Syntaxin 3B is essential for the exocytosis of synaptic vesicles in ribbon synapses of the retina.

Ribbon synapses of the vertebrate retina are specialized synapses that release neurotransmitter by synaptic vesicle exocytosis in a manner that is proportional to the level of depolarization of the cell. This release property is different from conventional neurons, in which the release of neurotransmitter occurs as a short-lived burst triggered by an action potential. Synaptic vesicle exocytosis is a calcium regulated process that is dependent on a set of interacting synaptic proteins that form the so-called SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex. Syntaxin 3B has been identified as a specialized SNARE molecule in ribbon synapses of the rodent retina. However, the best physiologically-characterized neuron that forms ribbon-style synapses is the rod-dominant or Mb1 bipolar cell of the goldfish retina. We report here the molecular characterization of syntaxin 3B from the goldfish retina. Using a combination of reverse transcription (RT) polymerase chain reaction (PCR) and immunostaining with a specific antibody, we show that syntaxin 3B is highly enriched in the plasma membrane of bipolar cell synaptic terminals of the goldfish retina. Using membrane capacitance measurements we demonstrate that a peptide derived from goldfish syntaxin 3B inhibits synaptic vesicle exocytosis. These experiments demonstrate that syntaxin 3B is an important factor for synaptic vesicle exocytosis in ribbon synapses of the vertebrate retina.

ATP is required at an early step in compensatory endocytosis in synaptic terminals.

Whole-terminal capacitance measurements were used to examine membrane retrieval that follows Ca(2+)-triggered exocytosis in single synaptic terminals. Exocytosis was followed by endocytosis only when the internal solution contained a hydrolyzable analog of ATP. ATP-gamma-S, a poorly hydrolyzable ATP analog, did not support endocytosis but instead produced a rapid and profound inhibition of membrane retrieval. Under similar conditions, the GTP analogs GTP-gamma-S and GDP-beta-S failed to block endocytosis, suggesting that ATP is the preferred substrate. Furthermore, the requirement for ATP was independent of the role of ATP in regulating intraterminal Ca(2+), and the role of Ca(2+) in endocytosis was different from that of ATP. The results suggest a direct, acute requirement for ATP hydrolysis in compensatory fast endocytosis in synaptic terminals. Given that the capacitance technique detects changes in membrane surface area, ATP must be required for the membrane fission step or at a step that is a prerequisite for membrane fission.

Comparison of secretory responses as measured by membrane capacitance and by amperometry.

We have compared capacitance and amperometric measurements in bovine chromaffin cells when secretion was elicited by flash photolysis of caged-calcium or step depolarizations. Total amperometric charge depended linearly on the amount of capacitance increase in both types of experiments. Furthermore, the properties of resolvable amperometric spikes after flashes were comparable to those observed after depolarizations, and their timing was compatible with the rate of capacitance increase. For a more detailed comparison, we used Monte Carlo simulations of multiple amperometric events occurring randomly over the surface of a sphere and summing together, to generate a reference amperometric signal for a given measured capacitance increase. Even after correction for endocytotic processes, the time courses of the integrated experimental records lagged behind the integrated Monte Carlo records by approximately 50 ms in flash and depolarization experiments. This delay was larger by approximately 40 ms than what can be expected from the "pre-foot delay" or the foot duration. Possible sources for the remaining delay could be diffusional barriers like the patch-pipette and the chamber bottom, which are not taken into account in the model. We also applied a novel type of fluctuation analysis to estimate the relative quantum size of an amperometric event. On average the estimates from experimental amperometric traces, in both flash and depolarization experiments, were 3-5 times smaller than estimates from simulated ones. This discrepancy can be due to contributions to the amperometric current from small vesicles, preferred release from cellular regions orientated toward the chamber bottom, or abundance of "foot-only" events. In conclusion, amperometric signals in flash and depolarization experiments displayed similar delayed average time courses and a lower estimate for the relative quantum size compared to the modeled amperometric signals. However, individual amperometric spikes were in agreement with expectations derived from capacitance signals.

Adenosine triphosphate and the late steps in calcium-dependent exocytosis at a ribbon synapse.

The ATP dependence of the kinetics of Ca2+-dependent exocytosis after flash photolysis of caged Ca2+ was studied by capacitance measurements with submillisecond resolution in single synaptic terminals of retinal bipolar neurons. After control experiments verified that this combination of techniques is valid for the study of exocytosis in synaptic terminals, a comparison was made between the Ca2+ dependence of the rate of exocytosis in synaptic terminals internally dialyzed with MgATP, MgATP-gamma-S, or no added Mg2+ or nucleotide. The Ca2+ threshold for release, the maximum rate of release, and the overall relationship between the rate of synaptic vesicle fusion and [Ca2+]i were found to be independent of MgATP. A decrease in the average rate at near-threshold [Ca2+]i was observed in terminals with MgATP-gamma-S, but due to the small sample size is of unclear significance. The Ca2+ dependence of the delay between the elevation of [Ca2+]i and the beginning of the capacitance rise was also found to be independent of MgATP. In contrast, MgATP had a marked effect on the ability of terminals to respond to multiple stimuli. Terminals with MgATP typically exhibited a capacitance increase to a second stimulus that was >70% of the amplitude of the first response and to a third stimulus with a response amplitude that was >50% of the first, whereas terminals without MgATP responded to a second stimulus with a response <35% of the first and rarely responded to a third flash. These results suggest a major role for MgATP in preparing synaptic vesicles for fusion, but indicate that cytosolic MgATP may have little role in events downstream of calcium entry, provided that [Ca2+]i near release sites is elevated above approximately 30 microM.

Calcium dependence of the rate of exocytosis in a synaptic terminal.

Rapid calcium-dependent exocytosis underlies neurotransmitter release from nerve terminals. Despite the fundamental importance of this process, neither the relationship between presynaptic intracellular calcium ion concentration ([Ca2+]i) and rate of exocytosis, nor the maximal rate of secretion is known quantitatively. To provide this information, we have used flash photolysis of caged Ca2+ to elevate [Ca2+]i rapidly and uniformly in synaptic terminals, while measuring membrane capacitance as an index of exocytosis and monitoring [Ca2+]i with a Ca(2+)-indicator dye. When [Ca2+]i was abruptly increased to > 10 microM, capacitance rose at a rate that increased steeply with [Ca2+]i. The steepness suggested that at least four calcium ions must bind to activate synaptic vesicle fusion. Half-saturation was at 194 microM, and the maximal rate constant was 2,000-3,000 s-1. A given synaptic vesicle can exocytose with high probability within a few hundred microseconds, if [Ca2+]i rises above 100 microM. These properties provide for the extremely rapid signalling required for neuronal communication.

Presynaptic inhibition by GABA is mediated via two distinct GABA receptors with novel pharmacology.

Mechanisms of presynaptic inhibition were examined in giant presynaptic terminals of retinal bipolar neurons, which receive GABAergic feedback synapses from amacrine cells. Two distinct inhibitory actions of GABA are present in the terminals: a GABAA-like Cl conductance and a GABAB-like inhibition of voltage-dependent Ca current. Both of the receptors underlying these actions have unusual pharmacology that fits neither GABAA nor GABAB classifications. The GABA-activated Cl conductance was not blocked by the classical GABAA antagonist bicuculline, while the inhibition of Ca current was neither mimicked by the GABAB agonist baclofen nor blocked by the GABAB antagonist 2-hydroxysaclofen. The "GABAC" agonist cis-4-aminocrotonic acid (CACA) both activated the Cl conductance and inhibited Ca current, but the inhibition of Ca current was observed at much lower concentrations of CACA (< 1 microM) than was the activation of the Cl conductance (K1/2 = 50 microM). Thus, by the criterion of being insensitive to both bicuculline and baclofen, both GABA receptors qualify as potential GABAC receptors. However, it is argued on functional grounds that the two GABA receptors coupled to Cl channels and to Ca channels are best regarded as members of the GABAA and GABAB families, respectively.

Dopamine enhances Ca2+ responses in synaptic terminals of retinal bipolar neurons.

The effect of dopamine on depolarization-induced Ca2+ influx was studied using the fluorescent Ca2+ indicator fura-2 in synaptic terminals of bipolar neurons from gold-fish retina. Dopamine reversibly enhanced the rise in intracellular Ca2+ elicited by elevated external potassium. The enhancement was slowly reversible. The effect of dopamine was mimicked by forskolin and CPT-cAMP, a membrane-permeant analog of cAMP. However, 1,9-dideoxyforskolin, a forskolin analog that does not activate adenylyl cyclase, was ineffective. This suggests that dopamine, via cAMP, regulates the rise in presynaptic Ca2+ concentration in response to depolarization, potentially enhancing transmitter release.

Calcium influx and calcium current in single synaptic terminals of goldfish retinal bipolar neurons.

1. The calcium influx pathway in large synaptic terminals of acutely isolated bipolar neurons from goldfish retina was characterized using Fura-2 measurements of intracellular calcium and patch-clamp recordings of whole-cell calcium current. 2. Depolarization of bipolar cells with high [K+]o resulted in a sustained, reversible increase in [Ca2+]i in both synaptic terminals and somata. Removal of external calcium abolished the response, as did the addition of 200 microM-cadmium to the bathing solution, indicating that the rise in [Ca2+]i was due to entry of external calcium. Dihydropyridine blockers of voltage-gated Ca2+ channels also blocked the influx, and the Ca2+ channel agonist Bay K 8644 potentiated influx, implicating voltage-activated, dihydropyridine-sensitive channels in the influx pathway. 3. Under voltage clamp, depolarization from a holding potential of -60 mV evoked a slowly inactivating inward current that began to activate at -50 to -40 mV and reached a maximal amplitude between -20 and -15 mV. This current was identified as a calcium current because it decreased when the extracellular calcium concentration was lowered, increased when barium was the charge carrier, and was blocked by 200 microM-external cadmium. The current was substantially blocked by 1 microM-nitrendipine and potentiated by 0.1 microM-Bay K 8644, as expected for L-type Ca2+ channels; it was unaffected by omega-conotoxin. No evidence for transient or rapidly inactivating Ca2+ current was found. 4. At a given level of potassium depolarization, both the amplitude and the speed of increase in [Ca2+]i were greater in synaptic terminals than in somata. For instance, depolarization by 32.6 mM-potassium caused an increase in intracellular calcium of 400 +/- 23 nM in terminals and 180 +/- 20 nM in somata (mean +/- S.E.M., n = 73 terminals, n = 30 somata), with maximal rates of change of 40 +/- 3 and 12 +/- 2 nM/s, respectively. 5. The contribution of terminal and somatic currents to the total whole-cell Ca2+ current was determined under voltage clamp by local application of calcium or of blocking agents. While there was no qualitative difference between currents in terminals and somata, synaptic terminals accounted for 64 +/- 3% (mean +/- S.E.M., n = 12) of the total whole-cell calcium current, and somata accounted for 39 +/- 2%. Thus, the density of Ca2+ current was higher in the terminal, accounting for the greater magnitude and speed of Ca2+ influx observed in terminals in Fura-2 experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of calcium influx and calcium current by gamma-aminobutyric acid in single synaptic terminals.

Inhibition of Ca influx and Ca current by gamma-aminobutyric acid (GABA) was studied in single synaptic terminals of isolated retinal bipolar neurons. Measurements of intracellular Ca concentration [( Ca]i) using the fluorescent Ca indicator fura-2 showed that GABA potently inhibited Ca influx into the terminal elicited by high extracellular K concentration ([K]o). This inhibition was attributed to GABA type A (GABAA) receptor-activated chloride ion conductance that prevented bipolar neurons from depolarizing sufficiently to activate the Ca current, even in response to increased [K]o. Patch-clamp recordings of the Ca current revealed a second effect of GABA: GTP-dependent inhibition of the Ca current. This inhibition was not mediated by GABAA receptors, but baclofen, which binds to the GABA type B (GABAB) receptor and is known to inhibit the Ca current in other systems, was not able to mimic the action of GABA. This suggests the involvement of a different type of GABAB-like receptor in the inhibition of Ca current by GABA. GABA did not cause an overall suppression of the Ca current; rather, the voltage-dependence of Ca-channel activation was shifted to more depolarized potentials. Thus, maximal inhibition of the Ca current by GABA occurred in the physiological range of potential.