Ion interaction at the pore of Lc-type Ca2+ channel is sufficient to mediate depolarization-induced exocytosis

The coupling of voltage-gated Ca2+ channel (VGCC) to exocytotic proteins suggests a regulatory function for the channel in depolarization-evoked exocytosis. To explore this possibility we have examined catecholamine secretion in PC12 and chromaffin cells. We found that replacing Ca2+ with La3+ or other lanthanide ions supported exocytosis in divalent ion-free solution. Cd2+, nifedipine, or verapamil inhibited depolarization-evoked secretion in La3+, indicating specific binding of La3+ at the pore of L-type VGCC, probably at the poly-glutamate (EEEE) locus. Lanthanide efficacy was stringently dependent on ionic radius with La3+>Ce3+>Pr3+, consistent with a size-selective binding interface of trivalent cations at the channel pore. La3+ inward currents were not detected and the highly sensitive La3+/fura-2 imaging assay (approximately 1 pm) detected no La3+ entry, cytosolic La3+ build-up, or alterations in cytosolic Ca2. These results provide strong evidence that occupancy of the pore of the channel by an impermeable cation leads to a conformational change that is transmitted to the exocytotic machinery upstream of intracellular cation build-up (intracellular Ca2+ concentration). Our model allows for a tight temporal and spatial coupling between the excitatory stimulation event and vesicle fusion. It challenges the conventional view that intracellular Ca2+ ion build-up via VGCC permeation is required to trigger secretion and establishes the VGCC as a plausible Ca2+ sensor protein in the process of neuroendocrine secretion.     

PKC theta activity maintains normal quantal size in chromaffin cells

Protein kinase C (PKC) activity mediates multiple neurosecretory processes, but these are poorly understood due in part to the existence of at least 12 PKC isoforms. Using amperometry to record quantal catecholamine release from chromaffin cells, we found that both broad spectrum PKC antagonists and rottlerin, a selective inhibitor of the novel isoforms PKC θ and PKC δ, decreased quantal size and the number of secretory events recorded per stimulus. In contrast, drugs that selectively inhibit the atypical and conventional PKC isoforms had no effect on these parameters. While both PKC θ and δ were expressed in chromaffin cells, mice deficient for PKC θ, but not for PKC δ, exhibited lower quantal size than wild-type and were insensitive to rottlerin. Finally, an inhibitory PKC θ pseudosubstrate produced rottlerin-like responses in wild-type mice, indicating that the lack of rottlerin response in the PKC θ mutants was not the result of a form of compensation. These findings demonstrate neurosecretory regulation by a novel PKC isoform, PKC θ, and should contribute to defining mechanisms of activity-dependent regulation of neurosecretion.     

Lysophospholipids regulate excitability and exocytosis in cultured bovine chromaffin cells

Bioactive lysophospholipids (LPLs) are released by blood cells and can modulate many cellular activities such as angiogenesis and cell survival. In this study, the effects of sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) on excitability and exocytosis in bovine chromaffin cells were investigated using the whole-cell configuration of the patch-clamp. Voltage-gated Ca(2+) current was inhibited by S1P and LPA pre-treatment in a concentration-dependent manner with IC(50)s of 0.46 and 0.79 mumol/L, respectively. Inhibition was mostly reversible upon washout and prevented by suramin, an inhibitor of G-protein signaling. Na(+) current was inhibited by S1P, but not by LPA. However, recovery of Na(+) channels from inactivation was slowed by both LPLs. The outward K(+) current was also significantly reduced by both LPLs. Chromaffin cells fired repetitive action potentials in response to minimal injections of depolarizing current. Repetitive activity was dramatically reduced by LPLs. Consistent with the reduction in Ca(2+) current, exocytosis elicited by a train of depolarizations and the ensuing endocytosis were both inhibited by LPL pre-treatments. These data demonstrate the interaction between immune and endocrine systems mediated by the inhibitory effects of LPLs on the excitability of adrenal chromaffin cells.     

Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

This study examines the Cav1 isoforms expressed in mouse chromaffin cells and compares their biophysical properties and roles played in cell excitability and exocytosis. Using immunocytochemical and electrophysiological techniques in mice lacking the Cav1.3α1 subunit (Cav1.3(-/-) ) or the high sensitivity of Cav1.2α1 subunits to dihydropyridines, Cav1.2 and Cav1.3 channels were identified as the only Cav1 channel subtypes expressed in mouse chromaffin cells. Cav1.3 channels were activated at more negative membrane potentials and inactivated more slowly than Cav1.2 channels. Cav1 channels, mainly Cav1.2, control cell excitability by functional coupling to BK channels, revealed by nifedipine blockade of BK channels in wild type (WT) and Cav1.3(-/-) cells (53% and 35%, respectively), and by the identical change in the shape of the spontaneous action potentials elicited by the dihydropyridine in both strains of mice. Cav1.2 channels also play a major role in spontaneous action potential firing, supported by the following evidence: (i) a similar percentage of WT and Cav1.3(-/-) cells fired spontaneous action potentials; (ii) firing frequency did not vary between WT and Cav1.3(-/-) cells; (iii) mostly Cav1.2 channels contributed to the inward current preceding the action potential threshold; and (iv) in the presence of tetrodotoxin, WT or Cav1.3(-/-) cells exhibited spontaneous oscillatory activity, which was fully abolished by nifedipine perfusion. Finally, Cav1.2 and Cav1.3 channels were essential for controlling the exocytotic process at potentials above and below -10 mV, respectively. Our data reveal the key yet differential roles of Cav1.2 and Cav1.3 channels in mediating action potential firing and exocytotic events in the neuroendocrine chromaffin cell.     

Real-time imaging of plasma membrane deformations reveals pre-fusion membrane curvature changes and a role for dynamin in the regulation of fusion pore expansion

Assays for real-time investigation of exocytosis typically measure what is released from the granule. From this, inferences are made about the dynamics of membrane remodeling as fusion progresses from start to finish. We have recently undertaken a different approach to investigate the fusion process, by focusing not primarily on the granule, but rather its partner in exocytosis - the plasma membrane. We have been guided by the idea that biochemical interactions between the granule and plasma membranes before and during fusion, cause changes in membrane conformation. To enable study of membrane conformation, a novel imaging technique was developed combining polarized excitation of an oriented membrane probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI) with total internal reflection fluorescence microscopy (pTIRFM). Because this technique measures changes in membrane conformation (or deformations) directly, its usefulness persists even after granule cargo reporter (catecholamine, or protein), is no longer present. In this mini-review, we first summarize the workings of pTIRFM. We then discuss the application of the technique to investigate deformations in the membrane preceding fusion, and later, during fusion pore expansion. Finally, we discuss how expansion of the fusion pore may be regulated by the GTPase activity of dynamin.     

Local electrostatic interactions determine the diameter of fusion pores

In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least 2 mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium; and/or directly, by altering local cation concentration, which affects fusion pore geometry likely via electrostatic interactions. By monitoring membrane capacitance, we investigated how extracellular cation concentration affects fusion pore diameter in pituitary cells and astrocytes. At low extracellular divalent cation levels predominantly transient fusion events with widely open fusion pores were detected. However, fusion events with predominately narrow fusion pores were present at elevated levels of extracellular trivalent cations. These results show that electrostatic interactions likely help determine the stability of discrete fusion pore states by affecting fusion pore membrane composition.     

Characterization of Exocytotic Events From Single PC12 Cells: Amperometric Studies in Native PC12h, DA-Loaded PC12h and Bovine Adrenal Chromaffin Cells

Exocytotic events from rat pheochromocytoma (PC12) cells were characterized by amperometric analysis. For single-cell amperometric recordings, PC12h cells cultured onto poly-L-lysine corted glass-base dish were incubated with 1 mM dopamine (DA) for 60 min. Amperometric recordings, with a carbon fiber microelectrode (5 μm diameter), of catecholamine release from the individual cells were conducted under an inverted microscope at 25 ^∘C. To characterize a single exocytotic event that is detected as a single spike current, the spike number, spike parameters (rise time, middle width and area) and spike shape were analyzed. Exposure of DA-loaded PC12h cells to 60 mM KCl (1000 hps) for 5 min and for 4 s evoked a train of events with the event number of 114± 19 (spikes/response for 5 min) and 12± 3 (spikes/response for 15 s), respectively. We observed distinctive kinetics in the events (rise time = 0.83± 0.19 ms, middle width = 2.89± 0.62 ms, area = 62± 7.6 fC and the spikes with a “foot” = 15.4± 2.7% of total spikes). The number and mean height of the events were 3- to 4-fold higher than that in DA-unloaded cells, and the values of rise time and middle width in DA-loaded PC12h cells were approx. 5- and 10-fold less than those observed in cultured adrenal chromaffin cells. The successful application of amperometry to monitor DA released from secretory vesicles in DA-loaded PC12h cell suggest that this technique is applicable to characterize exocytotic events in neurons.     

Local electrostatic interactions determine the diameter of fusion pores

In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least 2 mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium; and/or directly, by altering local cation concentration, which affects fusion pore geometry likely via electrostatic interactions. By monitoring membrane capacitance, we investigated how extracellular cation concentration affects fusion pore diameter in pituitary cells and astrocytes. At low extracellular divalent cation levels predominantly transient fusion events with widely open fusion pores were detected. However, fusion events with predominately narrow fusion pores were present at elevated levels of extracellular trivalent cations. These results show that electrostatic interactions likely help determine the stability of discrete fusion pore states by affecting fusion pore membrane composition.     

The permeability transition pore as a mitochondrial calcium release channel: A critical appraisal

Mitochondria from a variety of sources possess an inner membrane channel, the permeability transition pore. The pore is a voltage-dependent channel, activated by matrix Ca²⁺ and inhibited by matrix H⁺, which can be blocked by cyclosporin A, presumably after binding to mitochondrial cyclophilin. The physiological function of the permeability transition pore remains unknown. Here we evaluate its potential role as a fast Ca²⁺ release channel involved in mitochondrial and cellular Ca²⁺ homeostasis. We (i) discuss the theoretical and experimental reasons why mitochondria need a fast, inducible Ca²⁺ release channel; (ii) analyze the striking analogies between the mitochondrial permeability transition pore and the sarcoplasmic reticulum ryanodine receptor-Ca²⁺ release channel; (iii) argue that the permeability transition pore can act as a selective release channel for Ca²⁺ despite its apparent lack of selectivity for the transported speciesin vitro; and (iv) discuss the importance of mitochondria in cellular Ca²⁺ homeostasis, and how disruption of this function could impinge upon cell viability, particularly under conditions of oxidative stress.     

Effects of purified myosin light chain kinase on myosin light chain phosphorylation and catecholamine secretion in digitonin-permeabilized chromaffin cells

Many non-muscle cells including chromaffin cells contain actin and myosin. The 20,000 dalton light chain subunits of myosin can be phosphorylated by a Ca²⁺/calmodulin-dependent enzyme, myosin light chain kinase. In tissues other than striated muscle, light chain phosphorylation is required for actin-induced myosin ATPase activity. The possibility that actin and myosin are involved in catecholamine secretion was investigated by determining whether increased phosphorylation in the presence of [-³²P]ATP of myosin light chain by myosin light chain kinase enhances secretion from digitonin-treated chromaffin cells. In the absence of exogenous myosin light chain kinase, 1 M Ca²⁺ caused a 30–40% enhancement of the phosphorylation of a 20 kDa protein. This protein was identified on 2-dimensional gels as myosin light chain by its comigration with purified myosin light chain. Purified myosin light chain kinase (400 g/ml) in the presence of calmodulin (10 M) caused little or no enhancement of myosin light chain phosphorylation in the absence of Ca²⁺ in digitonin-treated cells. In the presence of 1 M Ca²⁺, myosin light chain kinase (400 g/ml) caused an approximately two-fold increase in myosin light chain phosphorylation in digitonin-treated cells in 5 min. The phosphorylation required permeabilization of the cells by digitonin and occurred within the cells rather than in the medium. Myosin light chain kinase-induced phosphorylation of myosin light chain was maximal at 1 M. Ca²⁺. Under identical conditions to those of the phosphorylation experiments, secretion was unaltered by myosin light chain kinase. The experiments indicate that the phosphorylation of myosin light chain by myosin light chain kinase is not a limiting factor in secretion in digitonin-treated chromaffin cells and suggest that the activation of myosin is not directly involved in secretion from the cells. The experiments also demonstrate the feasibility of investigation of effects of exogenously added proteins on secretion in digitonin-treated cells.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - KGEPM solution containing potassium glutamate, EGTA, PIPES and MgCl₂ - NE norepinephrine - PIPES piperazine-N,-N-bis-(2-ethanesulfonic acid) - PSS physiological salt solution     

SNARE‐mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle

Constitutive membrane fusion within eukaryotic cells is thought to be controlled at its initial steps, membrane tethering and SNARE complex assembly, and to rapidly proceed from there to full fusion. Although theory predicts that fusion pore expansion faces a major energy barrier and might hence be a rate‐limiting and regulated step, corresponding states with non‐expanding pores are difficult to assay and have remained elusive. Here, we show that vacuoles in living yeast are connected by a metastable, non‐expanding, nanoscopic fusion pore. This is their default state, from which full fusion is regulated. Molecular dynamics simulations suggest that SNAREs and the SM protein‐containing HOPS complex stabilize this pore against re‐closure. Expansion of the nanoscopic pore to full fusion can thus be triggered by osmotic pressure gradients, providing a simple mechanism to rapidly adapt organelle volume to increases in its content. Metastable, nanoscopic fusion pores are then not only a transient intermediate but can be a long‐lived, physiologically relevant and regulated state of SNARE‐dependent membrane fusion.     

Membrane fusion in cells: molecular machinery and mechanisms

Membrane fusion is a sine qua non process for cell physiology. It is critical for membrane biogenesis, intracellular traffic, and cell secretion. Although investigated for over a century, only in the last 15 years, the molecular machinery and mechanism of membrane fusion has been deciphered. The membrane fusion event elicits essentially three actors on stage: anionic phospholipids - phosphatidylinositols, phosphatidyl serines, specific membrane proteins, and the calcium ions, all participating in a well orchestrated symphony. Three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs) have been implicated in membrane fusion. Target membrane proteins, SNAP-25 and syntaxin (t- SNARE) and secretory vesicle-associated membrane protein (v-SNARE) or VAMPwere discovered in the 1990's and suggested to be the minimal fusion machinery. Subsequently, the molecular mechanism of SNARE-induced membrane fusion was discovered. It was demonstrated that when t-SNARE-associated lipid membrane is exposed to v-SNARE-associated vesicles in the presence of Ca(2+), the SNARE proteins interact in a circular array to form conducting channels, thus establishing continuity between the opposing bilayers. Further it was proved that SNAREs bring opposing bilayers close to within a distance of 2-3 Angstroms, allowing Ca(2+) to bridge them. The bridging of bilayers by Ca(2+) then leads to the expulsion of water between the bilayers at the contact site, allowing lipid mixing and membrane fusion. Calcium bridging of opposing bilayers leads to the release of water, both from the water shell of hydrated Ca(2+) ions, as well as the displacement of loosely coordinated water at the phosphate head groups in the lipid membrane. These discoveries provided for the first time, the molecular mechanism of SNARE-induced membrane fusion in cells. Some of the seminal discoveries are briefly discussed in this minireview.     

Alpha-latrotoxin Triggers Extracellular Ca^2＋-dependent Exocytosis and Sensitizes Fusion Machinery in Endocrine Cells

α-Latrotoxin from the venom of black widow spider induces and augments neurotransmitterand hormone release by way of extracellular Ca~(2 ) influx and cellular signal transduction pathways.By usingwhole cell current and capacitance recording,the photolysis of card Ca~(2 ),and Ca~(2 ) microfluorometry andamperometry,we investigated the stimulating effect and mechá(?)ism of α-latrotoxin on exocytosis in ratpancreatic β cells,LβT2 cells and latrophilin plasmid-transfected INS-1 cells.Our data indicated that:(1)α-latrotoxin increased cytosolic Ca~(2 ) concentration through the formation of cation-permitting pores and sub-sequent Ca~(2 ) influx with the presence of extracellular Ca~(2 );(2)α-latrotoxin stimulated exocytosis in normalbath solution and its stimulating effect on secretion was eradicated in Ca~(2 )-free bath solution; and (3)α-latrotoxin sensitized the molecular machinery of fusion through activation of protein kinase C and increasedthe response of cells to Ca~(2 ) photolysed by a flash of ultraviolet light.In summary,α-latrotoxin inducedexocytosis by way of Ca~(2 ) influx and accelerated vesicle fusion by the sensitization of fusion machinery.     

A comparison of bradykinin,angiotensin II and muscarinic stimulation of cultured bovine adrenal chromaffin cells

Bradykinin, angiotensin II and a mascarnic agonist, acetyl-B-methacholine (methacholine) were all found to elict catecholamine release from cultured bovine adrenal chromaffin cells. Bradykinin was the most potent of these secretagogues and methacholine the weakest, with angiotenin II intermediate in efficacy. All three secretagogues were much less effective than nicotinic stimulation. The three secretagogues all produced a rise in cytoplasmic free calcium concentration ([Ca²⁺]_i), measured with the fluorescent indicator fura2, which was partially independent of external calcium. In the case of bradykinin the full rise in ([Ca²⁺]_i) may involve a component of calcium entry in addition to release of calcium from an internal store. Secretion was also found to be partially independent of external calcium. The different efficacies of the three secretagogues in elicting secretion were correlated with the rise in ([Ca²⁺]_i) produced. The differeing efficacies of the three secretagogues may be due to the extent of release of calcium from an intracellular store which itself is less effective in eliciting secretion than a rise in [Ca²⁺]_i following calcium entry due to nicotine. Bradykinin also stimulates calcium entry, and this may increase the efficacy of the initial rise in [Ca²⁺]_i. Treatment with pertussis toxin resulted in an enhancement of secretion in response to all of the secretagogues.Abbreviations ([Ca²⁺]_i) cytoplasmic free calcium concentration - EGTA ethylene glycol bis (-amino ethyl ether)-N,N,N,N,-tetraacetic acid - Hepes 4-(2-hydroxy ethyl)-1-piperazinethanesulphonic acid - TPA 12-O-tetradecanoylphorbol-13-acetate - DAG diacyl glycerol - IP₃ inositol-1,4,5-trisphosphate - PIP₂ phosphatidylinositol-4,5-bisphosphate     

Characterization of a delayed rectifier K+ channel in NG108-15 neuroblastomaX glioma cells: Gating kinetics and the effects of enrichment of membrane phospholipids with arachidonic acid

Summary The calcium sensitivity of exocytosis from electroper-meabilized chromaffin cells is increased by activators of protein kinase C, such as TPA and certain phorbol esters, diacylglycerols, and mezerein. A range of putative inhibitors of protein kinase C block both the phorbol ester-sensitive component of secretion and also the underlying insensitive component. These inhibitors are also shown to inhibit medulla protein kinase C activity in vitro. The extent of secretion is reduced when electropermeabilized cells are exposed to Ca²⁺ levels much in excess of 50 m. The onset of inhibition is faster than the relatively slow rate of Ca-dependent exocytosis and is insensitive to inhibitors of proteolysis. Adrenal medulla protein kinase C activity is also irreversibly inhibited by high Ca²⁺ concentrations. Both the secretory response and the protein kinase C activity in vitro have similar nucleotide and cation specificities. Although these data do not definitely establish an involvement of protein kinase C in exocytosis, none argue against it.Deceased     

The Use of Permeabilized Cells to Assay Protein Phosphorylation and Catecholamine Release

A number of approaches can be used to determine the protein kinases and protein phosphatases acting on particular phosphoproteins in vivo. Cell permeabilization represents one such approach. In this overview we discuss the different permeabilization procedures used in bovine adrenal chromaffin cells and in particular the use of digitonin. The effect of various factors on the extent of digitonin-permeabilization, protein phosphorylation and catecholamine release are also discussed. The factors include the permeabilization medium, the ions such as calcium, and the second messengers, such as cAMP, IP₃, cADPR and calmodulin. The effect of specific peptide inhibitors of protein kinases on tyrosine hydroxylase phosphorylation is illustrated. Advantages and disadvantages of cell permeabilization procedures are discussed throughout the text.     

Physical Link and Functional Coupling of Presynaptic Calcium Channels and the Synaptic Vesicle Docking/Fusion Machinery

N- and P/Q-type calcium channels are localized in high density in presynaptic nerve terminals and are crucial elements in neuronal excitation–secretion coupling. In addition to mediating Ca²⁺ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. As outlined in the preceding article, these calcium channels can be purified from brain as a complex with SNARE proteins which are involved in exocytosis. In addition, N-type and P/Q-type calcium channels are co-localized with syntaxin in high-density clusters in nerve terminals. Here we review the role of the synaptic protein interaction (synprint) sites in the intracellular loop II–III (L_II–III) of both _1B and _1A subunits of N-type and P/Q-type calcium channels, which bind to syntaxin, SNAP-25, and synaptotagmin. Calcium has a biphasic effect on the interactions of N-type calcium channels with SNARE complexes, stimulating optimal binding in the range of 10–20 M. PKC or CaM KII phosphorylation of the N-type synprint peptide inhibits interactions with native brain SNARE complexes containing syntaxin and SNAP-25. Introduction of the synprint peptides into presynaptic superior cervical ganglion neurons reversibly inhibits EPSPs from synchronous transmitter release by 42%. At physiological Ca²⁺ concentrations, synprint peptides cause an approximate 25% reduction in transmitter release of injected frog neuromuscular junction in cultures, consistent with detachment of 70% of the docked vesicles from calcium channels based on a theoretical model. Together, these studies suggest that presynaptic calcium channels not only provide the calcium signal required by the exocytotic machinery, but also contain structural elements that are integral to vesicle docking, priming, and fusion processes.