Neurotransmitter release is a multistep process that is coordinated by a large number of synaptic proteins and depends on proper protein-protein interactions. Using morphological, capacitance, and amperometric measurements, we investigated the effect of tomosyn, a Syntaxin-binding protein, on the different kinetic components of exocytosis in adrenal chromaffin cells. Overexpression of tomosyn decreased the release probability and led to a 50% reduction in the number of fusion-competent vesicles. The number of docked vesicles and the fusion kinetics of single vesicles were not altered suggesting that tomosyn inhibits the priming step. Interestingly, this inhibition is partially relieved at elevated calcium concentration. Calcium ramp experiments supported the latter finding and indicated that the reduction in secretion is caused by a shift in the calcium-dependence of release. These results indicate that secretion is not entirely blocked but occurs at higher calcium concentrations. We suggest that tomosyn inhibits the priming step and impairs the efficiency of vesicle pool refilling in a calciumdependent manner. C ommunication between neurons occurs by the release of neurotransmitter into the synaptic cleft and the subsequent activation of postsynaptic receptors. Neurotransmitter release is mediated by the fusion of synaptic vesicles and is restricted to presynaptic active zones (1). The vesicles in the synapse undergo a multistep cycle that includes vesicles docking at the active zone, priming (the formation of fusion-competent vesicles), fusion, and recycling (2-4). To achieve rapid and efficient synaptic transmission, synapses contain specific protein machinery that mediates these processes. The modulation of these steps is believed to account for several forms of short-term synaptic plasticity (5, 6).Although numerous proteins have been implicated in the synaptic vesicle cycle, the molecular mechanisms underlying defined steps are only beginning to emerge (7-10). Among the well characterized proteins associated with the synaptic vesicle cycle are Syntaxin, Synaptobrevin (also known as VAMP), and synaptosome-associated protein of 25 kDa (SNAP-25). These proteins form the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which plays an essential role in priming and͞or in the fusion reaction itself (11)(12)(13)(14). Changes in the quantity or availability of SNARE complexes directly affect the number of fusion-competent vesicles and neurotransmitter release (15)(16)(17)(18)(19).Under resting conditions, the major restriction for SNARE complex formation is the availability of its different components. For example, high affinity binding of Munc-18 to Syntaxin can prevent assembly of the core complex (10,20). Recent studies provide accumulating evidence regarding the involvement of a previously uncharacterized protein family in regulation of the SNARE complex (21-25). Tomosyn, a brain-specific member of this family, was identified as a binding partner for Syntaxin. Tomosyn can d...
Neurotransmitter release involves two consecutive Ca(2+)-dependent steps, an initial Ca(2+) binding to the selectivity filter of voltage-gated Ca(2+) channels (VGCC) followed by Ca(2+) binding to synaptic vesicle protein. The unique Ca(2+)-binding site of the VGCC is located within the alpha(1) subunit of the Ca(2+) channel. The structure of the selectivity filter allows for the binding of Ca(2+), Sr(2+), Ba(2+), and La(3+). Despite its cell impermeability, La(3+) supports secretion, which is in contradistinction to the commonly accepted mechanism in which elevation of cytosolic ion concentrations ([Ca(2+)](i)) and binding to synaptotagmin(s) trigger release. Here we show that a Cav1.2-mutated alpha(1)1.2/L775P subunit which does not conduct Ca(2+) currents supports depolarization-evoked release by means of Ca(2+) binding to the pore. Bovine chromaffin cells, which secrete catecholamine almost exclusively via nifedipine-sensitive Cav1.2, were infected with the Semliki Forest Virus, pSFV alpha(1)1.2/L775P. This construct also harbored a second mutation that rendered the channel insensitive to nifedipine. Depolarization of cells infected with alpha(1)1.2/L775P triggered release in the presence of nifedipine. Thus, the initial Ca(2+) binding at the pore of the channel appeared to be sufficient to trigger secretion, indicating that the VGCC could be the primary Ca(2+) sensor protein. The 25% lower efficiency, however, implied that additional ancillary effects of elevated [Ca(2+)](i) were essential for optimizing the overall release process. Our findings suggest that the rearrangement of Ca(2+) ions within the pore of the channel during membrane depolarization triggers secretion prior to Ca(2+) entry. This allows for a tight temporal coupling between the depolarization event and exocytosis of vesicles tethered to the channel.
Large numbers of neurons are eliminated by apoptosis during nervous system development. For instance, in the mouse dorsal root ganglion (DRG), the highest incidence of cell death occurs between embryonic days 12 and 14 (E12-E14). While the cause of cell death and its biological significance in the nervous system is not entirely understood, it is generally believed that limiting quantities of neurotrophins are responsible for neuronal death. Between E12 and E14, developing DRG neurons pass through tissues expressing high levels of axonal guidance molecules such as Semaphorin 3A (Sema3A) while navigating to their targets. Here, we demonstrate that Sema3A acts as a death-inducing molecule in neurotrophin-3 (NT-3)-, brain-derived neurotrophic factor (BDNF)-and nerve growth factor (NGF)-dependent E12 and E13 cultured DRG neurons. We show that Sema3A most probably induces cell death through activation of the c-Jun Nterminal kinase (JNK)/c-Jun signaling pathway, and that this cell death is blocked by a moderate increase in NGF concentration. Interestingly, increasing concentrations of other neurotrophic factors, such as NT-3 or BDNF, do not elicit similar effects. Our data suggest that the number of DRG neurons is determined by a fine balance between neurotrophins and Semaphorin 3A, and not only by neurotrophin levels.
The role of the L-type calcium channel (Cav1.2) as a molecular switch that triggers secretion prior to Ca 2؉ transport has previously been demonstrated in bovine chromaffin cells and rat pancreatic beta cells. Here, we examined the effect of specific Cav1.2 allosteric modulators, BayK 8644 (BayK) and FPL64176 (FPL), on the kinetics of catecholamine release, as monitored by amperometry in single bovine chromaffin cells. We show that 2 M BayK or 0.5 M FPL accelerates the rate of catecholamine secretion to a similar extent in the presence either of the permeable Ca 2؉ and Ba 2؉ or the impermeable charge carrier La 3؉ . These results suggest that structural rearrangements generated through the binding of BayK or FPL, by altering the channel activity, could affect depolarization-evoked secretion prior to cation transport. FPL also accelerated the rate of secretion mediated by a Ca 2؉ -impermeable channel made by replacing the wild type ␣ 1 1.2 subunit was replaced with the mutant ␣ 1 1.2/ L775P. Furthermore, BayK and FPL modified the kinetic parameters of the fusion pore formation, which represent the initial contact between the vesicle lumen and the extracellular medium. A direct link between the channel activity and evoked secretion lends additional support to the view that the voltagegated Ca 2؉ channels act as a signaling molecular switch, triggering secretion upstream to ion transport into the cell.The kinetic properties of voltage-gated Ca 2ϩ channels (VGCC) 2 are determined by the conformational changes induced at the channel during membrane depolarization. The kinetics of the L-type VGCC Cav1 is modulated also by allosteric agonists, which include BayK 8644 (BayK), a 1,4-dihydropyridine, FPL64176 (FPL), and CGP 48506 (1-9). BayK, FPL, and CGP 48506, which are structurally unrelated, interact with the cardiac Cav1.2 channel through binding within discrete sites at the transmembrane and extracellular loops of the ␣ 1 1.2 pore-forming subunit (10 -14). BayK enhances macroscopic currents (15) by increasing the rate of transition to "mode 2" single channel behavior (16,17) and the single channel currents by means of lengthening the channel open time (17)(18)(19)(20). BayK binding at selective Cav1.2 regions alters indirectly the selectivity filter, in turn affecting ion permeability (21). The changes observed in channel deactivation and inactivation are sensitive to the type of the cation used as the charge carrier (17).Like BayK 8644 (1), FPL is coupled to the cation binding at the selectivity filter of Cav1.1 acting as an allosteric regulator (22). In neonatal rat ventricular myocytes and rat ventricular cells, FPL enhances Ca 2ϩ influx and slows both the activation and the inactivation kinetics of the Cav1.2 (23). In isolated rat ventricular myocytes, FPL slowed the transition of the channel to a closed or inactivated state (24). In GH3 cells, FPL increased Cav1.2 current amplitude and shifted the current-voltage relationship to negative voltages (25). Single channel analysis showed that FPL increased both the...
The activation of the ryanodine Ca(2+) release channels (RyR2) by the entry of Ca(2+) through the L-type Ca(2+) channels (Cav1.2) is believed to be the primary mechanism of excitation-contraction (EC) coupling in cardiac cells. This proposed mechanism of Ca(2+)-induced Ca(2+) release (CICR) cannot fully account for the lack of a termination signal for this positive feedback process. Using Cav1.2 channel mutants, we demonstrate that the Ca(2+)-impermeable α(1)1.2/L775P/T1066Y mutant introduced through lentiviral infection into neonate cardiomyocytes triggers Ca(2+) transients in a manner independent of Ca(2+) influx. In contrast, the α(1)1.2/L775P/T1066Y/4A mutant, in which the Ca(2+)-binding site of the channel was destroyed, supports neither the spontaneous nor the electrically evoked contractions. Ca(2+) bound at the channel selectivity filter appears to initiate a signal that is conveyed directly from the channel pore to RyR2, triggering contraction of cardiomyocytes prior to Ca(2+) influx. Thus, RyR2 is activated in response to a conformational change in the L-type channel during membrane depolarization and not through interaction with Ca(2+) ions diffusing in the junctional gap space. Accordingly, termination of the RyR2 activity is achieved when the signal stops upon the return of the L-channel to the resting state. We propose a new model in which the physical link between Cav1.2 and RyR2 allows propagation of a conformational change induced at the open pore of the channel to directly activate RyR2. These results highlight Cav1.2 as a signaling protein and provide a mechanism for terminating the release of Ca(2+) from RyR2 through protein-protein interactions. In this model, the L-type channel is a master regulator of both initiation and termination of EC coupling in neonate cardiomyocytes.
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