The cytoplasmic domain of rat synaptotagmin I enhances synaptic transmission

Abstract

Synaptotagmin, an integral membrane protein of synaptic vesicles, functions as a calcium sensor in the temporal control of neurotransmitter release. Although synaptotagmin facilitates lipid membrane fusion in biochemical experiments, overexpression of synaptotagmin inhibits neurotransmission. A facilitatory effect of synaptotagmin on synaptic transmission was never observed. To determine whether synaptotagmin may accelerate synaptic transmission in vivo, we injected the cytoplasmic domain of rat synaptotagmin I (CD-syt) into crayfish motor axons and tested the effect of CD-syt on synaptic response. We confirmed that CD-syt accelerates neuromuscular transmission. The injected preparation had larger synaptic potentials with shorter rise time. Experiments with varying calcium concentrations showed that CD-syt increased the maximum synaptic response of the neuromuscular synapses. Further tests on short-term plasticity of neuromuscular synapses revealed that CD-syt increases the release probability of the release-ready vesicles.

Fig. 1

Recombinant rat synaptotagmin I cytoplasmic domain (CD-syt) recognized by a synaptotagmin antibody. a Illustration of synaptotagmin I on synaptic vesicle membrane. CD-syt (96-421) covers most of the cytoplasmic portion of synaptotagmin. b Western blot of recombinant CD-syt (7.2 μg), samples of crayfish ganglia (labelled as “Crayfish”), and rat cerebrum lysate (labelled as “Rat”) with an antibody of rat synaptotagmin I. The continuous bands of large molecular weight in the rat brain sample may be synaptotagmin dimmers or synaptotagmin in protein complex, which has been reported in other studies (Perin et al. 1991). c SDS-PAGE of CD-syt after gel filtration

Fig. 2

CD-syt accelerates crayfish synaptic transmission. a Effect of CD-syt on the EPSP amplitude of crayfish muscle cells. Representative EPSPs before and 2 h after CD-syt injection are overlapped for comparison. b EPSPs before and 2 h after injection of Troponin. Downward stimulus artifact transients were blanked out. c CD-syt increased EPSP amplitude. Data were from 11 experiments. The increase in standard error with time reflects the large variation in the latency of CD-syt effect in different experiments. d Mean EPSP amplitude in 8 experiments of Troponin injection. In (c) and (d), the EPSP amplitude was normalized to that at time zero, when the 2 h of injection started. Data are presented as mean ± standard error

Fig. 3

CD-syt reduces the rise time of crayfish neuromuscular synaptic potentials. a, b Effects of CD-syt and Troponin on EPSP rise time, respectively. The same traces in Fig. 2a, b are displayed at higher temporal resolution, with the EPSP amplitude adjusted, to highlight the EPSP rise time. Arrows point to the peak of EPSPs. The two EPSPs in (b) overlap. Downward stimulus artifact transients were blanked out in (b). c EPSP rise time reduced after CD-syt injection. The mean EPSP rise time from the same experiments in Fig. 2c was plotted. d EPSP rise time was constant in Troponin injections. Data were from the same experiments in Fig. 2d. In (c) and (d), the EPSP rise time was normalized to that at time zero, when the 2 h of injection started. Data are presented as mean ± standard error

Fig. 4

EPSP rise time reduced at low extracellular calcium concentrations. The rise time of EPSPs is presented as a fraction of that in normal crayfish saline (13.5 mM Ca²⁺). Each data point is an average of 5 experiments, presented as mean ± standard deviation

Fig. 5

CD-syt increased the maximum synaptic response. All data were taken after injections of CD-syt or Troponin as labelled. Mean maximum derivative of EPSPs in 10 different [Ca²⁺] was plotted. The maximum derivative of EPSPs was normalized to that of the same muscle fiber in standard crayfish saline (Ca²⁺: 13.5 mM) and then normalized to that of Troponin injection in 13.5 mM Ca²⁺. Each dot is the average of 6–9 experiments. Error bars are standard errors

Fig. 6

Effect of CD-syt on short-term plasticity of synaptic transmission. a Normalized amplitude of eight EPSPs at 30 Hz in experiments without injection. b Normalized rise time of EPSPs in (a). c Normalized amplitude of eight EPSPs after CD-syt injection. d Normalized rise time of EPSPs in (c). Each data represents mean of six experiments. All data were normalized to that of the first EPSP. Error bars are standard deviations