neurotransmitter release is characterized by a steep relationship between vesicular release rate constant and intracellular ca concentration ( [ ca]i ) ( dodge and rahamimoff , 1967 ; bollmann et al . , 2000 ;
this relationship has a slope of 35 in a double - logarithmic plot , indicating the minimal number of ca - binding steps required to elicit vesicular fusion .
likewise , there is an inverse relationship between the secretory delay and [ ca]i , such that higher [ ca]i leads to a shorter delay . keeping the delay
short is critical for ensuring that secretion will take place during the duration of an evoked [ ca]i signal . for these reasons
it is assumed that ca - dependent triggering of exocytosis is a precisely regulated process , which is performed by a protein or a protein complex generally referred to as the calcium sensor for exocytosis ( augustine , 2001 ) .
the best studied candidate for this ca sensor is the vesicular protein synaptotagmin 1 , which contains two ca- and phospholipid - binding c2 domains , c2a and c2b ( perin et al . , 1990 ; brose et al . , 1992 ) and is anchored in the vesicle membrane via a transmembrane domain .
ca binding was first demonstrated for the c2a domain ( davletov and sdhof , 1993 ; chapman and jahn , 1994 ) , and for several years , the c2b domain was suggested to have other functions ( ca - dependent multimerization , sugita et al . , 1996 ; chapman et al . , 1996 ;
schiavo et al . , 1996 ; binding to endocytic proteins , zhang et al .
, 1994 ; von poser et al . , 2000 ; or ca channels , sheng et al . , 1997 ; kim and catterall , 1997 ) .
recent studies have shown that the c2b domain also participates directly in ca - dependent phospholipid binding ( fernandez et al . , 2001 ) , an observation which in itself does not rule out any of the other proposed functions of the c2b domain .
synaptotagmin 1 binds to the snare ( soluble n - ethylmaleimide - sensitive fusion protein receptor ) proteins syntaxin 1 ( bennett et al . , 1992 ; chapman et al . , 1995 ;
li et al . , 1995 ) , and snap-25 ( synaptosomal protein of 25 kd ) ( gerona et al . , 2000 ;
zhang et al . , 2002 ) , providing a link between the putative calcium sensor and the fusion machinery ( the snare complex consisting of syntaxin 1 , synaptobrevin 2 , and snap-25 ) . in deletion studies it was found that synaptotagmin 1 is required for fast release of neurotransmitter in various organisms ( littleton et al . , 1993 ; nonet et al . , 1993 ; geppert et al . ,
1994 ) and it was suggested that synaptotagmin 1 may be the calcium sensor for fast release ( geppert et al . , 1994 ) .
recent experiments have indicated that ca binding to the c2b domain of synaptotagmin 1 is a prerequisite for fast release in neurons ( mackler et al .
, 2002 ; yoshihara and littleton , 2002 ; shin et al . , 2003 ) .
furthermore , mutations that affected the ca - binding affinities of the c2a domain without affecting binding to the double c2a - c2b domain were without effect in vivo ( fernndez - chacn et al . , 2002 ) .
whereas these data show that ca binding to synaptotagmin is of paramount importance for function , they do not show whether this ca - binding step directly underlies the calcium dependence of exocytosis triggering . to make this point a mutation
the mutation r233q in the c2a domain fulfills this criterion ; it decreases the affinity of ca - dependent phospholipid binding to the double c2a - c2b domain by a factor of two and causes a concomitant twofold shift in the dependence of epsc ( excitatory postsynaptic current ) size on extracellular [ ca ] in hippocampal autaptic cultures ( fernndez - chacn et al .
this finding is widely accepted as proof of the role of synaptotagmin 1 as the ca sensor for release .
however , two problems with recording in hippocampal autaptic neurons prevent a more detailed interpretation : ( 1 ) since the [ ca ] at the release site probably deviates by 23 orders of magnitude ( being in the order of 1020 m in the calyx of held ; schneggenburger and neher , 2000 ) from the extracellular [ ca ] ( assayed in the range 112 mm ; fernndez - chacn et al . ,
2001 ) extrapolating a ca dependence between extracellular and local intracellular [ ca ] can lead to apparent shifts along the [ ca]-axis for a number of reasons .
for instance , it is well known that there is a nonlinear relationship between the extracellular [ ca ] and the [ ca ] at the release site which could cause distortion of the release - ca relationship .
( 2 ) synaptic vesicles in neurons ( including hippocampal neurons ) are known to have heterogeneous release probabilities ( rosenmund et al .
, 1993 ; wu and borst , 1999 ; burrone and lagnado , 2000 ; sakaba and neher , 2001 ) .
the observed effects could be explained by more or less stabilization or recruitment of the vesicles with the higher release probability depending on whether wild - type synaptotagmin 1 or the r233q mutant is expressed .
it is not known whether in neurons the heterogeneous release probability is a result of different intrinsic properties of the vesicles ( e.g. , due to differences in the maturation state ) , or different localization of the vesicles with respect to calcium channels .
the observation that synaptotagmin 1 binds to the synprint site of calcium channels led to the suggestion that synaptotagmin promotes high release probability by positioning vesicles near ca - channels ( sheng et al .
, 1997 ; kim and catterall , 1997 ) . to overcome these alternative explanations ,
a demonstration of ca sensor function should preferably involve measurement of intracellular [ ca ] at the release site as a prerequisite for determining the relation between the biophysical properties of the calcium sensor for exocytosis and the biochemical properties of synaptotagmin 1 .
particularly , it would be important to demonstrate that a change in the response to ca caused by a mutation in synaptotagmin 1 is not caused by an overall increase or decrease in release , but a change in ca affinity of the release apparatus .
even though primed vesicles in chromaffin cells have a heterogeneous release probability , this heterogeneity is well - defined and can be accurately assayed .
furthermore , a detailed biophysical model of calcium triggered exocytosis has been derived ( voets , 2000 ) .
the response to a step elevation of [ ca]i ( caused by flash photolysis of caged - ca ) typically consists of a triphasic capacitance increase .
the two faster components ( collectively referred to as the exocytotic burst ) represent the heterogeneity of vesicular release probability in the sense that they comprise release of two types of vesicles that were in a ca - triggerable state at the time of the flash ( voets , 2000 ) .
the fast exocytotic burst phase represents fusion of vesicles residing in the readily releasable pool ( rrp , high release probability ) , whereas the slow exocytotic burst phase represents fusion of vesicles from a pool of slowly releasable vesicles ( srp , low release - probability ) .
the slowest phase of the flash response (  of seconds ) is called the sustained phase and is thought to represent priming of new vesicles ( i.e. , refilling of srp and rrp ) , followed by fusion as long as the [ ca]i remains high ( voets , 2000 ) . in chromaffin cells from synaptotagmin knock - out mice , the fastest exocytotic burst phase  and
therefore the rrp  was missing , but the slow burst phase and the sustained phase were still present ( voets et al . , 2001 ) .
the conclusion from that study was therefore that synaptotagmin 1 is required for the formation or stabilization of the rrp , or for triggering fusion from the rrp .
we examine secretion from chromaffin cells from mutant mice carrying the r233q point mutation in the c2a - domain of synaptotagmin 1 ( fernndez - chacn et al . ,
it should be noted that the r233q point mutation , although localized to the c2a - domain , affects the overall ca affinity of the double c2a - c2b domain fragment in which both the c2a and the c2b domain bind ca ( fernndez - chacn et al . , 2001 ) .
we wanted to test the hypothesis that ca - dependent lipid binding to synaptotagmin 1 is the event that triggers exocytosis .
if this is the case a concomitant shift in the intracellular ca dependence of secretion should be apparent in mutant cells . alternatively ,
if synaptotagmin stabilized an intrinsically fast and highly ca - sensitive state of the release apparatus one would expect a smaller relative amplitude of the readily releasable component ( rrp ) for the r233q mutant , since its stabilizing action should be weaker .
for preparation of cells we took advantage of a newly developed method for enzymatic ( papain - based ) isolation of chromaffin cells from the adrenals of a single mouse ( srensen et al . , 2003 ) .
the method was developed for single mouse embryos , but was used in the current work on young adult mouse adrenals .
the only modifications required for adult mice were to rip open the adrenals by tweezers before the papain treatment , and increase the duration of this treatment from 40 min to 4575 min .
all analysis were performed on cell preparations prepared in parallel from homozygous wild - type ( wt ) and r233q littermates derived from matings between heterozygotes of wt / r233q knock - in ( fernndez - chacn et al . ,
experimental procedures related to whole - cell patch - clamp and capacitance measurements were performed essentially as described ( voets , 2000 ) .
rapp optoelektronik , hamburg , germany ) and coupled to the epifluorescence port of an inverted zeiss 135 tv microscope equipped with a 40 fluar oil immersion objective ( na 1.3 ) ( zeiss ) .
fluorescence excitation light was generated by a monochromator ( polychrome ii ; till photonics ) and a two - port epifluorescence condensor was used for coupling 85% of the flash light and 15% of the monochromator light into the microscope ( till photonics ) .
np - egta was supplied by g. ellis - davies ( mcp hahnemann university , philadelphia ) .
cells were bathed in extracellular solution ( in mm : 150 nacl , 2.8 kcl , 2 cacl2 , 1 mgcl2 , 10 na - hepes , 2 mg / ml glucose , ph 7.2 , osmolarity 310 mosm ) and patched using a pipette solution containing the photolysable calcium chelator np - egta ( in mm : 109 cs - glutamate , 35 cs - hepes , 2 mg - atp , 0.3 na - gtp , 5 np - egta , 1 cacl2 , 400 m fura-4f ( molecular probes ) , 400 m furaptra ( molecular probes ) , ph 7.2 , osmolarity 300 mosm ) .
calcium measurements were performed by dual - wavelength ratiometric fluorimetry as described ( voets , 2000 ; srensen et al . , 2002 ) using a 1:1 mixture of the calcium - sensitive dyes fura-4f ( kd = 1 m ) and furaptra ( kd = 40 m ) .
the signal ( ratio of flurescence with excitation at 350 and 380 nm ) was calibrated intracellularly by dialyzing the cells in whole - cell configuration with eight different pipette solutions with known [ ca]i .
calcium ramps were elicited by the fluorescence excitation light , alternating between 350 and 380 nm , so that photolysis of np - egta could be combined with simultaneous measurement of [ ca]i .
individual capacitance traces were fitted with a triple exponential function : ( 1)\documentclass[10pt]{article } 
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 \begin{equation*}f \left \left(t\right ) \right = a_{0}+{{\sum^{3}_{i=1}}}a_{i}{\cdot } \left \left\{1-{\mathrm{exp } } \left \left[{- \left \left(t - t_{0}\right ) \right } /{{\mathrm{{\tau}}}_{i}}\right ] \right \right\ } \right { \mathrm{,}}\end{equation*}\end{document}for t > t0 , where a0 is the capacitance of the cell before the flash , t0 is the time of the flash .
the amplitudes ( ai ) and time constants ( i ) of the two faster exponentials define the size and release kinetics of the fast and the slow exocytotic burst , respectively .
the secretory delay ( time from the flash until the beginning of the capacitance increase ) was found by extrapolating the fit function backward in time until it crossed the baseline value ( cm= 0 , see fig .
the slowest exponential was included in order to correct for the sustained component of release during the fit , but it was not used to estimate any parameters , since the time constant of the sustained component is too slow to be measured accurately in this way .
the sustained component was in some cases fitted better by a straight line , in other cases an exponential with a time constant in the several - second range seemed more appropriate . by the fitting of eq .
1 the linear case was in practice fitted with a third - exponential with a very long time constant ( so that the exponential approaches a straight line ) . to obtain the rate of sustained release ( fig .
2 d , right ) we subtracted the amplitudes of the fast and slow burst ( a1 and a2 ) from the total amount of secretion up to 5 s after the flash and calculated the linear rate .
3 d ) contains 11 free parameters and is underdetermined by the secretion detected during a calcium ramp .
however , some of these parameters are specified by flash photolysis data and using this information it is possible to determine some other parameters or parameter combinations .
for instance , the sustained component ( steady secretion at high [ ca]i after release of the pools ) will be given by k2 .
the size of the srp before the ramp ( assuming equilibrium ) will be proportional to the ratio k2/k2 , and the proportion that the rrp makes up of the entire releasable pool ( srp+rrp ) will be given by k1/(k1 + k1 ) , even though no information can be gained on each of the parameters , k1 and k1 .
the model was fitted to calcium ramp data by first solving the steady - state condition in order to identify pool sizes ( which depends on the ratios k2/k2 and k1/(k1 + k1 ) ) .
we varied k2 , k1 , k2 , and  during the fit while keeping k1 equal to the previously measured value ( 0.12 s ; voets , 2000 ) .
the model was then integrated forward in time using a fourth order runge - kutta routine with automatic step size control ( press et al .
given the order of magnitude of - and -values , as determined by flash experiments , the timing of the secretion of the pools will be governed by / ( which is equal to kd , the equilibrium constant for calcium binding ) , as shown in fig .
parameter optimization was obtained by performing repetitive simulations in a backward simplex scheme to minimize the sum of squared deviations between data and model ( press et al . , 1997 ) .
for analyzing flash - data a simplified model encompassing only one vesicle pool ( the srp or the rrp ) with a ca sensor was used for deriving rate constant - versus - calcium and delay - versus - calcium fits ( lines in fig .
the calcium - input was taken to be a step - function , jumping from zero to some set value .
the model was now integrated forward in time to simulate the fusion of a single pool of vesicles following the step ( see fig .
4 a ) . after the simulation a single exponential component was fitted to the fusion curve , and extrapolation to zero was used to determine the delay ( fig .
this procedure was repeated at different calcium concentrations between 2 and 100 m and the rate constant versus calcium and delay versus calcium relationships were drawn ( curves in fig .
computations were performed by macros written for igor pro version 3.16 ppc ( wavemetrics ) .
carbon fiber electrodes were prepared from 10-m diameter fibers ( amoco performance products ) as described previously ( schulte and chow , 1996 ) .
secretion was elicited by dialyzing the cells in the whole - cell configuration with a pipette solution containing 1 m calcium ( 88 mm cs - glutamate , 28 mm cs - hepes , 2 mm mg - atp , 0.3 mm na - gtp , 10 mm bapta , 810 mm cacl2 , 400 m fura-4f , 400 m furaptra , ph 7.2 ) .
the carbon fiber was held at 700 mv over the bath ag / agcl electrode by an epc-7 and pressed against the cell membrane .
the [ ca]i varied from 500 nm to several m due to differences in leak current and ca extrusion capacity of the cells .
cells which did not show secretion because of too low [ ca]i were depolarized in order to induce ca influx .
the carbon fiber electrodes were cut by a new scalpella blade against a clean glass surface between each measurement .
the electrodes themselves were used only a few times ( 14 times and when possible symmetrical between the two cell types ) in order to avoid introducing a significant between - electrode variation component into the dataset .
currents were filtered at 3-khz bandwidth and sweeps of 10-s measurements recorded by an epc-9 controlled by pulse software v. 8.31 ( heka elektronik ) . upon loading into igor pro ver .
3.16 ppc ( wavemetrics ) data were digitally filtered at 1 khz by a custom - written gaussian filter .
the rms noise of the background current was 0.40.7 pa . data analysis was performed as described in the text by a custom - written macro running under igor pro .
further details of this macro are described elsewhere ( srensen et al . , 2003 ) .
for preparation of cells we took advantage of a newly developed method for enzymatic ( papain - based ) isolation of chromaffin cells from the adrenals of a single mouse ( srensen et al . , 2003 ) .
the method was developed for single mouse embryos , but was used in the current work on young adult mouse adrenals .
the only modifications required for adult mice were to rip open the adrenals by tweezers before the papain treatment , and increase the duration of this treatment from 40 min to 4575 min .
all analysis were performed on cell preparations prepared in parallel from homozygous wild - type ( wt ) and r233q littermates derived from matings between heterozygotes of wt / r233q knock - in ( fernndez - chacn et al . ,
experimental procedures related to whole - cell patch - clamp and capacitance measurements were performed essentially as described ( voets , 2000 ) . flashes of uv light were generated by a flash lamp ( dr .
rapp optoelektronik , hamburg , germany ) and coupled to the epifluorescence port of an inverted zeiss 135 tv microscope equipped with a 40 fluar oil immersion objective ( na 1.3 ) ( zeiss ) .
fluorescence excitation light was generated by a monochromator ( polychrome ii ; till photonics ) and a two - port epifluorescence condensor was used for coupling 85% of the flash light and 15% of the monochromator light into the microscope ( till photonics ) .
np - egta was supplied by g. ellis - davies ( mcp hahnemann university , philadelphia ) .
cells were bathed in extracellular solution ( in mm : 150 nacl , 2.8 kcl , 2 cacl2 , 1 mgcl2 , 10 na - hepes , 2 mg / ml glucose , ph 7.2 , osmolarity 310 mosm ) and patched using a pipette solution containing the photolysable calcium chelator np - egta ( in mm : 109 cs - glutamate , 35 cs - hepes , 2 mg - atp , 0.3 na - gtp , 5 np - egta , 1 cacl2 , 400 m fura-4f ( molecular probes ) , 400 m furaptra ( molecular probes ) , ph 7.2 , osmolarity 300 mosm ) .
calcium measurements were performed by dual - wavelength ratiometric fluorimetry as described ( voets , 2000 ; srensen et al . , 2002 ) using a 1:1 mixture of the calcium - sensitive dyes fura-4f ( kd = 1 m ) and furaptra ( kd = 40 m ) .
the signal ( ratio of flurescence with excitation at 350 and 380 nm ) was calibrated intracellularly by dialyzing the cells in whole - cell configuration with eight different pipette solutions with known [ ca]i .
calcium ramps were elicited by the fluorescence excitation light , alternating between 350 and 380 nm , so that photolysis of np - egta could be combined with simultaneous measurement of [ ca]i .
individual capacitance traces were fitted with a triple exponential function : ( 1)\documentclass[10pt]{article } 
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 \usepackage{amsfonts } 
 \usepackage{amssymb } 
 \usepackage{amsbsy } 
 \usepackage{mathrsfs } 
 \usepackage{pmc } 
 \usepackage[euler]{upgreek } 
 \pagestyle{empty } 

 \oddsidemargin -1.0 in 

 \begin{document } 
 \begin{equation*}f \left \left(t\right ) \right = a_{0}+{{\sum^{3}_{i=1}}}a_{i}{\cdot } \left \left\{1-{\mathrm{exp } } \left \left[{- \left \left(t - t_{0}\right ) \right } /{{\mathrm{{\tau}}}_{i}}\right ] \right \right\ } \right { \mathrm{,}}\end{equation*}\end{document}for t > t0 , where a0 is the capacitance of the cell before the flash , t0 is the time of the flash .
the amplitudes ( ai ) and time constants ( i ) of the two faster exponentials define the size and release kinetics of the fast and the slow exocytotic burst , respectively .
the secretory delay ( time from the flash until the beginning of the capacitance increase ) was found by extrapolating the fit function backward in time until it crossed the baseline value ( cm= 0 , see fig .
the slowest exponential was included in order to correct for the sustained component of release during the fit , but it was not used to estimate any parameters , since the time constant of the sustained component is too slow to be measured accurately in this way .
the sustained component was in some cases fitted better by a straight line , in other cases an exponential with a time constant in the several - second range seemed more appropriate . by the fitting of eq .
1 the linear case was in practice fitted with a third - exponential with a very long time constant ( so that the exponential approaches a straight line ) . to obtain the rate of sustained release ( fig .
2 d , right ) we subtracted the amplitudes of the fast and slow burst ( a1 and a2 ) from the total amount of secretion up to 5 s after the flash and calculated the linear rate .
3 d ) contains 11 free parameters and is underdetermined by the secretion detected during a calcium ramp .
however , some of these parameters are specified by flash photolysis data and using this information it is possible to determine some other parameters or parameter combinations .
for instance , the sustained component ( steady secretion at high [ ca]i after release of the pools ) will be given by k2 .
the size of the srp before the ramp ( assuming equilibrium ) will be proportional to the ratio k2/k2 , and the proportion that the rrp makes up of the entire releasable pool ( srp+rrp ) will be given by k1/(k1 + k1 ) , even though no information can be gained on each of the parameters , k1 and k1 .
the model was fitted to calcium ramp data by first solving the steady - state condition in order to identify pool sizes ( which depends on the ratios k2/k2 and k1/(k1 + k1 ) ) .
we varied k2 , k1 , k2 , and  during the fit while keeping k1 equal to the previously measured value ( 0.12 s ; voets , 2000 ) .
the model was then integrated forward in time using a fourth order runge - kutta routine with automatic step size control ( press et al . , 1997 ) and with the calcium ramp providing the input .
given the order of magnitude of - and -values , as determined by flash experiments , the timing of the secretion of the pools will be governed by / ( which is equal to kd , the equilibrium constant for calcium binding ) , as shown in fig .
parameter optimization was obtained by performing repetitive simulations in a backward simplex scheme to minimize the sum of squared deviations between data and model ( press et al .
, 1997 ) . for analyzing flash - data a simplified model encompassing only one vesicle pool ( the srp or the rrp ) with a ca sensor was used for deriving rate constant - versus - calcium and delay - versus - calcium fits ( lines in fig .
the calcium - input was taken to be a step - function , jumping from zero to some set value .
the model was now integrated forward in time to simulate the fusion of a single pool of vesicles following the step ( see fig .
4 a ) . after the simulation a single exponential component was fitted to the fusion curve , and extrapolation to zero was used to determine the delay ( fig .
this procedure was repeated at different calcium concentrations between 2 and 100 m and the rate constant versus calcium and delay versus calcium relationships were drawn ( curves in fig .
computations were performed by macros written for igor pro version 3.16 ppc ( wavemetrics ) .
carbon fiber electrodes were prepared from 10-m diameter fibers ( amoco performance products ) as described previously ( schulte and chow , 1996 ) .
secretion was elicited by dialyzing the cells in the whole - cell configuration with a pipette solution containing 1 m calcium ( 88 mm cs - glutamate , 28 mm cs - hepes , 2 mm mg - atp , 0.3 mm na - gtp , 10 mm bapta , 810 mm cacl2 , 400 m fura-4f , 400 m furaptra , ph 7.2 ) .
the carbon fiber was held at 700 mv over the bath ag / agcl electrode by an epc-7 and pressed against the cell membrane .
the [ ca]i varied from 500 nm to several m due to differences in leak current and ca extrusion capacity of the cells .
cells which did not show secretion because of too low [ ca]i were depolarized in order to induce ca influx .
the carbon fiber electrodes were cut by a new scalpella blade against a clean glass surface between each measurement .
the electrodes themselves were used only a few times ( 14 times and when possible symmetrical between the two cell types ) in order to avoid introducing a significant between - electrode variation component into the dataset .
currents were filtered at 3-khz bandwidth and sweeps of 10-s measurements recorded by an epc-9 controlled by pulse software v. 8.31 ( heka elektronik ) . upon loading into igor pro ver .
3.16 ppc ( wavemetrics ) data were digitally filtered at 1 khz by a custom - written gaussian filter .
data analysis was performed as described in the text by a custom - written macro running under igor pro .
further details of this macro are described elsewhere ( srensen et al . , 2003 ) .
flash photorelease of caged calcium typically increased [ ca]i to > 10 m and resulted in robust secretion from both wt and r233q cells as assayed by capacitance increase ( fig .
when fitting a sum of exponential functions to the capacitance traces ( see materials and methods ) the inclusion of a rapid component (  = 20 ms ) was necessary for both r233q and wt cells .
therefore , in contrast to synaptotagmin knockout cells , in the r233q mutants the fast exocytotic burst component , and therefore the rrp , persisted .
( a ) after whole - cell dialysis of an example wt cell with a pipette solution containing a photolysable ca - cage , the [ ca]i was increased by a brief flash of uv light at 0.5 s ( top ) .
the simultaneous capacitance measurement with high time resolution shows a fast secretory response ( bottom ) .
( b ) similar experiment as in a shown for an example r233q cell where the post - flash [ ca]i reached comparable levels . note that the difference in the sustained rate of secretion seen between the two examples was not a general feature ( compare fig .
( c ) the first 200 ms after the flash . by plotting the two example responses on the same time scale
fitting of a sum of three exponential components ( gray lines ) to the capacitance increase was possible in both cases .
fast burst secretion from the r233q cell displayed a longer time delay ( 13.5 ms instead of 4.3 ms ) and a slower fast time constant ( 32.5 ms instead of 22.9 ms ) than the wt cell .
closer inspection of the time course of wt and r233q secretion revealed a difference in kinetics , with the r233q secretion being slower than wt secretion for uv flashes resulting in comparable post - flash [ ca]i ( fig .
this difference was caused by both a longer time constant of fast burst secretion in the r233q cells and by a longer secretory delay ( fig .
plotting the rate constant ( = 1/time constant ) for fast burst secretion and the secretory delay against the post - flash [ ca]i ( fig .
2 , a and b )   showed that increasing [ ca]i resulted in increasing rate constants for release and shorter secretory delays , as described previously ( voets , 2000 ) .
the relationships for the r233q mutant cells were displaced toward higher [ ca]i ( fig .
2 , a and b ) , indicating a difference in the ca dependence of fusion . considering only those points where post - flash [ ca]i >
post - flash [ ca]i was similar for wild - type cells ( 26  2 m ) and r233q cells ( 25  2 m ) . a similar plot of the rate constant for the slow exocytotic burst against post - flash [ ca]i revealed no striking difference , even though rates did seem slower at low [ ca]i in r233q cells ( fig .
2 c ) . finally , we considered the pool sizes ( the amplitudes in the exponential fits ) .
we found significantly larger pool sizes , both for the rrp ( amplitude of fast burst component ) and the srp ( amplitude of slow burst component ) in the r233q mutant cells ( fig .
the rate of the sustained component of release was also on average higher in r233q mutant cells , but this was not significant .
slow - down of secretion in r233q cells is caused by a decrease in fast secretory rate constant and a increase in secretory delay in a ca - dependent manner .
( a ) rate constant ( = 1/time constant ) of the fast burst component as a function of post - flash [ ca]i for wt ( open circles ) and r233q ( closed squares ) cells ( left ) .
the mean ( sem ) rates in r233q cells ( filled bar ) were significantly lower than wt cells ( open bar ) when considering cells with post - flash [ ca]i > 10 m ( right ) .
( b ) secretory delay as a function of post - flash [ ca]i ( left ) .
the secretory delays in r233q cells ( filled bar ) were significantly longer than wt cells ( open bar ) when considering cells with post - flash [ ca]i > 10 m ( right ) .
( a and b ) the black lines are simulation of a 4-ca binding site model with parameters obtained by fitting to calcium ramp data ( full line , wt ; dashed line , r233q . for parameters
( c ) rate constant of the slow burst component as a function of post - flash [ ca]i ( left ) and mean  sem rates ( right ) .
line is the simulation of a 3-ca binding site model according to voets ( 2000 ) .
mean  sem of the amplitudes of the fast and slow burst component ( left ) , and rate of sustained release ( right ) for wt ( open bars ) and r233q ( closed bars ) cells .
* , p < 0.05 ; * * , p < 0.01 ( mann - whitney test ) .
these data show that a mutation in the c2a domain that changes ca - dependent phospholipid binding , alters the calcium dependence of the fast fusion rate constant and the secretory delay .
the effect on the pool sizes was not expected and points to the possibility that the c2a domain of synaptotagmin may also act during priming ( the process filling the two releasable vesicle pools , the srp and rrp ) .
another possibility is that due to defective secretion in these cells the pool sizes are up - regulated either passively ( by the accumulation of unfused granules ) or actively by a regulatory mechanism .
it should be noted that the relative proportion of the releasable vesicles pools that was made up of the rrp ( e.g. , the quantity rrp/(rrp+srp ) , where srp and rrp denotes pool sizes ) was not changed in the r233q mutant .
therefore , the r233q mutation and ( by inference ) ca - dependent phospholipid binding of synaptotagmin 1 does not influence the equilibrium between the srp and rrp .
if the effect of the r233q mutation is to decrease the calcium - affinity ( increase the kd ) of the calcium sensor for exocytosis , as would be expected from the biochemical data , then this should be even more noticeable at lower [ ca]i , where the calcium sensor would be far from saturation .
we therefore stimulated the cells using a calcium - ramp protocol , where calcium was liberated from the calcium cage by light from the fluorescence light source alternating between 350 and 380 nm , combining slow ca release with measurement of [ ca]i ( srensen et al .
simultaneous capacitance measurements showed sigmoid - shaped capacitance increases during the calcium - ramp protocol ( fig .
3 , a and b , show responses from wt and r233q cells chosen for display because the shape of the calcium ramp was almost identical between the two cells , allowing for easy comparison . note that these cells had uncharacteristic large responses . inspecting
the ramp responses showed that r233q cells started secretion at a significantly higher ca - threshold than wt cells ( fig .
a higher ca threshold for secretion in r233q cells can be explained by an increase in the kd of the ca - sensor for secretion .
( a and b ) example calcium ramps ( top ) and simultaneously measured capacitance changes ( bottom ) for a wt ( a ) and a r233q ( b ) cell . superimposed is shown a regression line ( straight dashed line ) used for deriving the ca - threshold for secretion ( c ) , and the fit of a model of secretion ( d ) to the ramp data ( gray curve ) together with the assumed exocytosis timecourse from the rrp and srp ( gray dashed curves ) .
( c ) mean  sem of the ca - threshold for secretion from wt ( open bar , n = 22 cells ) and r233q ( closed bar , n = 25 ) cells .
model for secretion from the srp and rrp pools . fixed parameters during the fit were : s = 4 s , s = 20 s,f = 56 s , f = 1450 s , k1 = 0.12 s ( voets , 2000 ) .
( e ) mean  sem of the fitted kd ( = / ) for the fast and slow calcium sensor for wt ( open bar , n = 22 cells ) and r233q ( closed bar , n = 25 ) * * * , p < 0.001 ( mann - whitney test ) . to obtain a more quantitative description we next considered a biophysical model of the calcium sensor for exocytosis ( fig .
3 d ) , which in its essential features was previously shown to describe exocytosis from chromaffin cells ( voets et al .
the model assumes that a number of ca ions bind to ca - binding sites on the calcium sensor with association rate constant (  on - rate  )
 , and dissociation rate constant (  off - rate  ) . once all binding sites are occupied the vesicle can undergo exocytosis in an irreversible ( and ca - independent ) process with rate constant . in the model in fig .
3 d we have included separate calcium sensors ( with separate  ,  , and s ) for the rrp - associated calcium sensor ( right part of the scheme ) , and the srp - associated calcium sensor ( left part of the scheme ) . the previous model ( voets , 2000 ) assumed three ca - binding sites for both the rrp - associated and the srp - associated calcium sensor . for reasons that will become apparent below we assumed four ca - binding sites for the rrp - associated calcium sensor , and we also included a cooperativity factor ( b ) that increases the affinity of subsequent ca - binding steps dependent on how many ca ions that have already bound .
we further allowed for priming and unpriming through the rate k2 , and the rate constant k2 , respectively , and for movement of vesicles between the rrp and srp ( but for simplicity only in the ca - unbound state ) through rate constants k1 and k1 .
the values of k2 , k1 , and k1 were measured previously ( voets , 2000 ) , and k2 was found to be calcium dependent , modeled as k2 = ( rmax [ ca])/([ca ] + kd ) , where kd = 2.3 m and rmax = 55 ff / s ( voets , 2000 ) .
we therefore first examined the model itself to see what one can conclude from flash photolysis and ca - ramp experiments , respectively . during flash photolysis the [ ca]i changes in a step - like manner from a relatively low to a high value .
this causes rapid fusion of vesicles in the rrp , and also the vesicles in the srp will fuse , albeit somewhat slower . in this case
the calcium binding and fusion reactions ( governed by  ,  , and  ) are much faster than the movement of vesicles between pools ( governed by k1 and k1 ; k2 and k2 ) . for purposes of flash photolysis experiments
, therefore , one can perceive each of the releasable vesicle pools ( srp and rrp ) as fusing in isolation .
thus , k1 , k1 , k2 , and k2 set the initial pool sizes and can be ignored subsequently .
therefore , we constructed a mathematical model that encompasses a single vesicle pool ( for both the srp and the rrp ) , each fusing according to its  ,  ,  , and b after a step elevation of [ ca]i ( voets , 2000 ) .
a shows that under these conditions the fusion of the rrp follows a near - exponential time course , except for some delay . to derive fusion rate constants and delays , we fitted these simulations with a single exponential component and used extrapolation to zero to derive the exocytotic delay ( fig .
4 a ) , exactly as we had done when analyzing experimental capacitance data ( fig .
this was repeated with calcium steps to several values between 2 and 100 m and the rate constant versus calcium and the delay versus calcium relationships were displayed in fig .
4 , c and d ( thick black line is simulation using parameters for wt cells ) . the assumption that each pool can be seen in isolation may break down at the lower levels of [ ca ] seen during calcium ramps . for these experiments
we therefore simulated the entire model ( fig . 3 d ) using the measured calcium ramp as an input .
4 b shows a typical calcium ramp together with the simulation of the model ( thick black line is a simulation using the parameters found for wt cells ) .
we next asked which parameters of the fast calcium sensor can be distinguished using flash photolysis and ramp measurements .
we therefore repeated simulations while changing one of the parameters of the fast calcium sensor at a time in the direction that would tend to cause slower secretion ( for  this would be a reduction , for  an increase , for  a decrease , and for b an increase , see color coded traces in fig .
the result from flash simulation showed the changes that one would expect ; for instance if  was decreased by a factor of four the rates were indistinguishable at lower [ ca]i and only at higher concentrations were a slightly lower rate and a longer delay observed .
increasing  ( dissociation rate constant for ca ) caused markedly lower rates at low [ ca]i , whereas the delays were similar .
notably , only if  ( association rate constant for ca ) was decreased were lower rates and longer delays found at lower [ ca]i . when comparing these simulations to the kinetic data obtained with the r233q mutation and wt cells ( fig .
2 , a and b ) , it is clear that the observed shift in calcium - dependence over the assayed range of [ ca]i can be explained within the framework of a sequential ca - binding model only by a decrease of  in the r233q mutant .
analysis of the model for secretion presented in fig . 3 d. ( a ) simulation of fusion of the rrp after flash stimulation to 23 m calcium ( black curve ) .
illustrated is the fitting of a single exponential component to the fusion curve ( red curve ) and the determination of the delay .
( b ) simulation of fusion of both rrp and srp ( including a sustained component ) according to the model in fig .
the capacitance units were normalized to the sum of the srp and rrp pools ( which had the same size ) .
parameters used for the thick black curve are those found for wt cell in the present paper .
color coded is shown the effect of decreasing  ( by a factor of 2 ) , increasing  ( by a factor of 2.4 ) , decreasing  ( by a factor of 4 ) , or increasing b ( from 0.55 to 1 ) .
( c and d ) delay ( c ) and rate ( d ) versus post - flash calcium ( determined as shown in a ) for rrp fusion using the parameters found for wt cells ( thick black line ) , or following the changes of  ( decrease by a factor of 2 ) ,  ( decrease by a factor of 2.4 ) ,  ( increase by a factor of 4 ) , or b ( from 0.55 to 1 ) . the model simulation show that only a decrease in  cause simultaneous decrease of rates at low calcium and increase of delays . from the ramp
experiments it is not possible to gain information about whether  is decreased or  increased , since in both cases is the capacitance increase displaced toward later times or higher calcium concentrations ( fig . 4 b ) .
the reason for this is that the ca ramp is so slow that the sensor is in a 
quasi steady - state  during the ramp , i.e. , it will be almost in equilibrium with [ ca]i , and therefore information about on- and off - rates can not be obtained .
however , the ramp shape will depend critically on the ratio between  and  , i.e. , the kd ( = / ) for calcium binding .
also a change in cooperativity ( b ) will change secretion since it will modify the mean affinity of the calcium - binding step .
finally , secretion is going to depend only very weakly on f ( as long as f is relatively large ) since the calcium concentrations during the ramp will not lead to saturation of the sensor ( green trace , fig . 4 b ) .
3 ) would therefore be qualitatively in agreement with the decrease in  observed in flash experiments . to check for quantitative agreement between the two stimulation regimes we fitted the model to ramp data by varying  for both calcium sensors ( srp and rrp ) between conditions ( wt or r233q cells ) , while keeping  and  constant at the previously estimated values ( f = 56 s , f = 1450 s , voets , 2000 ) .
the resulting differential equation model was first solved in the steady - state at resting [ ca]i in order to find the initial pool sizes and then integrated forward in time using the calcium ramp measurements to drive the model ( materials and methods ) .
it was found that the model could fit ramp capacitance increases quite satisfactorily ( fig .
one of the advantages of ramp stimulation is that each cell is assayed over a range of calcium concentrations , rather than at only one concentration ( as by flash photolysis stimulation ) .
therefore , by fitting the model an estimate of  and hence of the kd for calcium binding to the calcium sensor is obtained for each cell .
thus , an accurate estimate of the mean kd can be obtained by measuring in several cells . by fitting several ramp responses we estimated the kd for the fast ca sensor to 11.4  0.9 m in wt cells ( corresponding to f = 4.9
ms ) whereas in r233q cells it was 23.3  1.6 m ( p < 0.001 , corresponding to f = 2.4 ms ) ( fig .
we also found that the kd for the slow ca sensor was increased ( from 5.0  0.7 m to 9.1  0.7 m , p < 0.001 ) ( fig .
3 e ) ; however , we are less confident about this result because fusion of the srp is temporally  sandwiched  between the fusion of the rrp and the sustained component .
we therefore can not exclude that the apparent difference in kd for the slow ca sensor is due to a difference in kinetics of the sustained component .
however , the increase in kd for the srp sensor would be in agreement with the difference in kinetics at lower [ ca ] seen in fig .
2 c. the result for the rrp ca sensor is more solid , because the initiation of fusion from the rrp is well resolved in both flash and ramp data . after the fitting to ramp data we simulated flash experiments using the fitted values of  and the ( fixed ) values of  and  without further fitting or parameter modification .
the simulations agreed well with flash data from wt and r233q cells ( lines in fig .
2 , a and b ) , indicating that the two stimulation regimes ( flash and calcium ramps ) are in agreement . the model for the ca sensor for exocytosis in previous work on chromaffin cells assumed three noncooperative ( i.e. , b = 1 ) ca - binding steps for both the fast and the slow calcium sensor ( voets , 2000 ) .
this did not change the conclusion of a twofold shift in the kd of the fast calcium sensor ( unpublished data ) , and the fit to rate - calcium data were essential unchanged ( gray lines in fig .
2 a ) . however , in the present study we found that with that model the long delays after flashes found in the r233q mutant , especially at low [ ca]i , could not be readily accounted for ( gray lines in fig .
similar observations led to the assumption of cooperative ca - binding in secretion models for retinal bipolar cells ( heidelberger et al .
, 1994 ) , the calyx of held synapse ( schneggenburger and neher , 2000 ) , and cochlear inner hair cells ( beutner et al . , 2001 ) .
in our work , the cooperativity coefficient was found to be 0.55 ( this value was used through - out , also when fitting ramp data ) , which means that the first ca ion binds with a kd of / = 11 m for wt cells , whereas the last ca ion bind with a kd of b 
it must be stressed that there is not a substantial or principal difference between the three or four calcium binding site models , and because of the considerable scatter in this kind of data it is possible that differences in recording conditions can lead to the preference of different models .
it has been reported that overexpression of synaptotagmin 1 in pc12 cells increased the duration of the amperometric 
signal that is thought to reflect the formation of a fusion pore preceding full vesicle fusion ( wang et al . ,
we therefore investigated whether the synaptotagmin 1 mutation r233q changed the characteristics of single fusion events . in these experiments ,
the cells were patched in the whole - cell configuration and dialyzed with a relatively low [ ca]i ( 1 m ) , where the calcium sensor would be far from saturation and the effect , if any , of the r233q mutation therefore noticeable .
detailed analysis of spike kinetics was performed by a semiautomatic software routine which identified the spikes and 5090% risetime , peak amplitude , halfwidth ( duration at 50% peak amplitude ) , and charge ( fig .
5 b ) . to inspect the statistical structure of the data we displayed the cumulative distribution of each parameter for each cell investigated .
5 c shows the distributions of halfwidths from 10 randomly selected wt cells and 10 randomly selected r233q cells .
this was confirmed by a hierarchic ( nested ) analysis of variance ( anova ) , which showed highly significant among - cell ( subgroup ) variability regardless of the parameter tested ( p < 0.0001 , peak amplitude , risetime , halfwidth , and foot duration were tested after logarithmic transformation , cubic - root of the charge was tested without transformation ) .
this result means that the spikes are not statistical independent but their characteristics depend strongly on the cell from which the spike was recorded , as has previously been described ( colliver et al . , 2000 ) .
the consequence for the analysis of two different populations of cells is that pooling all spikes for a two - sample test assuming independence is not a valid test for the null hypothesis of no difference between the two cell populations .
instead , one can use the spikes to calculate one statistic for each cell , then compare this statistic between populations of cells ( colliver et al . , 2000 ) .
furthermore , we averaged the cumulative distributions of each parameter between cells and displayed the mean distribution ( in these distributions each cell has the same weight , regardless of the number of spikes obtained from that cell ) of the cubic - root of the charge ( fig .
5 d , the cubic - root of the charge is expected to be proportional to the diameter of the fused vesicle ) , the 5090% risetime ( fig .
5 e ) , the foot duration ( fig . 5 f ) , the halfwidth ( fig .
5 g ) , and the peak amplitude ( fig . 5 h ) for wt and r233q cells , respectively .
the resulting mean distributions showed no striking differences between wt and r233q spikes ( fig . 5 , d  h ) .
the cell medians for each parameters were averaged across cells and the mean ( sem ) for each cell population displayed as the insets in fig . 5 , d  h ( for the foot duration
the 80% fractile was chosen , because close to 40% of the cells did not have any detectable foot signal ) .
mann - whitney tests between medians confirmed that there was no statistical significant differences ( p > 0.1 ) between wt and r233q cells in any of the parameters investigated .
the above - mentioned hierarchic anova also leads to a test of the hypothesis of no difference between wt and r233q cell populations ( groups ) based on moments .
we conclude that the mutation r233q in our hands do not cause detectable changes in single - spike characteristics .
( b ) extraction of single event characteristics : a semiautomatic routine detected the spikes , performed baseline subtraction , identified the halfwidth ( thick line with two crosses across the peak ) , the risetime , peak amplitude , duration of the foot signal ( line with two crosses connects the beginning and end of the foot signal ) , and integrated the spike ( gray area ) to derive the charge .
( c ) cell - distributions of halfwidths for 10 randomly selected wt cells ( full lines ) and 10 randomly selected r233q cells ( dashed lines ) .
note the large cell - to - cell variations in the position of the distribution .
( d ) averaged cumulative distribution of cubic - root of the charge for wt cells ( full lines ) , and r233q cells ( dashed lines ) .
a total of 2,436 spikes from 22 wt cells ( 41236 spikes pr cell ) and 2,086 spikes from 22 r233q cells ( 19165 spikes per cell ) were analyzed .
our data indicate that synaptotagmin 1 acts as a ca - trigger during vesicle fusion . in the introduction
we hypothesized that if ca - dependent phospholipid binding to the c2 domains of synaptotagmin 1 was the parameter that determines the rate of release , then changing the kd for this binding reaction should shift the intracellular ca dependence of the vesicle release rate along the [ ca]i - axis .
this prediction was born out by experiments on chromaffin cells from the r233q mutant , which showed that mutant cells had lower secretory rates ( fig . 2 a ) , and longer secretory delays ( fig . 2 b ) for a given [ ca]i , and a higher ca threshold of release ( fig .
3 , a  c ) . in a mathematical model of the calcium sensor , which in its essential features was previously shown to describe secretion from chromaffin cells ( voets , 2000 ) ,
all of these changes were consistent with a change in the kd of the fast calcium - sensor for exocytosis by a factor of two ( figs .
the alternative interpretations set out in the introduction , which would predict a smaller amplitude of the fast rrp could clearly be rejected .
our assay system bypassed calcium influx through calcium channels and instead stimulated cells by photorelease of caged calcium .
thus , a function of synaptotagmin in colocalization of vesicles with calcium channels can not be the basis for the observed difference in exocytosis triggering .
this , of course , does not rule out that synaptotagmin may influence the spatial relationship between calcium channels and release sites and , thereby , lead to additional effects on release during depolarizations .
our amperometric measurements ruled out another alternative hypothesis : the synaptotagmin mutation did not alter the rate of fusion pore expansion .
together , our results show that ca binding of synaptotagmin 1 is unlikely to regulate events immediately upstream of fusion triggering ( transitions between vesicles pools , colocalization with calcium channels ) , and also does not seem to be involved in events immediately following triggering ( regulating fusion pore expansion ) . in biochemical measurements the r233q mutations induced a twofold lower affinity for ca - dependent lipid binding , whereas the binding to syntaxin was unchanged ( fernndez - chacn et al . , 2001 ) .
this supports the view that ca - dependent lipid binding , rather than ca - dependent binding to components of the snare complex , is the event that triggers exocytosis ( see also shin et al . , 2003 ) .
it should be noted , though , that a shift in the ca dependence of exocytosis could also come about without changing properties of the ca sensor itself .
this could be the case if the ca - sensor coupled allosterically to exocytosis by shifting the equilibrium between two conformational states (  relaxed  and  tense  , which may correspond to conformations of the snare complex ) , of which only one ( tense ) supports exocytosis .
ca would bind more tightly to the ca sensor in the tense state . in such a scheme
a change of the energy level of any of the two states could lead to a change in the apparent ca sensitivity of exocytosis , even though the ca sensor per se was left unaffected .
examples for such effects can be found in the literature on ca - dependent k channels ( e.g. , cui et al . , 1997 ) .
however , in the current work we can correlate changes in apparent affinity of the ca sensor with observed biochemical changes of ca binding , suggesting that the effect we see is on the ca sensor itself .
recently , data obtained with the mutation of an aspartate residue in the c2a - domain of synaptotagmin 1 in drosophila were proposed to suggest that the c2a domain of synaptotagmin 1 may not be involved in ca triggering ( robinson et al . , 2002 ) .
we have found that similar mutations have a severe effect on ca - dependent phospholipid binding only on the isolated c2a domain , but not on the double c2-domain fragment ( fernndez - chacn et al . ,
in vivo , these mutations show no major changes in synaptic transmission consistent with the finding that the two c2-domains cooperate and may be partially redundant in ca - dependent phospholipid binding ( fernndez - chacn et al . , 2002 ) .
this is in contrast to the r233q mutation studied here which , although the site is localized on the c2a - domain too , alters the apparent ca - affinity of the entire c2a / b - domain fragment ( fernndez - chacn et al . , 2001 ) .
recent mutagenesis data on the c2a and c2b domain may indeed suggest that ca - binding to the c2b domain is the prevailing event leading to phospholipid binding ( mackler et al .
these and other results ( fernndez - chacn et al . , 2002 ) suggest that the c2a domain contributes to the apparent ca affinity of the c2b domain , but that actual ca binding to the c2a domain may not be essential for triggering release . whether this is correct or not is of no consequence for the interpretation of the data presented here . it has been argued that synaptotagmin 1 acts not only to promote fusion at higher [ ca]i , but also to prevent release at lower [ ca]i , and that these functions may be mechanistically distinct ( yoshihara and littleton , 2002 ) .
this idea was based on the observations that a synaptotagmin - null mutant displayed increased secretion during sustained ca increases ( yoshihara and littleton , 2002 ) , and that mutated synaptotagmins defective in triggering had an even more severe phenotype than the complete absence of synaptotagmin ( mackler et al . , 2002 ) .
we note that in a simple sequential ca - binding model of the calcium sensor the steep dependence of the fusion rate on the [ ca]i can be described simultaneously as promotion of fusion at high-[ca]i and inhibition of secretion at low [ ca]i . within this framework
, we suggest that the increased secretion in synaptotagmin - null mutants may result from functional substitution with another calcium sensor with a higher affinity for calcium and lower maximal triggering rates
. this calcium sensor could be one of several other synaptotagmin isoforms , for instance synaptotagmins 37 or 9 which are expressed in neuroendocrine cells and neurons ( mizuta et al .
, 1994 ; li et al . , 1995 ; sugita et al . , 2001 ;
of these , synaptotagmins 3 , 5 , and 7 have a higher calcium - affinity than synaptotagmin 1 ( sugita et al . , 2001 , 2002 ) , whereas calcium binding and affinities of synaptotagmins 4 , 6 , and 9 are at present unknown .
thus , the action of synaptotagmin as a  fusion clamp  would result simply from its ability to displace the high - affinity sensor .
this interpretation removes the potentially confusing discussion of whether the action of the r233q mutant should be described as an increase in fusion clamping activity at low [ ca]i ( for instance in ramp experiments ) , or whether the function is to impair fusogenic function at higher [ ca]i ( flash data ) . in a sequential ca - binding model of the ca sensor
overexpression of synaptotagmin 1 in pc12 cells was found to increase the duration of the fusion pore , whereas overexpression of synaptotagmin 4 decreased fusion pore duration ( wang et al . , 2001 ) .
we did not find differences in any amperometric parameter with the r233q mutation , even though vesicles were brought to fuse at relatively low [ ca]i , where a difference in synaptotagmin function due to the mutation should have been noticed .
the most likely explanation for this is that the r233q mutation regulates only the ( calcium dependent ) probability that a vesicle will fuse ; however , once the initial fusion pore is formed the function of synaptotagmin 1 in triggering release is terminated .
the interpretation of the overexpression approach is quite different since in this case the amount of synaptotagmin 1 in the vesicle membrane will increase over wild - type levels .
this may cause fusion with a different stochiometry of calcium sensors and lead to interference with fusion pore dilation at a time after exocytosis triggering .
although our data provide direct evidence that synaptotagmin 1 functions in the ca - triggering step as opposed to immediately preceding or following steps , the data also raise many questions .
we currently have no explanation for the increase in the total number of vesicles in the releasable pools in the r233q mutant .
furthermore , our data do not provide insight into the nature of the ca sensors that are responsible for release from the srp .
critical to addressing these questions will be the analysis of additional synaptotagmin 1 mutants , as well as mutants in other candidate ca - sensor proteins .
 we tested the long - standing hypothesis that synaptotagmin 1 is the ca2 + sensor for fast neurosecretion by analyzing the intracellular ca2 + dependence of large dense - core vesicle exocytosis in a mouse strain carrying a mutated synaptotagmin c2a domain . 
 the mutation ( r233q ) causes a twofold increase in the kd of ca2 + -dependent phospholipid binding to the double c2a - c2b domain of synaptotagmin . using photolysis of caged calcium and capacitance measurements we found that secretion from mutant cells had lower secretory rates , longer secretory delays , and a higher intracellular ca2 + -threshold for secretion due to a twofold increase in the apparent kd of the ca2 + sensor for fast exocytosis . 
 single amperometric fusion events were unchanged . 
 we conclude that ca2 + -dependent phospholipid binding to synaptotagmin 1 mirrors the intracellular ca2 + dependence of exocytosis . 
INTRODUCTION
MATERIALS AND METHODS
Preparation of Chromaffin Cells
Capacitance and Calcium Measurements
Mathematical Model
Amperometry
RESULTS
DISCUSSION