Synaptotagmin 7 functions as a Ca2+-sensor for synaptic vesicle replenishment
Chemical communication at synapses in the central nervous system is subject to short- and long-term changes in strength ( Malenka , 1994 ) . For example , the replenishment of synaptic vesicles ( SV ) in the readily releasable pool ( RRP ) , which can be exhausted during high frequency stimulation ( HFS ) , plays a critical role in determining the rate and degree of short-term synaptic depression ( Wang and Kaczmarek , 1998 ) . It has been reported that Ca2+ plays an important role in the replenishment step ( Dittman and Regehr , 1998; Wang and Kaczmarek , 1998; Hosoi et al . , 2007; Sakaba , 2008 ) . Thus , identification of Ca2+ sensors for SV replenishment has emerged as a critical issue , and the ubiquitous Ca2+-binding protein CaM has been implicated in this process ( Sakaba and Neher , 2001; Hosoi et al . , 2007 ) , by interacting with Munc13-1 ( Lipstein et al . , 2013 ) . Synaptotagmins represent another well-known family of Ca2+-binding proteins; some isoforms function as Ca2+-sensors for rapid SV exocytosis ( Chapman , 2008 ) . Of the 17 syt isoforms in the mammalian genome , only syt 1 , 4 , and 7 appear to be expressed in all metazoans ( Barber et al . , 2009 ) , suggesting that they are involved in key , evolutionarily conserved , membrane trafficking pathways . In neurons , functions for syt 1 and 4 have been established . Syt 1 serves as a Ca2+ sensor for fast synchronous SV release ( Geppert et al . , 1994; Koh and Bellen , 2003; Nishiki and Augustine , 2004; Liu et al . , 2009 ) , and syt 4 modulates transmission by regulating neurotrophin release ( Dean et al . , 2009 , 2012b ) and also regulates exocytosis in the posterior pituitary ( Zhang et al . , 2009 ) . One group reported that Syt7 knock-out ( KO ) mice ( hereafter termed KO ) had normal synaptic transmission ( Maximov et al . , 2008 ) , but in a more recent study from the same group concluded that syt 7 functions as Ca2+ sensor for asynchronous release ( Bacaj et al . , 2013 ) . It was proposed that this contradiction was due to the use of KO mice in the former study , as opposed to the more acute knock-down ( KD ) approach used in the latter study . However , KD of syt 7 in otherwise WT neurons had no effect ( Bacaj et al . , 2013 ) , so the physiological function of syt 7 in the mammalian central nervous system remains an open issue , whereas there is evidence that syt 7 regulates asynchronous synaptic transmission at the Zebrafish neuromuscular junction ( Wen et al . , 2010 ) . The apparent discrepancy regarding the physiological function of syt 7 between mice and Zebrafish is likely due to species differences , analogous to the different functions of syt 4 in Drosophila vs mice ( Yoshihara et al . , 2005; Dean et al . , 2009; Wang and Chapman , 2010 ) . Here , we used high frequency stimulation , and other methods , to study synaptic transmission in cultured hippocamal neurons from KO mice and discovered that syt 7 , via a highly specific interaction with CaM , functions as a Ca2+-sensor that regulates Ca2+-dependent SV replenishment .
We first carried out whole-cell voltage clamp recordings using primary hippocampal neurons obtained from wild-type ( WT ) and KO mice and found that spontaneous SV release , measured in the presence of TTX , was unaffected in the KO neurons ( Figure 1A–E ) . We then carried out paired recordings by stimulating one neuron with a bipolar glass electrode and recording from a second synaptically connected neuron , as previously described ( Liu et al . , 2009 ) , to examine evoked excitatory postsynaptic currents ( EPSCs ) triggered by single action potentials ( AP ) . The EPSC amplitudes ( Figure 1F–I , K–L ) , and the charge transfer kinetics ( Figure 1J ) , were also unchanged in the KOs , confirming that syt 7 does not function as Ca2+ sensor for asynchronous release in hippocampal neurons ( Maximov et al . , 2008 ) . We also examined the Ca2+-sensitivity of single AP-evoked SV release , by recording at different concentrations of extracellular Ca2+ , and found that it was the same between WT and KO neurons ( Figure 1K–M ) . 10 . 7554/eLife . 01524 . 003Figure 1 . Normal spontaneous and single AP evoked EPSCs in KO neurons . ( A ) Representative miniature EPSC ( mEPSC; recorded in the presence of TTX ) traces from WT and KO hippocampal neurons . ( B ) Representative mEPSCs from WT ( grey ) and KO ( red ) neurons . ( C–E ) mEPSC amplitude ( C , WT: 20 . 78 ± 1 . 26 pA; KO: 20 . 46 ± 2 . 38 pA ) , charge ( D , WT: 116 ± 12 fC; KO: 119 ± 18 fC ) and frequency ( E , WT: 2 . 32 ± 0 . 44 Hz; KO: 2 . 83 ± 0 . 63 pA ) were the same between WT and KO neurons . ( F–J ) Analysis of single AP evoked EPSCs recorded from WT and KO neurons in the presence of 10 mM [Ca2+]o . ( F ) Representative EPSCs recorded from WT and KO neurons . ( G–I ) EPSC amplitude ( G , WT: 0 . 25 ± 0 . 05 nA; KO: 0 . 24 ± 0 . 03 nA ) , peak latency ( H , WT: 17 . 03 ± 2 . 22 ms; KO: 18 . 27 ± 2 . 29 ms ) and rise time ( I , WT: 4 . 8 ± 0 . 53 ms; KO: 5 . 21 ± 0 . 74 ms ) were the same between WT and KO neurons . ( J ) The normalized cumulative total charge was fitted with a double exponential function ( solid line ) . WT and KO neurons had identical charge transfer kinetics . ( K ) Representative single AP evoked EPSCs recorded from WT and KO neurons in the presence of the indicated [Ca2+]o . ( L–M ) EPSC amplitudes ( L ) and relative amplitudes ( M ) plotted vs [Ca2+]o . The n values indicate the number of neurons from two independent litters of mice; these values are provided in the bars in all figures in this study . All data represent mean ± SEM . Statistical significance was analyzed using the Student’s t test . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 003 We next examined short-term synaptic plasticity in WT and KO neurons by examining the paired pulse ratio ( PPR ) , calculated by dividing the second EPSC ( EPSC2 ) by the first EPSC ( EPSC1 ) ( 50 ms interval; Figure 2A ) . We also measured synaptic depression during trains of HFS ( 20 Hz/2 . 5 s , Figure 3A ) . The PPR was the same between WT and KO neurons ( Figure 2B ) , but KO neurons displayed faster depression during HFS ( Figure 3B ) . Importantly , expression of WT syt 7 , but not the 4D/N Ca2+-ligand mutant ( which was properly folded but failed to bind Ca2+ ( Figure 3—figure supplement 1 ) ) , rescued the depression phenotype ( Figure 3B ) . We note that WT syt 7 , and the 4D/N mutant , were both present in synapses but were not well colocalized with the SV marker , synapsin , as compared to syt 1 ( Figure 3—figure supplement 2 ) . The surface fractions , as well as the internal fractions , were similar between syt 1 and 7 measured by using a pHluorin tag ( Figure 3—figure supplement 3 ) , consistent with a previous report ( Dean et al . , 2012a ) . 10 . 7554/eLife . 01524 . 004Figure 2 . Loss of syt 7 does not affect the PPR . ( A ) Representative EPSCs recorded in paired-pulse experiments , with 50 ms inter-stimulus intervals , from WT , KO , or KO neurons expressing either WT ( KO + syt 7 ) or the Ca2+ ligand mutant form ( KO + syt 7 4D/N ) of syt 7 . ( B ) The PPRs were the same among all groups ( WT: 0 . 83 ± 0 . 04; KO: 0 . 75 ± 0 . 03; KO + syt 7: 0 . 84 ± 0 . 05; and KO + syt 7 4D/N: 0 . 76 ± 0 . 03 ) . The n values indicate the number of neurons , obtained from three independent litters of mice , used for these experiments . All data represent mean ± SEM . Statistical significance was analyzed by one-way ANOVA . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 00410 . 7554/eLife . 01524 . 005Figure 3 . SV replenishment is reduced in KO neurons . ( A ) Representative EPSC traces ( 20 Hz/2 . 5 s ) recorded from WT ( black ) , KO ( red ) , or KO neurons expressing either WT ( KO + syt 7 , gray ) or the 4D/N mutant form of syt 7 ( KO + syt 7 4D/N , blue ) . The phasic and tonic components of transmission are indicated within the representative WT trace . ( B ) The peak amplitude of each EPSC was divided by the peak of the first response and plotted vs time . Inset: expansion of the last 10 responses . ( C ) Plot of cumulative total phasic charge transfer vs time . Data points from the last ten EPSCs were fitted with a linear function to calculate the RRP size ( y-intercept in panel D ) , and SV replenishment rate ( the slope divided by the RRP size; panel E ) . Inset: expansion of the last ten responses . ( D ) The RRP size is the same among all groups ( WT: 38 . 70 ± 4 . 78 pC; KO: 33 . 49 ± 2 . 48 pC; KO + syt 7: 37 . 58 ± 3 . 02 pC; and KO + syt 7 4D/N: 34 . 16 ± 3 . 63 pC ) . ( E ) The SV replenishment rate was reduced in KO ( 0 . 26 ± 0 . 03 s−1 ) as compared to WT neurons ( 0 . 52 ± 0 . 06 s−1 ) . WT ( 0 . 47 ± 0 . 05 s−1 ) , but not 4D/N mutant syt 7 ( 0 . 24 ± 0 . 02 s−1 ) , rescued SV replenishment . The n values indicate the number of neurons , obtained from three independent litters of mice , used for these experiments . All data shown represent mean ± SEM . ***p<0 . 001 , analyzed by one-way ANOVA , compared to WT . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 00510 . 7554/eLife . 01524 . 006Figure 3—figure supplement 1 . Ca2+-ligand mutations disrupt Ca2+ binding , but not proper folding , of syt 7 . ( A ) CD spectra of WT ( syt 7 C2AB; solid lines ) and the 4D/N mutant ( syt 7 C2AB 4D/N , dashed line ) versions of syt 7 C2AB domains; the spectra were identical . ( B ) ITC experiments utilizing the isolated C2-domains of WT ( syt 7 C2A , syt 7 C2B ) and mutant forms of each domain in which two putative acidic Ca2+ ligands were changed to Asn ( syt 7 C2A 2D/N , syt 7 C2B 2D/N ) . Ca2+-binding to C2A and C2B was endothermic and exothermic , respectively; neither of the mutant C2-domains appeared to bind Ca2+ under these experimental conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 00610 . 7554/eLife . 01524 . 007Figure 3—figure supplement 2 . Syt 7 is not localized to SVs . ( A ) A c-myc tag with a signal sequence was fused to the N-terminus of syt 7 ( ss-myc-syt 7 ) , syt 7 4D/N ( ss-myc-syt 7 4D/N ) , and 1 ( ss-myc-syt 1 ) as described in 'Materials and methods' , and the resultant fusion protein was expressed in KO hippocampal neurons using lentivirus; cells were immunostained with antibodies against c-myc , the SV marker synapsin , or against GFP that was expressed via an independent promoter . ( B ) Analysis of the c-myc signals relative to the synapsin signals revealed that syt 1 ( 0 . 48 ± 0 . 03 ) , but not syt 7 ( 0 . 18 ± 0 . 02 ) and syt 7 4D/N ( 0 . 19 ± 0 . 01 ) , co-localized with SVs . Average colocalization , from 12 fields of view ( ∼100 boutons per field ) from six coverslips , two coverslips each from three independent litters of mice . All data shown represent mean ± SEM . ***p<0 . 001 , analyzed by one-way ANOVA , compared to WT . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 00710 . 7554/eLife . 01524 . 008Figure 3—figure supplement 3 . Relative distribution of syt 7 in the plasma membrane , vs internal compartments , in hippocampal neurons . ( A ) Average fluorescence signals from pHluorin-syt 1 ( black ) and pHluorin-syt 7 ( red ) during perfusion with a low pH solution ( pH 5 . 5 ) to quench surface fluorescence , and during perfusion with NH4Cl to neutralize acidic internal compartments and thus reveal internal pHluorin signals . ( B ) Quantitation of the surface fraction of syt 7 and syt 1; the majority of the signal is internal , but significant fractions of each protein are present in the plasma membrane . Data are averages from ∼300 boutons; 50 boutons from each of six coverslips , two coverslips per mouse , three independent litters of mice . All data shown represent mean ± SEM . Statistical significance was analyzed by the Student's t test . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 00810 . 7554/eLife . 01524 . 009Figure 3—figure supplement 4 . Postsynaptic receptor desensitization and saturation do not contribute to the KO phenotype . ( A ) Representative EPSC traces ( 20 Hz/2 . 5 s ) recorded from WT ( black ) and KO ( red ) neurons , in the presence of both CTZ and KYN . ( B ) The peak amplitude of each EPSC was normalized to the peak of the first response and plotted vs time . The n values indicate the number of neurons , obtained from three independent litters of mice , used for these experiments . All data shown represent mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 00910 . 7554/eLife . 01524 . 010Figure 3—figure supplement 5 . Replenishment of the tonic component of transmission is reduced in KO neurons . ( A ) Representative EPSC traces from WT neurons illustrating both phasic and tonic release as shown in Figure 3A . ( B ) Plot of cumulative total tonic charge transfer vs time . Data were fitted and analyzed as described in Figure 3C . ( C ) SV replenishment was reduced in KO ( 2 . 19 ± 0 . 22 s−1 ) as compared to WT neurons ( 3 . 95 ± 0 . 61 s−1 ) . WT ( 3 . 93 ± 0 . 31 s−1 ) , but not the 4D/N mutant form ( 1 . 88 ± 0 . 26 s−1 ) of syt 7 , rescued replenishment . The n values indicate the number of neurons , obtained from three independent litters of mice , used for these experiments . All data shown represent mean ± SEM . ***p<0 . 001 , analyzed by one-way ANOVA , compared to WT . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 010 Five mechanisms could potentially contribute to the faster depression observed in KO neurons: changes in postsynaptic receptor density or kinetic properties; saturation or desensitization of postsynaptic receptors; a smaller RRP size which fails to supply sufficient SVs; defective SV replenishment; or slower SV recycling/refilling . Below , we address each of these possibilities and demonstrate that defective SV replenishment underlies the increased rate of depression in KO neurons . The unaltered quantal release properties in KO neurons ( Figure 1A–E ) rule out changes in postsynaptic components , such as the density or kinetic properties of AMPA receptors . To further exclude the role of desensitization or saturation of postsynaptic receptors during HFS , we monitored transmission , during HFS , in the presence of cyclothiazide ( CTZ , 50 µM ) and kynurenic acid ( KYN , 100 µM ) , as described previously ( Neher and Sakaba , 2001; Figure 3—figure supplement 4A ) . CTZ and KYN reduce AMPA receptor desensitization and saturation , respectively . KO neurons still displayed faster depression ( Figure 3—figure supplement 4B ) . Next , we integrated the phasic charge from the HFS experiments ( Figure 3A ) and plotted the cumulative charge vs time ( Figure 3C ) . The last ten EPSCs were best fitted by a linear function ( solid line in Figure 3C; linearity is established in the inset ) , as described in previous reports utilizing hippocampal neurons ( Stevens and Williams , 2007 ) and the calyx of Held ( Schneggenburger et al . , 1999; Hosoi et al . , 2007 ) , to calculate RRP size , release probability , and SV replenishment rates . The size of the RRP ( the y-intercept ) was similar between WT and KO neurons ( Figure 3D ) . Since single AP evoked EPSCs were identical between WT and KO neurons ( Figure 1F ) , SV release probability ( calculated by dividing the EPSC charge by the RRP size ) was unchanged in the KOs ( 0 . 21 ± 0 . 03 for KO vs 0 . 24 ± 0 . 02 for WT ) . The slope of the linear function was divided by the y-intercept in a given cell pair to calculate the SV replenishment rate . Strikingly , we found that the replenishment rate was reduced by ∼50% for phasic release in KO neurons ( 0 . 26 ± 0 . 03 s−1 for KO vs 0 . 52 ± 0 . 06 s−1 for WT ) ( Figure 3E ) . WT syt 7 , but not the 4D/N mutant , fully rescued SV replenishment ( Figure 3C , E ) . The size of the RRP was unchanged in the rescue experiments ( Figure 3D ) . We also applied similar methods as described in Figure 3C to investigate SV replenishment during tonic transmission ( Figure 3—figure supplement 5A , B ) ; the replenishment rate was , again , reduced by ∼50% in KO neurons ( 2 . 19 ± 0 . 22 s−1 for KO vs 3 . 95 ± 0 . 61 s−1 for WT ) , and WT syt 7 , but not the 4D/N mutant , rescued this defect ( Figure 3—figure supplement 5C ) . These results are consistent with a previous report indicating that tonic transmission is maintained by Ca2+-dependent SV replenishment during activity ( Otsu et al . , 2004 ) . To further confirm the replenishment defect in KO neurons , we investigated the recovery kinetics of phasic release after HFS . Recovery was calculated by measuring the total charge transfer during phasic release at various time intervals , between bouts of HFS , where the second response was divided by the first response ( Figure 4A ) . A double exponential function was used to fit the recovery time-course , revealing fast and slow components of recovery . Notably , fast recovery ( Figure 4B ) was delayed by ∼60% in KO neurons ( 1 . 32 ± 0 . 22 s ) as compared to WT neurons ( 0 . 55 ± 0 . 17 s ) , while slow recovery ( Figure 4C ) was unaffected by loss of syt 7 ( WT: 15 . 72 ± 2 . 85 s; KO: 14 . 13 ± 2 . 24 s ) . WT syt 7 ( 0 . 52 ± 0 . 12 s ) , but not the 4D/N mutant ( 1 . 28 ± 0 . 26 s ) , rescued fast recovery ( Figure 4A , B ) , indicating that syt 7 regulates this process in a Ca2+-dependent manner during phasic release . Surprisingly , the recovery of total tonic transmission during HFS also displayed the same two recovery components ( Figure 4—figure supplement 1A–C ) . WT syt 7 , but not the 4D/N mutant , rescued fast recovery of tonic transmission ( Figure 4—figure supplement 1A , B ) . Thus , recovery of phasic and tonic release might involve the same Ca2+-dependent pathway . 10 . 7554/eLife . 01524 . 011Figure 4 . Impaired fast recovery of phasic transmission in KO neurons . ( A ) Time course for the recovery of phasic release ( Δt = 0 . 5 , 1 , 3 , 5 , 9 , 17 , 33 and 65 s ) . Data were fitted using a double exponential function , yielding fast ( B ) and slow ( C ) recovery . The upper diagram is the stimulation protocol . Inset: expansion of the early phase of the plot . ( B ) The fast recovery time constant was increased in KO ( 1 . 32 ± 0 . 22 s ) as compared to WT neurons ( 0 . 55 ± 0 . 17 s ) . WT syt 7 ( 0 . 52 ± 0 . 12 s ) , but not the 4D/N mutant ( 1 . 28 ± 0 . 26 s ) , rescued the SV replenishment rate in KO neurons . ( C ) The slow recovery time constant was invariant among all groups . ( WT: 15 . 72 ± 2 . 85 s; KO: 14 . 13 ± 2 . 24 s; KO + syt 7: 17 . 03 ± 3 . 24 s; and KO + syt 7 4D/N: 18 . 88 ± 4 . 45 s ) . The n values indicate the number of neurons , obtained from three independent litters of mice , used for these experiments . All data shown represent mean ± SEM . ***p<0 . 001 , analyzed by one-way ANOVA , compared to WT . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 01110 . 7554/eLife . 01524 . 012Figure 4—figure supplement 1 . The fast recovery component of tonic transmission is impaired in KO neurons . ( A ) Time course for the recovery of tonic release . Data were fitted and analyzed as described in Figure 4A . Upper panel depicts the HFS protocol ( Δt = 0 . 5 , 1 , 3 , 5 , 9 , 17 , 33 and 65 s ) . ( B ) The fast recovery time constant for tonic release was increased in KO ( 1 . 20 ± 0 . 24 s ) as compared to WT neurons ( 0 . 54 ± 0 . 05 s ) . WT ( 0 . 61 ± 0 . 15 s ) , but not the 4D/N mutant form ( 1 . 4 ± 0 . 22 s ) of syt 7 , rescued SV replenishment . ( C ) The slow recovery time constant for tonic release was the same among all groups . ( WT: 18 . 52 ± 2 . 43 s; KO: 17 . 43 ± 3 . 39 s; KO + syt 7: 17 . 42 ± 3 . 65 s; and KO + syt 7 4D/N: 16 . 07 ± 4 . 15 s ) . The n values indicate the number of neurons , obtained from three independent litters of mice , used for these experiments . All data shown represent mean ± SEM . ***p<0 . 001 , analyzed by one-way ANOVA , compared to WT . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 012 To address potential defects in SV recycling/refilling in KO neurons during ongoing transmission , we utilized synaptophysin-pHluorin ( sypHy ) to measure SV exocytosis driven by HFS ( 20 Hz ) in the presence of bafilomycin ( 1 µM ) , an inhibitor of the vesicular proton pump ( Figure 5A–C ) , to measure first fusion events . Under these conditions , in which SV recycling cannot play a role in ongoing synaptic transmission , KO neurons still displayed a significant reduction in SV release ( Figure 5B ) . Importantly , KO neurons exhibited reductions in SV release as early as the second imaging time point ( i . e . , 1 sec , 20 Hz stimulation ) ( Figure 5C ) , consistent with electrophysiological recordings in Figure 3C . Moreover , we also examined first release events during HFS by recording synaptic currents in the presence of bafilomycin and , again , significantly faster depression ( Figure 5D ) , reduced cumulative release ( Figure 5E ) , unchanged RRP size ( Figure 5F ) , and slower SV replenishment ( Figure 5G ) , were observed in the KOs . These data indicate that SV recycling does not contribute to the observed reduction in SV release in KO neurons during HFS . 10 . 7554/eLife . 01524 . 013Figure 5 . The reduction in SV release in KO neurons during HFS is independent of vesicle recycling . ( A ) Representative image showing the sypHy signal from the same synapse upon stimulation in the absence , and presence , of bafilomycin . ( B ) Averaged traces of sypHy in WT ( black ) and KO ( red ) neurons in response to stimulation in the presence of bafilomycin . KO neurons displayed a significant reduction in sustained SV release as compared to WT neurons . Data were collected for ∼300 boutons per condition as follows: ∼50 boutons per coverlip , from six coverslips ( two each from three independent litters of mice ) . All data shown represent mean ±SEM . ***p<0 . 001 analyzed by two-way ANOVA . The initial response , indicated by the tan rectangle , is expanded in panel C . ( C ) KO neurons display reduced sypHy responses early in the train . *p<0 . 05 , analyzed using the Student’s t test . ( D–G ) ESPC recordings , in response to HFS , in the presence of bafilomycin . ( D ) The peak amplitude of each EPSC was normalized to the peak of the first response and plotted vs time . ( E ) Plot of cumulative total phasic charge transfer vs time . Data were fitted and analyzed as described in Figure 3C . ( F ) The size of the RRP is the same in WT and KO neurons ( WT: 35 . 42 ± 5 . 07 pC; KO: 32 . 07 ± 4 . 54 pC ) . ( G ) Reduction in the SV replenishment rate in KO neurons in the presence of bafilomycin ( WT: 0 . 49 ± 0 . 06 s−1; KO: 0 . 23 ± 0 . 03 s−1 ) . The n values indicate the number of neurons , obtained from three independent litters of mice , used for these experiments . All data shown represent mean ± SEM . ***p<0 . 001 , analyzed using the Student’s t test . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 013 Previous studies demonstrated that Ca2+ plays a critical role in the replenishment of SVs ( Dittman and Regehr , 1998; Wang and Kaczmarek , 1998; Hosoi et al . , 2007; Sakaba , 2008 ) . The syt 7 4D/N mutant , which fails to bind Ca2+ , was unable to rescue SV replenishment in KO neurons ( Figure 3 ) , suggesting that syt 7 functions as Ca2+-sensor during replenishment . To test whether Ca2+ is essential for the function of syt 7 , we measured recovery of the RRP by applying paired pulses of hypertonic sucrose ( 500 mM , each for 10 s ) , with varying interpulse time intervals , under Ca2+-free conditions ( replacement of [Ca2+]o with EGTA , incubation with BAPTA-AM , and addition of cyclopazonic acid [CPA] to empty internal stores [Liu et al . , 2009] ) ( Figure 6A ) . For comparison , we also measured RRP recovery under physiological [Ca2+]o ( 2 mM; Figure 6A ) . 10 . 7554/eLife . 01524 . 014Figure 6 . Syt 7 functions as a Ca2+-sensor for Ca2+-dependent SV replenishment . ( A ) Upper panel illustrates the sucrose application protocol ( Δt = 7 , 14 , 21 , 28 and 60 s ) . Lower panel shows typical responses to sequential application of sucrose ( 7 s interval ) from different groups in 2 mM [Ca2+]o or Ca2+-free conditions . ( B ) The RRP size , calculated from the first sucrose response , was same among the all groups ( in 2 mM [Ca2+]o , WT: 0 . 86 ± 0 . 21 nC; KO: 0 . 72 ± 0 . 14 nC; KO + syt 7: 0 . 78 ± 0 . 09 nC; and KO + syt 7 4D/N: 0 . 85 ± 0 . 08 nC; in Ca2+-free conditions , WT: 0 . 83 ± 0 . 18 nC; KO: 0 . 76 ± 0 . 2 nC ) . ( C ) The time course of RRP recovery in 2 mM [Ca2+]o conditions between applications of hypertonic sucrose . Data were fitted with a single exponential function to calculate the recovery time constant in panel E . ( D ) The recovery of the RRP in Ca2+-free conditions . Data were fitted with a single exponential function to calculate the recovery time constant in panel E . ( E ) In 2 mM [Ca2+]o , recovery of the RRP was significantly slowed in KO ( 18 . 03 ± 2 . 87 s ) neurons as compared to WT ( 7 . 29 ± 1 . 05 s ) neurons , and was rescued by WT syt 7 ( 8 . 30 ± 0 . 79 s ) , but not the 4D/N mutant ( 20 . 07 ± 2 . 63 s ) . In Ca2+-free conditions , RRP recovery in WT neurons ( 17 . 42 ± 2 . 11 s ) was slowed to the same level as in KO neurons ( 19 . 2 ± 3 . 95 s ) . The n values indicate the number of neurons , obtained from three independent litters of mice , used for these experiments . All data shown represent mean ± SEM . ***p<0 . 001 , analyzed using one-way ANOVA , compared to WT . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 014 Consistent with the same RRP size measured by HFS among different groups ( Figure 3D ) , the RRP size , calculated from the first response to sucrose , was the same between WT , KO , and KOs expressing WT or the syt 7 4D/N mutant , in physiological [Ca2+]o ( Figure 6B ) , excluding a role for syt 7 in SV priming . Moreover , the RRP size was the same between the Ca2+-free and physiological [Ca2+]o conditions ( Figure 6B ) . Of note , there was a significant difference in the absolute size of the RRP as measured using hypertonic sucrose ( Figure 6B; ∼8000 SVs , calculated by dividing the RRP charge by the quantal charge ) vs HFS ( Figure 3D; ∼300 SVs ) . This was expected , because during electrical stimulation , only the synapses formed between the recorded neuron and stimulated neuron are measured . In contrast , hypertonic sucrose activates all of the synapses that impinge on the recorded neuron . We then plotted the ratio of the second ( P2 ) relative to the first ( P1 ) sucrose response , as a function of the interpulse time interval , to calculate the recovery time course of the RRP in physiological [Ca2+]o ( Figure 6C ) and Ca2+-free conditions ( Figure 6D ) ; these data were best fitted by a single exponential function ( solid line in Figure 6C , D ) . In physiological [Ca2+]o , recovery of the RRP was slowed by ∼60% in the KOs ( 18 . 03 ± 2 . 87 s for KO vs 7 . 29 ± 1 . 05 s for WT ) ( Figure 6E ) , which is similar to the findings reported above using HFS ( Figure 4B ) . WT syt 7 ( 8 . 30 ± 0 . 79 s ) , but not the 4D/N mutant ( 20 . 07 ± 2 . 63 s ) , rescued this phenotype ( Figure 6C , E ) . Remarkably , in Ca2+-free conditions ( Figure 6D ) , the recovery of the RRP in WT neurons was reduced to the same level as observed for KO neurons , with or without expression of the 4D/N mutant in physiological [Ca2+]o conditions ( Figure 6E ) . Together , the results thus far suggest that there are Ca2+-dependent and Ca2+-independent pathways for replenishment , and that syt 7 functions as a Ca2+-sensor that regulates the Ca2+-dependent pathway . Hypertonic sucrose stimulates SV release without increasing [Ca2+]i ( Yao et al . , 2011 , 2012 ) . Therefore , the Ca2+-dependent component for recovery of the RRP , observed in the hypertonic sucrose experiments , is likely due to the resting [Ca2+]i . In vitro reconstitution experiments revealed that syt 7 begins to drive fusion at [Ca2+] > 100 nM ( [Ca2+]½ = 300 nM ) ( Bhalla et al . , 2005 ) . Hence , it is plausible that syt 7 can indeed sense resting [Ca2+]i to regulate Ca2+-dependent SV replenishment . The reduced SV replenishment phenotype in KO neurons is reminiscent of the effects of CaM antagonists on vesicle replenishment in the calyx of Held ( Sakaba and Neher , 2001; Hosoi et al . , 2007 ) , raising the possibility that there are links between CaM and syt 7; indeed , syt 1 has been reported to bind CaM ( Popoli , 1993; Perin , 1996 ) . We therefore conducted pull-down assays using immobilized CaM and the soluble cytoplasmic domains ( C2AB ) of syt 1 , 2 , 4 , 7 , 9 and 10 ( Figure 7A ) . Surprisingly , robust Ca2+-dependent binding to CaM was observed only for syt 7 , demonstrating the specificity of the interaction ( note: low levels of binding of syt 1 , or other syt isoforms , to CaM might have escaped detection ) . Moreover , the Ca2+-promoted interaction between syt 7 and CaM was abolished by the Ca2+-ligand mutations in the syt 7 4D/N mutant ( Figure 7A ) and was specific for Ca2+ as Mg2+ was without effect ( Figure 7B ) . Together , these biochemical findings are consistent with a model in which the Ca2+-sensor for replenishment is actually a complex formed between these two conserved Ca2+-binding proteins , syt 7 and CaM . 10 . 7554/eLife . 01524 . 015Figure 7 . Syt 7 cooperates with CaM to regulate Ca2+-dependent SV replenishment . ( A ) Immobilized CaM efficiently pulled-down the C2AB domain of syt 7 , but not syt 1 , 2 , 4 , 9 or 10 , in the presence , but not the absence , of Ca2+; the syt 7 4D/N mutant failed to bind . E , 2 mM EGTA; Ca , 1 mM free Ca2+ . Spaces between gels indicate samples that were run separately . Input corresponds to 25% of the starting material; 50% of the bound material was loaded per lane; no binding was observed when CaM was omitted from the beads ( data not shown ) . ( B ) Mg2+ fails to promote binding of syt 7 C2AB to CaM . E , 2 mM EGTA; Ca , 1 mM free Ca2+; Mg , 1 mM free Mg2+ . Gels were loaded as in panel A . ( C ) Representative EPSCs traces ( 20 Hz/2 . 5 s ) recorded from WT ( black ) and KO ( red ) neurons in the presence of CDZ . ( D ) Plot of cumulative total phasic release charge vs time in the presence of CDZ . Data were analyzed as described in Figure 3C . ( E ) The size of the RRP was the same among all groups ( in control: WT: 38 . 70 ± 4 . 78 pC; KO: 33 . 49 ± 2 . 48 pC; in CDZ treated neurons: WT: 36 . 85 ± 5 . 35 pC; KO: 34 . 77 ± 6 . 74 pC ) . ( F ) CDZ treatment reduced SV replenishment in WT neurons ( 0 . 24 ± 0 . 04 s−1 vs 0 . 52 ± 0 . 06 s−1 for controls ) , but had no effect on KO neurons ( 0 . 21 ± 0 . 05 s−1 vs 0 . 26 ± 0 . 03 s−1 for controls ) . The n values indicate the number of neurons , obtained from three independent litters of mice , used for these experiments . All data shown represent mean ± SEM . ***p<0 . 001 , analyzed by one-way ANOVA , compared to WT . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 01510 . 7554/eLife . 01524 . 016Figure 7—figure supplement 1 . CDZ inhibits SV replenishment during tonic transmission in WT neurons . ( A ) Plot of cumulative total tonic charge transfer vs time from WT and KO neurons , in the presence and absence of CDZ , during HFS . Data were fitted and analyzed as described in Figure 3C . ( B ) During tonic transmission , CDZ treatment reduced SV replenishment in WT neurons ( 1 . 88 ± 0 . 32 s−1 vs 3 . 95 ± 0 . 61 s−1 for controls ) , but had no effect on the already compromised replenishment rate in KO neurons ( 1 . 73 ± 0 . 34 s−1 vs 2 . 19 ± 0 . 22 s−1 for controls ) . The n values indicate the number of neurons , obtained from three independent litters of mice , used for these experiments . All data shown represent mean ± SEM . ***p<0 . 001 , analyzed by one-way ANOVA , compared to WT . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 016 To address the potential contribution of CaM to SV replenishment in hippocampal neurons , we next analyzed the SV replenishment rate in WT and KO neurons in the presence and absence of calmidazolium ( CDZ; 20 µM ) , a cell-permeable CaM antagonist ( Sakaba and Neher , 2001; Figure 7C ) . Cells were incubated for 30 min prior to recording to ensure that CDZ had taken effect , and then HFS trains were applied . WT neurons displayed faster depression ( Figure 7D , upper ) and reduced cumulative phasic release ( Figure 7D , lower ) in the presence of CDZ , consistent with previous observations based on the calyx of Held ( Sakaba and Neher , 2001; Hosoi et al . , 2007 ) . Similarly , cumulative tonic transmission in WT neurons was also reduced by CDZ ( Figure 7—figure supplement 1A ) . Interestingly , both cumulative phasic and tonic release in WT neurons were reduced to the same levels as in the KO neurons , which were not affected by CDZ ( Figure 7C , Figure 7—figure supplement 1A ) . These experiments also revealed that: ( 1 ) CDZ does not change the size of the RRP ( Figure 7E ) , and ( 2 ) in WT neurons , the replenishment rates for phasic ( Figure 7F ) and tonic release ( Figure 7—figure supplement 1B ) were both reduced to the same levels observed for the KOs , with or without CDZ . We next examined the recovery of phasic ( Figure 8A ) and tonic release ( Figure 8—figure supplement 1A ) , as described in Figure 4 , in the presence of CDZ during HFS , in WT and KO neurons . The time course for fast recovery , during phasic ( Figure 8B ) and tonic ( Figure 8—figure supplement 1B ) transmission in WT neurons , was impaired , phenocopying the KO neurons which , in turn , were unaffected by CDZ . The time course for slow recovery was unaffected under all conditions tested ( Figure 8C , Figure 8—figure supplement 1C ) . 10 . 7554/eLife . 01524 . 017Figure 8 . CDZ inhibits RRP recovery in WT neurons , phenocopying the KO . ( A ) Time course for the recovery of phasic release upon HFS; data were collected and analyzed as described in Figure 4A . The inset panel shows the expanded early phase of the plot . ( B ) CDZ slowed the time course of fast recovery of the RRP in WT neurons ( 1 . 35 ± 0 . 37 s vs 0 . 55 ± 0 . 17 s for control ) , phenocopying the KO neurons , which were not affected by CDZ ( 1 . 22 ± 0 . 36 s vs 1 . 32 ± 0 . 22 s for control ) . ( C ) The time course of slow recovery of the RRP was the same between WT and KO neurons with or without CDZ ( WT: 16 . 02 ± 3 . 02 s vs 15 . 72 ± 2 . 85 s for controls; KO: 15 . 78 ± 2 . 5 s vs 14 . 13 ± 2 . 24 s for controls ) . ( D ) Typical responses to sequential ( 7 s ) applications of sucrose to WT and KO neurons in 2 mM [Ca2+]o in the presence of CDZ . The upper diagram illustrates the protocol used ( Δt = 7 , 14 , 21 , 28 and 60 s ) . ( E ) Time course for the recovery of the RRP after depletion with hypertonic sucrose in 2 mM [Ca2+]o . Data were fitted with a single exponential function to calculate the recovery time constant ( F ) . ( F ) CDZ increased the time constant for recovery of the RRP in WT neurons ( 20 . 69 ± 1 . 43 s vs 7 . 29 ± 1 . 05 s for controls ) , but had no effect on KO neurons ( 20 . 37 ± 2 . 1 s vs 18 . 03 ± 2 . 87 s for controls ) . The n values indicate the number of neurons , obtained from three independent litters of mice , used for these experiments . All data shown represent mean ±SEM . ***p<0 . 001 , analyzed by one-way ANOVA , compared to WT . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 01710 . 7554/eLife . 01524 . 018Figure 8—figure supplement 1 . CDZ impairs recovery of tonic transmission in WT neurons . ( A ) Time course of recovery of tonic transmission . Data were fitted and analyzed as described in Figure 4A . Upper diagram describes the stimulation protocol ( Δt = 0 . 5 , 1 , 3 , 5 , 9 , 17 , 33 and 65 s ) . Inset: expanded view of the initial section of the plot . ( B ) CDZ slowed the fast recovery component during tonic transmission in WT neurons ( 1 . 34 ± 0 . 35 s vs 0 . 54 ± 0 . 05 s for controls ) , but had no effect on KO neurons ( 1 . 18 ± 0 . 24 s vs 1 . 20 ± 0 . 24 s for controls ) . ( C ) The slow recovery time constant was the same between WT and KO neurons , with or without CDZ ( WT: 17 . 32 ± 2 . 64 s vs 18 . 52 ± 2 . 43 s for controls; KO: 15 . 83 ± 3 . 13 s vs 17 . 43 ± 3 . 39 s for controls ) . The n values indicate the number of neurons , obtained from three independent litters of mice , used for these experiments . All data shown represent mean ± SEM . ***p<0 . 001 , analyzed by one-way ANOVA , compared to WT . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 01810 . 7554/eLife . 01524 . 019Figure 8—figure supplement 2 . CDZ does not compete with syt 7 for binding to CaM . The cytoplasmic domains of syt 7 ( syt 7 C2AB ) and different concentrations of CDZ were simultaneously incubated with immobilized CaM for 2 hr at 37°C in the presence and absence of Ca2+ . CDZ had no effect on Ca2+-dependent binding of syt 7 to CaM . E , 2 mM EGTA; Ca , 1 mM free Ca2+ . DOI: http://dx . doi . org/10 . 7554/eLife . 01524 . 019 We also used hypertonic sucrose , in 2 mM [Ca2+]o and in the presence of CDZ , to measure the time course for recovery of the RRP in WT and KO neurons ( Figure 8D ) . CDZ impaired the RRP recovery kinetics in WT neurons , again phenocopying the KOs , which were not affected by the drug ( Figure 8E , F ) . The findings reported above indicate that CaM and syt 7 might act together to regulate vesicle replenishment . We therefore assayed the effect of CDZ on this interaction . CDZ , up to 100 µM , was simultaneously incubated with the syt 7 C2AB domain and immobilized CaM . Competition was not observed ( Figure 8—figure supplement 2 ) . We also incubated immobilized CaM with CDZ for 30 min prior to the addition of syt 7 C2AB . Again , CDZ had no effect on syt 7·CaM interactions ( data not shown ) . These findings indicate that syt 7 and CDZ occupy different sites on CaM to regulate SV replenishment . Thus , CaM might serve as an adaptor , linking syt 7 to another CDZ-sensitive effector in the Ca2+-dependent replenishment pathway .
Short term synaptic plasticity ( STP ) plays an important role in the central nervous system , including learning and memory ( Zucker and Regehr , 2002 ) . Changes in SV release probability , RRP size , SV replenishment , or SV recycling can impact STP . In the current report , we discovered that syt 7 contributes to STP by regulating SV replenishment . WT syt 7 , but not the 4D/N mutant that fails to bind Ca2+ , rescues the defects in SV replenishment observed in KO neurons , indicating that syt 7 functions as Ca2+ sensor for this process . We further distinguished a Ca2+-independent SV replenishment pathway from the Ca2+-dependent pathway by using hypertonic sucrose to drive secretion in the complete absence of Ca2+ ( Figure 6 ) . Ca2+-independent SV replenishment has been discussed in previous studies ( Stevens and Wesseling , 1998; Hosoi et al . , 2007 ) ; however , only the slow Ca2+ chelator , EGTA-AM , was used . In the current study , extracellular Ca2+ was removed and EGTA was included in the bath solution . Moreover , intracellular stores of Ca2+ were depleted using CPA and the fast Ca2+ chelator , BAPTA-AM . The finding that WT neurons , in Ca2+-free conditions , display a similar RRP recovery rate as compared to KO neurons in normal ( Ca2+ ) conditions , along with the inability of the syt 7 4D/N mutant to rescue recovery of the RRP in the presence of Ca2+ ( Figure 6 ) , strongly indicate that syt 7 is a Ca2+-sensor in the Ca2+-dependent SV replenishment pathway . CaM is a ubiquitous Ca2+-binding protein with multiple functions in the SV cycle , including SV replenishment ( Sakaba and Neher , 2001; Hosoi et al . , 2007 ) . Remarkably , when applied to WT hippocampal neurons , a CaM antagonist precisely phenocopies the defects in SV replenishment apparent in KO neurons . Moreover , we discovered an avid and direct Ca2+-promoted interaction between CaM and syt 7; in contrast , syt 1 , 2 , 4 , 9 and 10 failed to bind to CaM in response to Ca2+ , and loss of these latter isoforms has not been associated with enhanced synaptic depression ( Maximov and Sudhof , 2005; Pang et al . , 2006; Sun et al . , 2007; Xu et al . , 2007; Dean et al . , 2009; Liu et al . , 2009; Cao et al . , 2011; Dean et al . , 2012a ) . These findings underscore both the specificity of the syt 7•CaM interaction , as well as the specific role played by syt 7 during synaptic depression . Thus , we propose that the Ca2+-sensor for replenishment consists of a complex formed between two conserved Ca2+-binding proteins , syt 7 and CaM . From the data reported here , neuronal functions for the only three syt isoforms found in all metazoans ( isoforms 1 , 4 and 7; Barber et al . , 2009 ) have been determined . In presynaptic terminals , syt 1 acts to directly trigger rapid SV exocytosis ( Geppert et al . , 1994; Koh and Bellen , 2003; Nishiki and Augustine , 2004; Liu et al . , 2009 ) , syt 4 regulates the release of brain derived neurotrophic factor to modulate synaptic function ( Dean et al . , 2009 , 2012b ) , and syt 7 functions as a Ca2+ sensor for SV replenishment during and after HFS . In a previous study it was concluded that neurotransmission was completely normal in KO inhibitory neurons , and that syt 7 did not regulate asynchronous synaptic transmission ( Maximov et al . , 2008 ) . In that study , there was a slight reduction in synaptic transmission in KO neurons during high , but not low , frequency stimulation , suggestive of a defect in replenishment . This effect was less pronounced as compared to our observations; the reasons for this difference are unknown , but might involve differences between the inhibitory and excitatory neurons used in these studies . Moreover , a more recent report from the same group ( Bacaj et al . , 2013 ) , using a KD approach , reached the contradictory conclusion that syt 7 plays a role in asynchronous evoked release . However , loss of syt 7 , by either KD or KO , had no effect on asynchronous release ( Maximov et al . , 2008; Bacaj et al . , 2013 ) ; the only case where a loss of release was observed was when syt 7 was knocked down ( and not knocked out ) in a syt 1 KO background . So , the physiological relevance of this observation is unclear . Moreover , in Bacaj et al . ( 2013 ) , there was , again , a reduction in release only at late , but not early stages , in the stimulus train in KO neurons . We argue that this result should be interpreted as defective SV replenishment , because loss of asynchronous release should occur for all evoked responses , early and late . Furthermore , impaired replenishment of large dense core vesicles ( LDCV ) was also reported in KO chromaffin cells ( Schonn et al . , 2008 ) , consistent with our current study . Thus , syt 7 might serve as a ubiquitous Ca2+ sensor for replenishment of vesicles , including LDCVs and SVs . At present , it remains unknown as to how syt 7 and CaM regulate SV replenishment . We observed that c-myc tagged WT syt 7 , as well as the 4D/N mutant , were enriched in synapses , but were not co-localized with SV markers ( Figure 3—figure supplement 2 ) . These findings raise the possibility that syt 7 may be targeted to , and thereby act at the plasma membrane , as proposed by Sugita et al . ( 2001 ) , to drive replenishment by either conveying SVs from reserve pools to the RRP ( Kuromi and Kidokoro , 1998; Murthy and Stevens , 1999; Ikeda and Bekkers , 2009 ) , or clearing vesicle release sites ( Kawasaki et al . , 2000; Wu et al . , 2009; Neher , 2010 ) . Using a pHluorin tag , we found that syt 1 and 7 have similar cell surface fractions; while significant levels of each protein are present in the plasma membrane , the largest pools of syt 7 and syt 1 are on internal organelles that acidify ( Figure 3—figure supplement 3 ) , consistent with earlier work localizing syt 7 to LDCVs and lysosomes in neuroendocrine cells and cells in the immune system ( Fukuda et al . , 2004; Wang et al . , 2005; Czibener et al . , 2006 ) . Clearly , syt 7 is not selectively targeted to the plasma membrane ( though , again , like all syt isoforms , some fraction is present in the plasma membrane [Dean et al . , 2012a] ) . Moreover , pHluorin-syt 7 does not recycle with SV-like kinetics , even after repetitive stimulation ( Dean et al . , 2012a ) , excluding the possibility that syt 7 traffics to SVs before , during , or after stimulation . So , syt 7 is likely to regulate replenishment either from the plasma membrane , or from LDCVs or lysosomes . In molecular terms , the emerging view is that CaM regulates SV replenishment via interactions with both Munc13-1 ( Lipstein et al . , 2013 ) and syt 7 . Future studies are needed to determine whether CaM can physically interact with both proteins simultaneously , or whether CaM forms mutually exclusive complexes in the replenishment pathway .
KO mice were provided by NW Andrews ( College Park , MD ) . Primary hippocampal cultures were prepared as described previously ( Liu et al . , 2009 ) . Briefly , heterozygous KO mice were bred , and hippocampal neurons from newborn pups ( postnatal day 0 ) were isolated . Neurons were plated at low density ( ∼5000 cells/cm2 ) on poly-lysine coated coverslips ( Carolina Biologicals , Burlington , NC ) , and cultured in Neurobasal media supplemented with 2% B-27 and 2 mM Glutamax ( Life Technologies , Great Island , NY ) . Tails from KO pups were kept for genotyping , and electrophysiological recordings from KO and WT littermate neurons were compared . All procedures involving animals were performed in accordance with the guidelines of the National Institutes of Health; the protocol used ( M01221-0-06-11 ) was approved by the Institutional Animal Care and Use Committee of the University of Wisconsin–Madison . cDNA encoding rat syt 1 and syt 4 were provided by TC Südhof ( Stanford , CA ) and H . Herschman ( Los Angeles , CA ) , respectively . cDNA encoding mouse syt 2 , 7 , 9 , and 10 were provided by M Fukuda ( Saitama , Japan ) . We note that the D374 mutation in the original syt 1 cDNA was corrected by replacement with a glycine residue ( Desai et al . , 2000 ) . cDNA encoding sypHy was provided by E Kavalali ( Dallas , TX ) . The syt 7 4D/N mutant harbors a D225 , 227 , 357 , 359N quadruple mutation in which four acidic Ca2+-ligands ( two in each C2 domain ) were neutralized , thereby disrupting Ca2+-binding activity . Because specific , high affinity syt 7 antibodies are not currently available , a c-myc tag , TGGAGCAGAAGCTGATCAGCGAGGAGGACCTGAACGGAATT , was added to the N-terminus of WT and syt 7 4D/N mutant to detect and localize the exogenously expressed proteins . C-myc tagged syt 1 was used as a control . A signal sequence ( ss ) : ATGGACAGCAAAGGTTCGTCGCAGAAAGGGTCCCGCCTGCTCCTGCTGCTGGTGGTGTCAAATCTACTCTTGTGCCAGGGTGTGGTCTCCGA , was appended onto the N-terminus of the c-myc tag to ensure translocation of the fusion proteins ( ss-myc-syt 1 , ss-myc-syt 7 and ss-myc-syt 7 4D/N ) into the endoplasmic reticulum ( Dean et al . , 2012a ) . These constructs were subcloned into the pLox Syn-DsRed-Syn-GFP lentivirus vector ( provided by F Gomez-School [Spain] ) using BamH I and Not I , thereby replacing the DsRed sequence . The GFP was used to identify infected neurons . For pHluorin imaging , sypHy ( Kwon and Chapman , 2011 ) was subcloned into this vector , again using BamH I and Not I to replace DsRed . GFP in the pLox vector was replaced by mCherry to avoid spectral overlap with pHluorin . For electrophysiological recordings , WT syt 7 or the 4D/N mutant were expressed in KO neurons at 5 days in vitro ( DIV ) using lentivirus as described previously ( Dong et al . , 2006 ) . For the pHluorin imaging experiments , neurons were grown on 12 mm coverslips in 24-well plates and transfected using the calcium phosphate method at 3–4 days in vitro ( DIV ) as described previously ( Dresbach et al . , 2003; Dean et al . , 2012a ) . Prior to transfection , medium was removed , saved , and replaced with 500 µl Optimem ( Life Technologies , Great Island , NY ) and incubated for 30–60 min . 105 µl of transfection buffer ( 274 mM NaCl , 10 mM KCl , 1 . 4 mM Na2HPO4 , 15 mM glucose , 42 mM HEPES , pH 7 . 06 ) was added drop-wise to a 105 µl solution containing 7 µg of DNA and 250 mM CaCl2 , with gentle vortexing . This mixture was incubated for 20 min at room temperature ( RT ) ; 30 µl was added per well and neurons were further incubated for 60–90 min . Medium was removed , cells were washed three times in Neurobasal medium , and the saved medium was added back to the transfected cells . Neurons were used for imaging between 12–17 DIV . For the immunostaining experiments , neuronal cultures were immunostained with a mouse monoclonal antibody directed against c-myc ( EMD Millipore , Bilerica , MA ) to determine the localization of exogenously expressed c-myc-tagged syt 1 , syt 7 and the syt 7 4D/N mutant . A polyclonal rabbit anti-synapsin antibody ( Synaptic System , Germany ) was used to localize synaptic vesicles in presynaptic terminals . 12–17 DIV neurons were fixed with 4% paraformaldehyde in PBS , permeabilized with 0 . 15% Triton X-100 for 30 min , blocked in 10% goat serum plus 0 . 1% Triton X-100 , stained with primary antibodies for 2 hr , washed with PBS three times , and then incubated with either Cy3-tagged anti-mouse or Alexa 647-tagged anti-rabbit ( Jackson ImmunoResearch Laboratories , West Grove , PA ) secondary antibodies for 1 hr at room temperature . Coverslips were then mounted in Fluoromount ( Southern Biotechnology , Birmingham , AL ) and images of were acquired on an Olympus FV1000 upright confocal microscope ( Japan ) with a 60× 1 . 10 numerical aperture water-immersion lens . To quantify the degree colocalization , images were imported into ImageJ ( NIH ) for analysis . The Pearson’s coefficient for colocalization was calculated using the intensity correlation analysis plugin for ImageJ . As described previously ( Liu et al . , 2009 ) , whole-cell voltage-clamp recordings of mEPSCs , evoked EPSCs , and hypertonic sucrose responses were carried out at RT using an EPC-10/2 amplifier ( HEKA , Germany ) from neurons at 12-17 DIV . The pipette solution consisted of , in mM: 130 K-gluconate , 1 EGTA , 5 Na-phosphocreatine , 2 Mg-ATP , 0 . 3 Na-GTP , and 10 HEPES , pH 7 . 3 ( 290 mOsm ) . The extracellular solution consisted of ( mM ) : 140 NaCl , 5 KCl , 1 MgCl2 , 10 glucose , 10 HEPES , pH 7 . 3 ( 300 mOsm ) , 0 . 05 D-2-amino-5-phosphonopentanoate ( D-AP5 ) , 0 . 1 picrotoxin . For mEPSC recordings , 2 mM Ca2+ was included , and 1 µM tetrodotoxin ( TTX ) was added to the extracellular solution . For evoked EPSCs , 5 mM QX-314 ( lidocaine N-ethyl bromide ) was added to the pipette solution . Various concentrations of [Ca2+]o ( in mM: 0 . 5 , 2 , 5 and 10 ) were used for recording single AP evoked EPSCs . Ten mM [Ca2+]o was used for the HFS recordings in order to maximize the first response . To evoke EPSCs , a bipolar electrode was placed against the soma , and used to trigger action potentials via a 1 ms minus 20 V pulse , as described previously ( Liu et al . , 2009 ) . Release was recorded as a postsynaptic response via a whole-cell patch electrode in a connected neuron . Neurons were voltage clamped at −70 mV . Only cells with series resistances of <15 M , with 70–80% of this resistance compensated , were analyzed . Currents were acquired using PATCHMASTER software ( HEKA , Germany ) , filtered at 2 . 9 kHz , and digitized at 10 kHz . Data were analyzed using MiniAnalysis software ( Synaptosoft , Decatur , GA ) , Clampfit ( Molecular Devices , Sunnyvale , CA ) , and Igor ( Wavemetrics , Portland , OR ) . For hypertonic sucrose experiments , we puffed sucrose over the entire area viewed under a 40× objective lens , which includes virtually all presynaptic boutons contacting the patched cells . 500 mM sucrose was applied to neurons , using an air pressure system ( PicoSpritzer III , Parker Hannifin , Cleveland , OH ) , for 10 s to empty the RRP of vesicles . After a quick wash ( Masterflex C/L , Cole–Parmer , Vernon Hills , IL ) , a second puff of sucrose was applied for another 10 s at various interpluse intervals . The recovery ratio was calculated by dividing the total charge elicited by the second puff of sucrose by the total charge elicited by the first puff . For the sucrose experiments in the presence of physiological [Ca2+]o , the extracellular solution included 2 mM Ca2+ . For the Ca2+-free condition , [Ca2+]o was replaced with 5 mM EGTA , and 25 µM BAPTA-AM ( 30 mM stock in DMSO ) and 30 µM CPA ( 100 mM stock in DMSO ) were added to the bath solution; cultures were incubated for 30 min before recording to allow BAPTA-AM and CPA to exert their effects . For the recordings using bafilomycin , 1 µM bafilomycin ( 1 . 6 mM stock in DMSO ) was added to the bath solution , and incubated for 3 min prior to recording . Neurons that were treated with bafilomycin were used within 20 min . For the CDZ experiments , 20 µM CDZ ( 100 mM stock in DMSO ) was added to the bath solution and neurons were incubated for 30 min prior to recording . In the CTZ and KYN experiments , both 50 µM CTZ ( 100 mM stock in DMSO ) and 100 µM KYN ( 75 mM stock in DMSO ) were added to the bath . All chemicals were purchased from Sigma–Aldrich . PHluorin imaging was performed as described previously ( Kwon and Chapman , 2011; Dean et al . , 2012a ) . Neurons were transferred to a live cell imaging chamber ( Warner Instruments , Hamden , CT ) and continuously perfused with bath solution ( 140 mM NaCl , 5 mM KCl , 2 mM CaCl2 , 2 mM MgCl2 , 10 mM HEPES , 10 mM glucose , 0 . 1 mM picrotoxin , 50 μM D-AP5 , 10 μM CNQX; 310 mOsm , pH 7 . 4 ) at RT . The transfected cells were identified via mCherry fluorescence . For field-stimulation , 1 ms voltage pulses ( 70 V ) were delivered using an SD9 stimulator ( Natus Neurology , Warwick , RI ) connected to two parallel platinum wires spaced by 10 mm in the imaging chamber ( Warner Instruments , Hamden , CT ) . Time-lapse images were acquired at 1 s intervals , with 484ex/517em , on an inverted microscope ( Nikon TE300 , Japan ) with a 1 . 42 NA 100× immersion oil objective under illumination with a Xenon light source ( Lambda DG4 , Sutter Instrument , Novato , CA ) . Fluorescence changes at individual boutons were detected using a Cascade 512II EMCCD camera ( Roper Scientific , Trenton , NJ ) ; data were collected and analyzed offline using MetaMorph 6 . 0 software . A baseline of 10 images was collected before applying high-frequency field stimulation . A typical exposure time was 400 ms for pHluorin . Puncta that did not exhibit any lateral movement during image acquisition were chosen for analysis . For pHluorin-syt 7 and pHluorin-syt 1 quenching/de-quenching experiments , fast solution exchanges were achieved using an MBS-2 perfusion system ( MPS-2 , World Precision Instruments , Sarasota , FL ) . The acidic solution , with a pH of 5 . 5 , was prepared by replacing HEPES , in the bath saline used for perfusion , with 2-[N-morpholinoethane sulphonic acid ( pKa = 6 . 1 ) . An ammonium chloride solution ( pH 7 . 4 ) was prepared by substituting 50 mM NaCl in bath saline with NH4Cl , while all other components remained unchanged . Recombinant cytoplasmic domains of syt isforms were generated as either GST fusion proteins , or his6-tagged proteins . For GST fusion proteins , cDNA encoding the tandem C2 domains of syt 1 ( residues 96-421 ) , WT ( residues 134–403 ) or the 4D/N mutant form of syt 7 , and cDNA encoding the isolated C2A ( residues 134–263 ) or C2B ( residues 243–403 ) domain of WT ( syt 7 C2A; syt 7 C2B ) or the Ca2+-ligand mutant forms of each C2-domain ( syt 7 C2A 2D/N , D225 , 227N; syt 7 C2B 2D/N , D357 , 359N ) of syt 7 , were expressed as glutathione S-transferase ( GST ) fusion proteins and purified using glutathione-Sepharose beads ( GE healthcare ) , as described previously ( Bhalla et al . , 2008 ) . The GST tag was removed via thrombin cleavage . For his6-tagged proteins , cDNA encoding the tandem C2 domains of syt 1 ( residues 96-421 ) , syt 2 ( residues 139–423 ) , syt 4 ( residues 152–425 ) , syt 7 ( residues 134-403 ) , syt 9 ( residues 104–386 ) , and syt 10 ( residues 223–501 ) were subcloned into either pTrc-HisA ( Invitrogen ) or pET28a ( Novagen ) generating an N-terminal His6-tag . His6-tagged proteins were purified using Ni Sepharose ( GE healthcare ) , as described previously ( Bhalla et al . , 2008 ) Aliquots of CaM agarose ( EMD Millipore , Bilerica , MA ) , containing 20 μg of CaM , were mixed with 2 μM of the indicated syt fragment in 150 μl of Dulbecco’s Phosphate Buffered Saline ( DPBS ) plus 0 . 5% Triton X-100 in the presence of 2 mM EGTA or 1 mM free Ca2+ or 1 mM free Mg2+ . The mixture was incubated with rotation at 4°C for 1 hr , after which the CaM beads were collected via centrifugation at 2000×g for 50 s , and washed three times with DPBS . Beads were boiled in sample buffer for 5 min , and then subjected to SDS-PAGE . Proteins were visualized by staining with Coomassie blue . In Figure 8—figure supplement 2 , samples were incubated for 2 hr at 37°C . CD spectra were obtained using a Model 420 Circular Dichroism Spectrophotometer ( Aviv Biomedical ) at 20°C using a 1 mm path length cuvette in 50 mM Na2HPO4 ( pH 7 . 4 ) ; the protein concentration was 0 . 4 mg/ml . ITC was carried-out using a MicroCal iTC200 calorimeter ( GE Healthcare , Madison , WI ) . WT and the Ca2+-ligand ( 2D/N ) mutant form of each isolated C2-domain of syt 7 were dialyzed overnight against ITC buffer ( 50 mM HEPES , 200 mM NaCl , 10% glycerol , pH 7 . 4 ) in the presence of Chelex-100 resin . Heat of binding was measured from 11 serial injections of Ca2+ ( 3 . 5 µl of 7 mM Ca2+ for C2A and 15 mM Ca2+ for C2B; higher [Ca2+] was needed to saturate C2B ) into a sample cell containing 1 . 5 mg/ml ( 100 μM ) protein at 25°C . Data were corrected for heat of dilution . Electrophysiology and imagining data were obtained from three independent litters of mice , unless otherwise indicated . Data were pooled for statistical analysis , as described in previous studies ( Xu et al . , 2007; Liu et al . , 2009; Kwon and Chapman , 2011 ) . All data are presented as the mean ± SEM and significance was determined using the Student’s t test , one-way ANOVA , or two-way ANOVA ( *p<0 . 05 , **p<0 . 01 , and ***p<0 . 001 ) , as appropriate ( Prism 6 , Graphpad Software ) .
Synaptotagmin ( syt ) 7 is one of three syt isoforms found in all metazoans; it is ubiquitously expressed , yet its function in neurons remains obscure . Here , we resolved Ca2+-dependent and Ca2+-independent synaptic vesicle ( SV ) replenishment pathways , and found that syt 7 plays a selective and critical role in the Ca2+-dependent pathway . Mutations that disrupt Ca2+-binding to syt 7 abolish this function , suggesting that syt 7 functions as a Ca2+-sensor for replenishment . The Ca2+-binding protein calmodulin ( CaM ) has also been implicated in SV replenishment , and we found that loss of syt 7 was phenocopied by a CaM antagonist . Moreover , we discovered that syt 7 binds to CaM in a highly specific and Ca2+-dependent manner; this interaction requires intact Ca2+-binding sites within syt 7 . Together , these data indicate that a complex of two conserved Ca2+-binding proteins , syt 7 and CaM , serve as a key regulator of SV replenishment in presynaptic nerve terminals .
Neurons communicate with one another at junctions called synapses . The arrival of an electrical signal called an action potential at the first neuron triggers the release of chemicals called neurotransmitters into the synapse . These chemicals then diffuse across the gap between the neurons and bind to receptors on the second cell . The neurotransmitter molecules are stored in the first cell in packages known as vesicles , which release their contents by fusing with the cell membrane . Following a fusion event , neurons must replenish their vesicle stocks to ensure that they are ready for the arrival of the next action potential . This replenishment process is known to involve a calcium-dependent pathway and a calcium-independent pathway . A protein called calmodulin , that binds calcium ions , has an important role in the first of these pathways . Now , Liu et al . have shown that another protein , synaptotagmin 7 , also has a key role in the replenishment of synaptic vesicles , possibly as a sensor for calcium ions . Moreover , Liu et al . found that synaptotagmin 7 and calmodulin bind to each other to form a complex , which suggests that the calcium-dependent replenishment pathway is regulated by this complex . The synaptotagmins are a family of 17 proteins , three of which are present in all animals . Two of these were known to have roles in synapses , but the role of the third—synaptotagmin 7—had been unclear . In addition to providing a more complete understanding of the replenishment of synaptic vesicles , the work of Liu et al . also supplies the final piece of the jigsaw regarding the role of the synaptotagmins that are present in all animals .