 mM^ blue\ ðCa1¦Ł 1[3 mM\ ðK ¦Ł 4 mM^ red\ ðCa1¦Ł 09 nM\ ðK ¦Ł 39 mM^ black\ ðCa1¦Ł 1[3 mM\ ðK ¦Ł 39 mM[ Each trace presented here is the average of three repeated experiments performed in triplicate measurements and represents 539 data recorded in the 9[3 ms!099 s range of time[ Fitting an exponential decrease of percent ~uorescence intensity\ the average rate constants from the repeated experiments were similar within 201)[ With pooled data "2 experiments# similar rate constants with 23) precision were obtained\ suggesting that the rate of ~uorescence decrease was not dependent on the batch of native plasma membrane vesicle suspensions[
"ðK ¦Ł 39 mM^ ðCa1¦Ł 1[3 mM^ ðK ¦Ł 39 mM plus ðCa1¦Ł 1[3 mM# "Figure 1#[ In high!ðCa1¦Ł conditions "ðCa1¦Ł 1[3 mM^ ðK ¦Ł 4 and 39 mM# two major cycles were observed] a decrease followed by increase in percent change of ~uorescence intensity below on the order of 099 ms " fast!phase# and a second cycle of increase and decrease above 099 ms "slow!phase#[ Apparently\ there was a little change in decrease of percent change of ~uorescence in the 09Ð099 s range of time when high!ðCa1¦Ł and high!ðCa1¦Ł plus high!ðK ¦Ł conditions were compared[ However\ the fast! and slow! phase cycles obeyed di}erential sensitivity to high!ðK ¦Ł and high!ðCa1¦Ł conditions "Figure 2#[ Comparing fast! and slow!phase cycles\ the slow!phase cycle was strongly in~uenced by the presence of external ðCa1¦Ł 1[3 mM and was less a}ected by depolarization "Fig[ 2#\ whereas the occurrence of the fast!phase cycle showed similar sensitivity to high!ðCa1¦Ł and high!ðK ¦Ł conditions[ Fast! and slow!phase cycles dissected this way can be char! acterized by di}erent on and off rates of the dye with half!times in the order of 07 and 39 ms " fast!phase# and
~uorescence\ are illustrated "Figure 0#[ All of the percent FM0!32 ~uorescence data obtained under di}erent con! ditions showed a decrease with time at di}erent rates[ Characteristically\ these decrease rates were variable when the experimental condition was altered[ The activity!dependent ½4) decrease in percent ~uorescence intensity observed in native plasma membrane vesicle suspensions is rather small change compared to changes found in living motor nerve terminals "Betz et al[\ 0881#[ Using the sucrose!gradient puri_ed synaptosomal frac! tion "Kardos et al[\ 0873#\ our preliminary experiments gave similar results[ The small activity!releasable FM0! 32 component found with these brain homogenates can be due to "i# non!selectivity and:or incomplete washout of the dye^ "ii# incomplete recycling of brain synaptic vesicles[ Transformation of the percent ~uorescence data into the percent change of ~uorescence with the subtraction of background decrease "ðCa1¦Ł 09 nM with ðK ¦Ł 4 mM# showed the di}erences in rate of these decreases obtained under di}erent experimental conditions
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Fig[ 1[ Kinetics of the change of ~uorescence intensity by mixing FM0!32 stained native plasma membrane suspensions from the rat cerebral cortex and low! or high!ðK ¦Ł bu}er with and without Ca1¦ added\ at 29>C[ Background "low!ðCa1¦Ł\ low!ðK ¦Ł# subtracted traces with di}erent colours refer to di}erent conditions] red\ 39 mM KCl^ blue\ 1[3 mM CaCl1^ black\ 39 mM KCl plus 1[3 mM CaCl1[ Each traces are the average of three experiments performed in triplicate measurements[
in~ux by Ca1¦ ion in native plasma membrane vesicle suspensions from neural tissue at 26>C[ Therefore\ we supposed that Ca1¦!sensitive manifestations of trans! porter activities were due to the appearance of low!a.n! ity transporters "Mabjeesh and Kanner\ 0878# on the plasma membrane surface\ via exocytosis[ The temporal separation of changes of ~uorescence intensity in native plasma membrane vesicle suspensions\ containing synaptic vesicles labelled with the ~uorescent indicator FM0!32\ presented the possibility of identifying the associated processes in synaptic vesicle recycling] the increase of ~uorescence with the endocytosis\ and the decrease of ~uorescence with the exocytosis\ since the quantum yield for FM0!32 ~uorescence is much lower in water than in lipid[ Measuring the activity!releasable FM0!32 ~uorescence on a timescale of millisecond to minute\ the separate phases of synaptic vesicle recycling can be resolved on the basis of their di}erent half!times on exposure to high!ðCa1¦Ł and:or high!ðK ¦Ł conditions[ Distinguishable fast! "³099 ms# and slow!phase "×099 ms# components of synaptic vesicle recycling can be char! acterized further by their di}erential sensitivity to the above experimental conditions[ Since the fast!phase cycle is found sensitive to high!ðCa1¦Ł as well as to high!ðK ¦Ł conditions\ one can assume that the fast!phase com!
9[66 and 39 s "slow!phase#\ respectively[ The half!time of the latter response was reduced considerably "04 s# under high!ðK ¦Ł condition[
3[ Discussion In accordance with a decrease in ð2HŁNCC!217 binding a.nity\ the present results show that Ca1¦ induces dis! sociation of the bound ð2HŁNCC!217 from GABA trans! porter sites in Ca1¦!deprived native plasma membrane vesicle suspensions with a half!time similar to those that were found for the enhancements of GABA ~ux by Ca1¦ and for Ca1¦!induced increases of ð2HŁD!aspartate and ð2HŁglycine in~ux at 26>C\ also[ Transport enhancements displaying similar kinetics for di}erent neurotransmitters are characterized by a single activation energy[ Fur! thermore\ the Ca1¦!sensitive component of GABA trans! porter binding is conserved under high!ðK ¦Ł condition[ One of the possible explanations for the above _ndings that these processes are related[ The kinetics of glucose transporters| tra.cking in non!neural tissue at 26>C indi! cate that exocytosis occurs with a half!time of about 1 min "Yang et al[\ 0881# resembling the half!times obtained for the enhancements of GABA\ D!aspartate and glycine
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Fig[ 2[ The e}ects of the change in the external ðCa1¦Ł and ðK ¦Ł on the kinetics of the change of ~uorescence intensity in FM0!32 stained native plasma membrane suspensions from the rat cerebral cortex at 29>C[ Blue trace] di}erential kinetics obtained by subtracting kinetics of change of ~uorescence displayed in the ðK ¦Ł 39 mM condition from that of the ðK ¦Ł 39 mM plus ðCa1¦Ł 1[3 mM condition[ Red trace] di}erential kinetics obtained by subtracting kinetics of change of ~uorescence displayed in the ðCa1¦Ł 1[3 mM condition from that of the ðK ¦Ł 39 mM plus ðCa1¦Ł 1[3 mM condition[
Ca1¦!sensitive neurotransmitter uptake takes place after exocytosis has terminated[ The matching kinetics observed in Ca1¦!induced enhancements of the transmembrane ~ux of di}erent neurotransmitters\ GABA transporter binding\ and exocytosis in native neuronal plasma membrane vesicles at 26>C suggest a role for Ca1¦ in neurotransmitter trans! port by taking low!a.nity transporters to the plasma membrane surface via exocytosis[
ponent of synaptic vesicle recycling is due to the acti! vation of Ca1¦ ion permeable\ voltage!dependent N! methyl!D!aspartate "NMDA# receptor channel by endogenous glutamate[ Subsequent to NMDA receptor activation\ the neurotransmitter release inherent of exocytosis may possibly be occurred[ Appeared under high external Ca1¦ condition with a rate accelerated by depolarization\ the major slow!phase cycle observed in native plasma membrane vesicle sus! pensions from the rat cerebral cortex in the 9[0 to 099 s range of time is supposed to be observed by optical monitoring of synaptic vesicle recycling in living nerve terminals "Betz and Bewick\ 0881^ Ryan et al[\ 0882^ Ryan and Smith\ 0884#\ although the minor capacity fast!phase cycle may also contributed to the response[ Reportedly\ one complete cycle of neurotransmitter release by exocytosis was estimated to take about 59 s "Betz and Bewick\ 0881#[ This cycle includes endocytic reuptake of vesicle membrane which lags exocytosis with a half!time of about 19 s as well as repriming of an endocytosed synaptic vesicle with a minimum time on the order of 04 s "Ryan and Smith\ 0884#[ In native plasma membrane vesicle suspensions from the rat cerebral cor! tex at 29>C\ the slow!phase exocytosis occurs over a time scale with a half!time of 39 s\ suggesting that the bulk of
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Acknowledgements J[K[ was supported by grants OTKA T 3929\ T 08292 and AKP 85:1!313 1\3 "Hungary#[ The skillful assistance of Mrs Erzsebet Fekete!Kuti is gratefully acknowledged[
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Kardos\ J[\ Kovacs\ I[\ Blandl\ T[\ Cash\ D[J[\ Simon!Trompler\ E[\ Luyen\ Ng[D[\ Dornyei\ G[\ Simonyi\ M[\ Blasko\ G[\ Szantay\ Cs[\ 0886[ Inhibition of g!aminobutyric acid uptake by bicuculline analogues[ Eur[ J[ Pharmacol[ 226\ 72Ð75[ Lowry\ O[H[\ Rosebrough\ N[J[\ Farr\ A[L[\ Randall\ R[J[\ 0840[ Pro! tein measurements with the folin phenol reagent[ J[ Biol[ Chem[ 082\ 154Ð164[ Mabjeesh\ N[J[\ Kanner\ B[I[\ 0878[ Low!a.nity g!aminobutyric acid transport in rat brain[ Biochemistry 17\ 6583Ð6588[ Ryan\ T[A[\ Reuter\ H[\ Wendland\ B[\ Schweizer\ F[E[\ Tsien\ R[W[\ Smith\ S[J[\ 0882[ The kinetics of synaptic vesicle recycling measured at single presynaptic boutons[ Neuron 00\ 602Ð613[ Ryan\ T[A[\ Smith\ S[J[\ 0884[ Vesicle pool mobilization during action potential _ring at hippocampal synapses[ Neuron 03\ 872Ð 878[ Serfo¼ zo¼ \ P[\ Cash\ D[J[\ 0881[ E}ect of a benzodiazepine "chlor! diazepoxide# on a GABA receptor from rat brain] requirement of only one bound GABA molecule for channel opening[ FEBS Lett[ 209\ 44Ð48[ Turner\ T[J[\ Goldin\ S[M[\ 0878[ Multiple components of syn! aptosomal ð2HŁ!g!aminobutyric acid release by a rapid superfusion system[ Biochemistry 17\ 475Ð482[ Yang\ J[\ Clark\ A[E[\ Harrison\ R[\ 0881[ Tra.cking of glucose!trans! porters in 2T2!L0 cells[ Inhibition of tra.cking by phenylarsine oxide implicates a slow dissociation of transporters from tra.cking protein[ Biochem[ J[ 170\ 798Ð706[
Matching kinetics of synaptic vesicle recycling and enhanced neurotransmitter in~ux by Ca1¦ in brain plasma membrane vesicles va Szarics\ Gabriella Nyitrai\ Tamas Blandl\ Julianna Kardos Ilona Kovacs\ E Group of Neurochemistry\ Institute of Chemistry\ Chemical Research Center\ Hungarian Academy of Sciences\ 0414 POB 06\ Pusztaszeri u t 48Ð56\ 0914 Budapest\ Hungary Received 07 March 0887^ accepted 01 June 0887
Abstract Using native plasma membrane vesicle suspensions from the rat cerebral cortex under conditions designed to alter intravesicular ðCa1¦Ł\ we found that Ca1¦ induced 3624) more in~ux of ð2HŁGABA\ ð2HŁD!aspartate and ð2HŁglycine at 26>C with half!times 0[629[4\ 0[229[3 and 0[229[3 min\ respectively[ We labelled GABA transporter sites with the uptake inhibitor\ ð2HŁ!"R\S#!N!ð3\3! bis"2!methyl!1!thienyl#but!2!en!0!ylŁnipecotic acid and found that Ca1¦ induced a partial dissociation of the bound inhibitor from GABA transporter sites with a similar half!time[ By means of rapid kinetic techniques applied to native plasma membrane vesicle suspensions\ containing synaptic vesicles stained with the amphipathic ~uorescent styryl membrane probe N!"2!triethylammonium! propyl#!3!ð3!"dibutylamino#styrylŁpyridinium dibromide\ we have measured the progress of the release and reuptake of synaptic vesicles in response to Ca1¦ and high!ðK ¦Ł depolarization in the 9[9993Ð099 s range of time[ Synaptic vesicle exocytosis\ strongly in~uenced by external ðCa1¦Ł\ appeared with the kinetics accelerated by depolarization[ These results are consistent with the potential involvement of Ca1¦ in taking low!a.nity transporters to the plasma membrane surface via exocytosis[ Þ 0887 Elsevier Science Ltd[ All rights reserved[
0[ Introduction It has been observed previously that Ca1¦ induced the enhancement of GABA ~ux across rat brain plasma membranes "Kardos et al[\ 0883#[ In the present study the e}ect of Ca1¦ on the transmembrane ð2HŁGABA\ ð2HŁD!aspartate and ð2HŁglycine ~ux was assessed at 26>C in native plasma membrane vesicle suspensions\ quickly prepared in physiologic salt solution containing protease inhibitors and antioxidant with minor preparative per! turbation[ By taking as an experimental model the native plasma membrane vesicle suspensions give results on GABAA receptor and GABA transport kinetics similar to the gradient!puri_ed synaptosomal fractions "Cash and Subbarao\ 0876a\ 0876b^ Kardos\ 0883# and neu! ronal cell cultures "Kardos\ 0882#[ Characterized at the EM level "Kardos et al[\ 0880#\ these native plasma mem! brane vesicle suspensions of resealed plasmalemma frag! ments and synaptosomes contains numerous synaptic vesicles[ Corresponding author[ Tel[] ¦25!0!214!8090^ Fax] ¦25!0!214!6443^ E!mail] jkardosÝcric[chemres[hu
Also\ we described the e}ect of Ca1¦ ion on the kinetics and equilibrium binding of the lypophilic derivative of the inhibitory nipecotic acid\ ð2HŁ!"R\S#!N!ð3\3!bis"2! methyl!1!thienyl#but!2!en!0!ylŁnipecotic acid "ð2HŁNNC! 217# to the GABA transporter sites[ Using the ~uorescent N!"2!triethylammoniumpropyl#!3!ð3! "dibutylamino#styrylŁ!pyridinium dibromide "FM0!32#\ whose quantum yield increases greatly upon insertion into membranes\ we have measured the exocytosis of synaptic vesicles in response to Ca1¦ addition under simi! lar conditions[
1[ Materials and methods 1[0[ Preparation of plasma membrane vesicle suspensions Native plasma membrane vesicle suspensions from the cerebral cortex of 3Ð5 weeks old male Wistar rat were prepared as described previously "Serfo¼ zo¼ and Cash\ 0881^ Kardos and Blandl\ 0883^ Kardos et al[\ 0883#[ The rat was killed by decapitation with a guillotine[ The
9086!9075:87:, ! see front matter Þ 0887 Elsevier Science Ltd[ All rights reserved[ PII] S 9 0 8 6 ! 9 0 7 5 " 8 7 # 9 9 9 2 8 ! 3
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cerebral cortex was homogenized by Ultraturrax "Kardos and Blandl\ 0883# in 29 ml of 09 mM HEPESÐNaOH bu}ered\ pH 6[4 9[21 M sucrose solution containing pro! tease inhibitors "phenyl!methanesulfonyl ~uoride\ 0 mM^ aprotinin\ 9[90 mg:ml^ antipain\ leupeptin\ pepstatin A\ 9[994 mg:ml each^ Serfo¼ zo¼ and Cash\ 0881#\ antioxidant "butylated hydroxy!toluene\ 9[91 mM^ Serfo¼ zo¼ and Cash\ 0881# and 0 mM ATP "Kardos et al[\ 0883# "all from Sigma#[ Thereafter 29 ml of bu}er A "in mM] NaCl\ 034^ KCl\ 4^ MgCl1\ 0^ NaHCO2\ 1^ D!glucose\ 09^ ami! nooxyacetate\ 9[0^ ATP\ 0^ HEPES\ 09^ EGTAÐTris\ 0^ pH 6[4^ ðCa1¦Ł 09 nM#^ Turner and Goldin\ 0878^ Kardos et al[\ 0883# or C "in mM] NaCl\ 034^ KCl\ 4^ MgCl1\ 0^ CaCl1\ 0[2^ D!glucose\ 09^ HEPES\ 09^ pH 6[4# was added and the suspension was centrifuged for 3 min\ 169g "Serfo¼ zo¼ and Cash\ 0881#[ The supernatant was centrifuged for 19 min\ 5499g followed by resuspension of the pellet in a glassÐTe~on homogenizer "19 up and down strokes by hand#[ Thereafter the suspension was centrifuged for 04 min\ 3499g "Serfo¼ zo¼ and Cash\ 0881#[ The _nal pellet resuspended as above in either the bu}er A or in the depolarizing\ bu}er B "in mM] NaCl\ 009^ KCl\ 39^ MgCl1\ 0^ NaHCO2\ 1^ D!glucose\ 09^ ami! nooxyacetate\ 9[0^ ATP\ 0^ HEPES\ 09^ EGTAÐTris\ 0^ pH 6[4^ ðCa1¦Ł 09 nM# was adjusted to 9[79 mg:ml of protein concentration assayed by the Folin reagent method "Lowry et al[\ 0840#[ Native plasma membrane vesicle suspension kept on ice for 2 h have retained the initial transport activity[
are presented as mean2S[E[M[ from two experiments using triplicate incubations[ The progress with time of 5[9 nM ð2HŁNNC!217 binding in native plasma membrane vesicle suspensions in bu}er A and B was followed up to the equilibrium at 26>C[ After 09 min\ 9[23 M CaCl1 solution was added "ðCa1¦Ł 1[3 mM after mixing# and the Ca1¦ ion!induced dissociation was measured until the new equilibrium was attained "09 min#[
1[1[ Neurotransmitter in~ux
In~ux data were analysed "Scientist\ MicroMath Version 1[92# according to a _rst!order kinetics^ the _rst!order overall rate constant "k\ mean2standard deviation of least squares _tting# and _nal in~ux "INFf# for ð2HŁGABA\ ð2HŁD!aspartate and ð2HŁglycine in~ux in low! ðCa1¦Ł "INFf−Ca# and high!ðCa1¦Ł "INFf¦Ca# conditions were determined and the corresponding half!times "ln 1:k# were derived[ Activation energy of transport was obtained from the slope "tg a# of the linearized log"k# f"T−0# Arrhenius equation in kcal:M] DE −1[292R tg a[ ð2HŁNCC!217 dissociation curves were calculated according to a _rst!order kinetics as above^ the _nal bound values "Bf−Ca and Bf¦Ca# and the overall rate constant for dissociation "kd\ mean2standard deviation of least squares _tting# in the virtual absence "ðCa1¦Ł 09 nM# and presence "ðCa1¦Ł 1[3 mM# of Ca1¦ ion in low! and high!ðK ¦Ł bu}er were determined and the corresponding half!times derived[ By taking traces of percent ~uorescence decrease mea! sured in the absence of Ca1¦ ion "ðCa1¦Ł 09 nM# as a background\ the di}erence of traces measured in the presence "ðCa1¦Ł 1[3 mM# and absence of Ca1¦ was calculated "percent change of FM0!32 ~uorescence inten! sity# and the on and off rates of the percent change of ~uorescence intensity oberved so far were analysed
The progress with time of 099 nM ð2HŁGABA "50 Ci: mmol\ 1[15 TBq:mmol^ Amersham#\ 099 nM ð2HŁD!aspar! tate "10 Ci:mmol\ 9[66 TBq:mmol^ Amersham# and 0 mM ð2HŁglycine "29 Ci:mmol\ 0[0 TBq:mmol\ Amersham# in~ux in native plasma membrane vesicle suspension in bu}er A was followed up to steady!state at di}erent temperatures "3\ 19\ 29 and 26>C# as described previously "Kardos et al[\ 0883\ 0886#[ Thereafter\ 9[23 M CaCl1 solution was added "1[3 mM _nal# and the time!course of Ca1¦!induced neurotransmitter in~ux was measured up to steady!state and compared[ 1[2[ ð2HŁNNC!217 binding and dissociation Native plasma membrane vesicle suspensions prepared in bu}er A in the presence "1[3 mM# or absence "09 nM# of Ca1¦ ion were incubated with increasing concentration of ð2HŁNNC!217 "34 Ci:mmol\ 0[54 TBq:mmol^ Amer! sham#[ At the end of incubation\ 0 ml suspension was _ltered "Whatman GF:B _lters#\ washed with 1×2 ml ice!cold bu}er and the radioactivity on the _lter was counted by standard liquid scintillation techniques[ Non! speci_c binding was determined in the presence of 099 mM GABA "Braestrup et al[\ 0889#[ Binding parameters
1[3[ FM0!32 ~uorescence Native plasma membrane vesicle suspensions prepared in bu}er C were stained by exposure for 4 min to 04 mM FM0!32 "Molecular Probes# dissolved in high!ðK ¦Ł "Ryan et al[\ 0882^ Betz et al[\ 0881# bu}er D "in mM] NaCl\ 019^ KCl\ 14^ MgCl1\ 0^ CaCl1\ 0[2^ D!glucose\ 09^ HEPES\ 09^ pH 6[4# at 29>C[ Thereafter the suspension was centrifuged for 04 min\ 2499g "Serfo¼ zo¼ and Cash\ 0881#[ The _nal pellet was washed gently three times and was resuspended in bu}er A[ With the application of rapid kinetic techniques using the SF!50DX1 Stopped! Flow UV:Fluorescence System "Hi!Tech# and varied mixing protocols\ traces of percent ~uorescence intensity as a function of time\ each comprising 539 data acquired automatically\ were recorded on a log time base[ Di}erent mixing protocols were characterized by di}erent traces representing running averages from three recordings[ 1[4[ Data analysis
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according to _rst!order kinetics[ First!order overall rate constants "kon and ko}# in low! and high!ðK ¦Ł conditions were determined and the corresponding half!times "ln 1:kon and ln 1:ko}# were derived[
2[ Results 2[0[ Neurotransmitter in~ux E}ects of Ca1¦ ions on transmembrane ð2HŁGABA\ ð2HŁD!aspartate and ð2HŁglycine in~ux in native plasma membrane vesicle suspensions prepared in low!ðCa1¦Ł condition "bu}er A\ ðCa1¦Ł 09 nM# were measured at 26>C "Table 0#[ By the addition of Ca1¦ ions to these plasma membrane vesicles\ enhancements of trans! membrane ð2HŁGABA\ ð2HŁD!aspartate and ð2HŁglycine ~uxes were observed and characterized by the average increase of _nal in~ux values\ 3624) "Table 0[#[ Onset kinetics for these Ca1¦!induced enhancements were alike\ within the precision of determination\ giving rise to the mean half!time of 0[329[1 min "Table 0[#[ The e}ect of the temperature "3Ð26>C# on the rate constants of Ca1¦! induced transmembrane ~uxes of ð2HŁGABA\ ð2HŁD! aspartate and ð2HŁglycine was characterized by a single activation energy of DE 3[929[4 kcal:M "given as mean2S[E[M[ of DE values\ obtained for GABA\ D! aspartate and glycine#[ 2[1[ ð2HŁNCC!217 binding and dissociation Speci_c binding of ð2HŁNCC!217 in native plasma mem! brane vesicle suspensions in low!ðCa1¦Ł condition "bu}er
A\ ðCa1¦Ł 09 nM# at 29>C was saturable\ data analysis showed no indication of multiple binding sites[ Equi! librium parameters for ð2HŁNCC!217 binding were Kd 4324 nM and Bmax 4[120[0 pmol:mg protein at 29>C[ By comparison\ in Tris!citrate bu}er\ containing 0 M NaCl\ a Kd value of 07 nM was obtained at 29>C with the use of the higher a.nity ð2HŁNO!217 enantiomer in rat forebrain membrane suspensions "Braestrup et al[\ 0889#[ The equilibrium binding of ð2HŁNCC!217 was alt! ered by the addition of Ca1¦ "external ðCa1¦Ł 1[3 mM# and characterized by a 0[6!fold increase of Kd "Kd 7426 nM# with Bmax value unchanged within the precision of determination "Bmax 4[520[0 pmol:mg protein#[ The addition of CaCl1 solution "1[3 mM after mixing# to native plasma membrane vesicle suspensions prepared in low!ðCa1¦Ł condition "bu}er A\ ðCa1¦Ł 09 nM# initiated a partial "1725)# dissociation of ð2HŁNCC!217 speci_cally bound to GABA transporter sites in these suspensions with a half!time of 0[729[3 min at 26>C[ Under high!ðK ¦Ł condition\ Ca1¦ induced a more com! plete dissociation "64203)# of the speci_c ð2HŁNNC! 217 binding in native plasma membrane vesicle sus! pensions with a similar half!time of 0[829[3 min at 26>C Table 0[ 2[2[ FM0!32 ~uorescence The e}ects of mixing suspensions of native plasma mem! brane vesicles containing FM0!32 stained synaptic ves! icles with bu}ers\ designed to alter intravesicular ðCa1¦Ł "external ðCa1¦Ł 09 nM and 1[3 mM after mixing# under low! and high!ðK ¦Ł "4 mM and 39 mM after mixing# conditions on the time dependence of percent FM0!32
Table 0 Comparison of the rate parametersa characteristic for Ca1¦!induced enhancements of ð2HŁGABA\ ð2HŁD!aspartate and ð2HŁglycine in~ux and dissociation of ð2HŁNCC!217 binding in native plasma membrane vesicles from the rat cerebral cortex at 26>C Dissociation of ð2HŁNCC!217
In~ux of
Half!time "min# INFf−Ca "pmol:mg# INFf¦Ca "pmol:mg# D"INFf¦Ca−INFf−Ca# ")# Bf−Ca "pmol:mg# Bf¦Ca "pmol:mg# D"Bf−Ca−Bf¦Ca# ")#
ð2HŁGABA
ð2HŁD!ASPb
ð2HŁGlycine
4 mM KCl
39 mM KCl
0[629[4 09621 05223c 4125 Ð Ð Ð
0[229[3 12323 225200c 3325 Ð Ð Ð
0[229[3 820 0220c 33211 Ð Ð Ð
0[729[3 Ð Ð Ð 0[0029[91 9[7929[94c 1725
0[829[3 Ð Ð Ð 9[1729[90 9[9629[92c 64203
a Data were analysed "Scientist\ MicroMath Version 1[92# according to a _rst!order kinetics^ the _rst!order overall rate constant "k#\ _nal in~ux "INFf# for ð2HŁGABA\ ð2HŁD!aspartate and ð2HŁglycine in~ux and _nal bound values "Bf# for ð2HŁNCC! 217 binding in native neuronal plasma membrane vesicle suspensions in the absence "ðCa1¦Ł 09 nM] INFf−Ca or Bf−Ca# and presence "ðCa1¦Ł 1[3 mM] INFf¦Ca or Bf¦Ca# of Ca1¦ ion were determined and the corresponding half!times "ln 1:k or ln 1:kd# were derived[ Data are given as mean2S[E[M[ from 2Ð4 kinetic experiments[ b 2 ð HŁD!ASP] ð2HŁD!aspartate[ c P³9[990\ when steady!state in~ux in low!ðCa1¦Ł bu}er "ðCa1¦Ł 09 nM# is compared "Student|s t!test#[
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Fig[ 0[ The e}ects of mixing suspensions of native plasma membrane vesicle suspensions from the rat cerebral cortex\ containing FM0!32 stained synaptic vesicles with bu}ers\ designed to alter intravesicular ðCa1¦Ł under low! and high!ðK ¦Ł conditions\ on the time dependence of percent FM0! 32 ~uorescence intensity at 29>C[ Traces with di}erent colours refer to bu}ers with di}erent ðCa1¦Ł and ðK ¦Ł] green "background#\ ðCa1¦Ł 09 nM\ ðK ¦Ł 4 mM^ blue\ ðCa1¦Ł 1[3 mM\ ðK ¦Ł 4 mM^ red\ ðCa1¦Ł 09 nM\ ðK ¦Ł 39 mM^ black\ ðCa1¦Ł 1[3 mM\ ðK ¦Ł 39 mM[ Each trace presented here is the average of three repeated experiments performed in triplicate measurements and represents 539 data recorded in the 9[3 ms!099 s range of time[ Fitting an exponential decrease of percent ~uorescence intensity\ the average rate constants from the repeated experiments were similar within 201)[ With pooled data "2 experiments# similar rate constants with 23) precision were obtained\ suggesting that the rate of ~uorescence decrease was not dependent on the batch of native plasma membrane vesicle suspensions[
"ðK ¦Ł 39 mM^ ðCa1¦Ł 1[3 mM^ ðK ¦Ł 39 mM plus ðCa1¦Ł 1[3 mM# "Figure 1#[ In high!ðCa1¦Ł conditions "ðCa1¦Ł 1[3 mM^ ðK ¦Ł 4 and 39 mM# two major cycles were observed] a decrease followed by increase in percent change of ~uorescence intensity below on the order of 099 ms " fast!phase# and a second cycle of increase and decrease above 099 ms "slow!phase#[ Apparently\ there was a little change in decrease of percent change of ~uorescence in the 09Ð099 s range of time when high!ðCa1¦Ł and high!ðCa1¦Ł plus high!ðK ¦Ł conditions were compared[ However\ the fast! and slow! phase cycles obeyed di}erential sensitivity to high!ðK ¦Ł and high!ðCa1¦Ł conditions "Figure 2#[ Comparing fast! and slow!phase cycles\ the slow!phase cycle was strongly in~uenced by the presence of external ðCa1¦Ł 1[3 mM and was less a}ected by depolarization "Fig[ 2#\ whereas the occurrence of the fast!phase cycle showed similar sensitivity to high!ðCa1¦Ł and high!ðK ¦Ł conditions[ Fast! and slow!phase cycles dissected this way can be char! acterized by di}erent on and off rates of the dye with half!times in the order of 07 and 39 ms " fast!phase# and
~uorescence\ are illustrated "Figure 0#[ All of the percent FM0!32 ~uorescence data obtained under di}erent con! ditions showed a decrease with time at di}erent rates[ Characteristically\ these decrease rates were variable when the experimental condition was altered[ The activity!dependent ½4) decrease in percent ~uorescence intensity observed in native plasma membrane vesicle suspensions is rather small change compared to changes found in living motor nerve terminals "Betz et al[\ 0881#[ Using the sucrose!gradient puri_ed synaptosomal frac! tion "Kardos et al[\ 0873#\ our preliminary experiments gave similar results[ The small activity!releasable FM0! 32 component found with these brain homogenates can be due to "i# non!selectivity and:or incomplete washout of the dye^ "ii# incomplete recycling of brain synaptic vesicles[ Transformation of the percent ~uorescence data into the percent change of ~uorescence with the subtraction of background decrease "ðCa1¦Ł 09 nM with ðK ¦Ł 4 mM# showed the di}erences in rate of these decreases obtained under di}erent experimental conditions
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Fig[ 1[ Kinetics of the change of ~uorescence intensity by mixing FM0!32 stained native plasma membrane suspensions from the rat cerebral cortex and low! or high!ðK ¦Ł bu}er with and without Ca1¦ added\ at 29>C[ Background "low!ðCa1¦Ł\ low!ðK ¦Ł# subtracted traces with di}erent colours refer to di}erent conditions] red\ 39 mM KCl^ blue\ 1[3 mM CaCl1^ black\ 39 mM KCl plus 1[3 mM CaCl1[ Each traces are the average of three experiments performed in triplicate measurements[
in~ux by Ca1¦ ion in native plasma membrane vesicle suspensions from neural tissue at 26>C[ Therefore\ we supposed that Ca1¦!sensitive manifestations of trans! porter activities were due to the appearance of low!a.n! ity transporters "Mabjeesh and Kanner\ 0878# on the plasma membrane surface\ via exocytosis[ The temporal separation of changes of ~uorescence intensity in native plasma membrane vesicle suspensions\ containing synaptic vesicles labelled with the ~uorescent indicator FM0!32\ presented the possibility of identifying the associated processes in synaptic vesicle recycling] the increase of ~uorescence with the endocytosis\ and the decrease of ~uorescence with the exocytosis\ since the quantum yield for FM0!32 ~uorescence is much lower in water than in lipid[ Measuring the activity!releasable FM0!32 ~uorescence on a timescale of millisecond to minute\ the separate phases of synaptic vesicle recycling can be resolved on the basis of their di}erent half!times on exposure to high!ðCa1¦Ł and:or high!ðK ¦Ł conditions[ Distinguishable fast! "³099 ms# and slow!phase "×099 ms# components of synaptic vesicle recycling can be char! acterized further by their di}erential sensitivity to the above experimental conditions[ Since the fast!phase cycle is found sensitive to high!ðCa1¦Ł as well as to high!ðK ¦Ł conditions\ one can assume that the fast!phase com!
9[66 and 39 s "slow!phase#\ respectively[ The half!time of the latter response was reduced considerably "04 s# under high!ðK ¦Ł condition[
3[ Discussion In accordance with a decrease in ð2HŁNCC!217 binding a.nity\ the present results show that Ca1¦ induces dis! sociation of the bound ð2HŁNCC!217 from GABA trans! porter sites in Ca1¦!deprived native plasma membrane vesicle suspensions with a half!time similar to those that were found for the enhancements of GABA ~ux by Ca1¦ and for Ca1¦!induced increases of ð2HŁD!aspartate and ð2HŁglycine in~ux at 26>C\ also[ Transport enhancements displaying similar kinetics for di}erent neurotransmitters are characterized by a single activation energy[ Fur! thermore\ the Ca1¦!sensitive component of GABA trans! porter binding is conserved under high!ðK ¦Ł condition[ One of the possible explanations for the above _ndings that these processes are related[ The kinetics of glucose transporters| tra.cking in non!neural tissue at 26>C indi! cate that exocytosis occurs with a half!time of about 1 min "Yang et al[\ 0881# resembling the half!times obtained for the enhancements of GABA\ D!aspartate and glycine
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Fig[ 2[ The e}ects of the change in the external ðCa1¦Ł and ðK ¦Ł on the kinetics of the change of ~uorescence intensity in FM0!32 stained native plasma membrane suspensions from the rat cerebral cortex at 29>C[ Blue trace] di}erential kinetics obtained by subtracting kinetics of change of ~uorescence displayed in the ðK ¦Ł 39 mM condition from that of the ðK ¦Ł 39 mM plus ðCa1¦Ł 1[3 mM condition[ Red trace] di}erential kinetics obtained by subtracting kinetics of change of ~uorescence displayed in the ðCa1¦Ł 1[3 mM condition from that of the ðK ¦Ł 39 mM plus ðCa1¦Ł 1[3 mM condition[
Ca1¦!sensitive neurotransmitter uptake takes place after exocytosis has terminated[ The matching kinetics observed in Ca1¦!induced enhancements of the transmembrane ~ux of di}erent neurotransmitters\ GABA transporter binding\ and exocytosis in native neuronal plasma membrane vesicles at 26>C suggest a role for Ca1¦ in neurotransmitter trans! port by taking low!a.nity transporters to the plasma membrane surface via exocytosis[
ponent of synaptic vesicle recycling is due to the acti! vation of Ca1¦ ion permeable\ voltage!dependent N! methyl!D!aspartate "NMDA# receptor channel by endogenous glutamate[ Subsequent to NMDA receptor activation\ the neurotransmitter release inherent of exocytosis may possibly be occurred[ Appeared under high external Ca1¦ condition with a rate accelerated by depolarization\ the major slow!phase cycle observed in native plasma membrane vesicle sus! pensions from the rat cerebral cortex in the 9[0 to 099 s range of time is supposed to be observed by optical monitoring of synaptic vesicle recycling in living nerve terminals "Betz and Bewick\ 0881^ Ryan et al[\ 0882^ Ryan and Smith\ 0884#\ although the minor capacity fast!phase cycle may also contributed to the response[ Reportedly\ one complete cycle of neurotransmitter release by exocytosis was estimated to take about 59 s "Betz and Bewick\ 0881#[ This cycle includes endocytic reuptake of vesicle membrane which lags exocytosis with a half!time of about 19 s as well as repriming of an endocytosed synaptic vesicle with a minimum time on the order of 04 s "Ryan and Smith\ 0884#[ In native plasma membrane vesicle suspensions from the rat cerebral cor! tex at 29>C\ the slow!phase exocytosis occurs over a time scale with a half!time of 39 s\ suggesting that the bulk of
CMYK Page 393
Acknowledgements J[K[ was supported by grants OTKA T 3929\ T 08292 and AKP 85:1!313 1\3 "Hungary#[ The skillful assistance of Mrs Erzsebet Fekete!Kuti is gratefully acknowledged[
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)
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