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, 147 (3), 666-77

Doc2 Is a Ca2+ Sensor Required for Asynchronous Neurotransmitter Release

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Doc2 Is a Ca2+ Sensor Required for Asynchronous Neurotransmitter Release

Jun Yao et al. Cell.

Abstract

Synaptic transmission involves a fast synchronous phase and a slower asynchronous phase of neurotransmitter release that are regulated by distinct Ca(2+) sensors. Though the Ca(2+) sensor for rapid exocytosis, synaptotagmin I, has been studied in depth, the sensor for asynchronous release remains unknown. In a screen for neuronal Ca(2+) sensors that respond to changes in [Ca(2+)] with markedly slower kinetics than synaptotagmin I, we observed that Doc2--another Ca(2+), SNARE, and lipid-binding protein--operates on timescales consistent with asynchronous release. Moreover, up- and downregulation of Doc2 expression levels in hippocampal neurons increased or decreased, respectively, the slow phase of synaptic transmission. Synchronous release, when triggered by single action potentials, was unaffected by manipulation of Doc2 but was enhanced during repetitive stimulation in Doc2 knockdown neurons, potentially due to greater vesicle availability. In summary, we propose that Doc2 is a Ca(2+) sensor that is kinetically tuned to regulate asynchronous neurotransmitter release.

Figures

Figure 1
Figure 1
PS is a critical Doc2α and β effector during regulated membrane fusion. (A) In vitro membrane fusion assays were performed in the presence of 3 μM syt, Doc2α, or Doc2β. All three proteins accelerated fusion upon addition of 1 mM Ca2+ (arrow). (B) A Ca2+ titration for each protein was performed using 15% PS vesicles. Data were fitted with sigmoidal dose-response curves to determine the [Ca2+]1/2 values for fusion (inset). (C) Syt or Doc2 titrations were carried out using vesicles with 0, 15 and 25% PS, and data were fit as in panel (B) to calculate EC50 values; (0% PS: not applicable; 15% PS: syt, 0.5 μM; Doc2α, 2 μM; Doc2β, 0.9 μM; 25%: syt, 0.6 μM; Doc2α, 0.3 μM; Doc2β, 0.2 μM). Each point represents the mean ± SEM from three independent experiments.
Figure 2
Figure 2
Doc2α and Doc2β can assemble syntaxin and SNAP-25 into functional SNARE complexes. (A) The ‘traditional’ fusion assay: vesicles harboring pre-assembled syntaxin•SNAP-25 heterodimers are incubated with syb-harboring vesicles and either syt, Doc2α or Doc2β. (B) Fusion assays were carried out using ‘split t-SNAREs’ in which syntaxin-alone was reconstituted into the t-SNARE vesicles and free SNAP-25 was subsequently added in a soluble form; under these conditions fusion is not observed unless Ca2+ and syt - and, as tested here, Doc2 - are added to drive folding of SNAP-25 onto syntaxin, resulting in fusion activity (Bhalla et al., 2006). (C) Fusion assays using syt (left panel), Doc2α (center panel), or Doc2β (right panel) were carried out with preassembled t-SNARE vesicles or syntaxin vesicles plus free SNAP-25. In the presence of Ca2+, all three proteins were able to drive assembly of active t-SNARE heterodimers capable of fusing with syb vesicles.
Figure 3
Figure 3
Doc2 is tuned to respond to changes in [Ca2+] with markedly slower kinetics than syt. Stopped-flow rapid mixing experiments were performed to analyze the kinetics of syt, Doc2α, and Doc2β interactions with vesicles that harbor PS. (A) Association kinetics were monitored via FRET between the endogenous aromatic residues of each protein and a dansyl-PE acceptor in the target vesicles. (B) Representative association traces for each protein showing the first 500 msec (full traces for Doc2α and Doc2β are shown in Figure S2A). (C) Each trace was fit with a double exponential function and the rate of the fast component was plotted as a function of the liposome concentration. Each point represents the mean ± SEM of at least 3 independent experiments. (D) The on-rates, off-rates, and dissociation constants were determined from the plots shown in panel C. (E) Disassembly of Ca2+ • Doc2 or syt from liposomes upon rapid mixing with excess EGTA to chelate all Ca2+. (F) The first 200 msec of a representative syt disassembly trace and (G) the first second of representative Doc2α and Doc2β traces are shown. The rate of disassembly was determined for syt by fitting the data with a single exponential function; Doc2 data were fit with a double exponential. (H) The data from at least 5 separate experiments was analyzed and the mean ± SEM for each kinetic component and the τ values were determined.
Figure 4
Figure 4
Doc2α KD reduces asynchronous SV release in syt I knock-out hippocampal neurons. (A and B) Immunoblots of Doc2α (A) or Doc2β (B) in cultured hippocampal neurons. Since Doc2β was not detected (B, left panel), the efficiency of Doc2β shRNA was examined in HEK-293T cells transfected with a Doc2β-GFP construct (B, right panel; vertical line indicates lanes that were removed). Doc2α and Doc2β were reduced ≥ 79% by shRNA KD. (C) Representative traces of evoked EPSCs recorded from syt I KO neurons (control) and lentivirus-infected KO neurons expressing Doc2α shRNA, Doc2β shRNA, or Doc2α shRNA plus Doc2β. (D) Bar graph showing that the total charge transfer over 0.5 s is significantly reduced in the neurons expressing Doc2α shRNA (control, n = 34; shRNA, n = 34), but not Doc2β shRNA (n = 24), or Doc2α shRNA plus Doc2β (n = 23). (E) Averaged total charge of 0.5 M sucrose-induced responses showing little difference between syt I KO neurons (n = 19) and KO neurons expressing Doc2α shRNA (n = 17). (F) Representative EPSC traces from syt I KO neurons, or KO neurons over-expressing wt Doc2α or Doc2α-CLM. (G) Bar graph showing the total charge transfer during EPSCs recorded from syt I KO neurons (n = 33), and KO neurons over-expressing wt Doc2α (n = 16) or Doc2α-CLM (n = 40). ** p<0.001. Error bars represent SEM.
Figure 5
Figure 5
Doc2α KD specifically reduces the asynchronous phase of SV release in wt hippocampal neurons. (A) Average traces of evoked EPSCs recorded from wt neurons and neurons pre-incubated with 25 μM AM-EGTA, as well as neurons expressing Doc2α shRNA alone or accompanied by expression of Doc2β. (B) Bar graphs summarizing the amplitude of EPSCs recorded from wt neurons (n = 29), neurons treated with AM-EGTA (n = 67), and neurons expressing Doc2α shRNA alone (n = 42) or Doc2α shRNA plus Doc2β (n = 25). (C) Average normalized cumulative EPSC charge transfer over 0.5 s demonstrating that neurons treated with AM-EGTA, or expressing Doc2α shRNA alone, significantly accelerate the decay kinetics of EPSCs, as compared to wt neurons and Doc2α KD neurons expressing Doc2β. (D and E) Bar graphs summarizing the charge (D) and the time constant (E) of the slow phase of transmission. (F and G) Bar graphs summarizing the charge (F) and the time constant (G) of the fast phase of transmission. (H) Average traces of evoked EPSCs recorded from wt neurons (n = 18) and Doc2α KO hippocampal neurons (n = 23). (I) Average normalized cumulative EPSC charge transfer over 0.5 s from wt neurons and Doc2α KO neurons. (J and K) Bar graphs comparing the charge (J) and the time constant (K) of the slow phase of transmission between wt neurons (n = 18) and Doc2α KO neurons (n = 23). (L and M) Bar graphs summarizing the charge (L) and the time constant (M) of the fast phase of transmission. * p<0.05, ** p<0.001. Error bars represent SEM.
Figure 6
Figure 6
Doc2α shRNA induces inverse changes in the synchronous and asynchronous components of transmission during high frequency train stimulation. (A) Representative EPSC traces evoked by 40 action potentials at 20 Hz. (B) Bar graph summarizing the RRP size measured by extrapolating the cumulative charge (upper small panel) recorded from wt neurons (n = 33), neurons treated with AM-EGTA (n = 84), or neurons expressing Doc2α shRNA (n = 56). (C) Bar graph showing that neurons treated with AM-EGTA or expressing Doc2α shRNA exhibited significant reductions in the fraction of total tonic charge transfer during the stimulus train. Top panel, diagram showing how the phasic and tonic charge were determined (Otsu et al., 2004). (D) Average normalized cumulative tonic charge transfer over 400 ms demonstrating that AM-EGTA or Doc2α KD significantly decelerated the accumulation of tonic charge. (E-G) Histograms summarizing the build-up of tonic current in wt neurons (E), neurons expressing Doc2α shRNA alone (F), and neurons treated with AM-EGTA (G). The data were fitted with Gaussian curves (red). Doc2α KD induced a left-shift in the distribution, compared to wt neurons and Doc2β rescued neurons. AM-EGTA neurons exhibited a disperse distribution and were not fitted. (H) Typical traces of the first EPSCs during the train showing the extent of decay prior to the next stimulation. The amplitudes were normalized for comparison. (I) Bar graph showing that both Doc2α KD and AM-EGTA treatment induce faster EPSC decay rates. (J) Doc2α KD induced slower depression of EPSC amplitudes at the beginning of the train compared to control neurons; AM-EGTA treatment gave rise to the same decrease in depression as Doc2α KD during the late stage of the train. ** p<0.001. Error bars represent SEM.
Figure 7
Figure 7
Doc2α KD reduces evoked reverberatory activity in high density cultures of hippocampal neurons. (A) Sample image of a high density hippocampal culture used to generate reverberatory activity. Scale bar, 100 μm. (B) Representative traces of evoked reverberation recorded from wt neurons (control) and neurons expressing Doc2α shRNA. (C and D) Histograms summarizing the number of reverberatory EPSCs that occurred within 2 s (C) and the duration of the reverberatory activity (D) in wt control (n = 22; left panels) and Doc2α KD neurons (n = 27; right panels).

Comment in

  • Dueling Ca2+ Sensors in Neurotransmitter Release
    M Xue et al. Cell 147 (3), 491-3. PMID 22036557.
    Ca(2+)-triggered neurotransmitter release is characterized by two kinetically distinct components: a fast synchronous phase and a slow asynchronous phase. Yao et al. (201 …

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