Voltage-driven Ca(2+) binding at the L-type Ca(2+) channel triggers cardiac excitation-contraction coupling prior to Ca(2+) influx.

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.

Conformational changes induced in voltage-gated calcium channel Cav1.2 by BayK 8644 or FPL64176 modify the kinetics of secretion independently of Ca2+ influx.

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 microm BayK or 0.5 microm 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 alpha(1)1.2 subunit was replaced with the mutant alpha(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 voltage-gated Ca(2+) channels act as a signaling molecular switch, triggering secretion upstream to ion transport into the cell.

The voltage-gated Ca(2+) channel is the Ca(2+) sensor protein of secretion.

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.

Semaphorin 3A and neurotrophins: a balance between apoptosis and survival signaling in embryonic DRG neurons.

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 N-terminal 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.

Tomosyn inhibits priming of large dense-core vesicles in a calcium-dependent manner.

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 calcium-dependent manner.