Determinants of postsynaptic Ca2+ signaling in Purkinje neurons

Neuronal integration in Purkinje neurons involves many forms of Ca2+ signaling. Two afferent synaptic inputs, the parallel and the climbing fibers, provide a major drive for these signals. These two excitatory synaptic inputs are both glutamatergic. Postsynaptically they activate alpha-amino-3-hydroxy-5-methyl-4-propionic acid (AMPA) receptors (AMPARs) and metabotropic glutamate receptors (mGluRs). Unlike most other types of central neurons, Purkinje neurons do not express NMDA (N-methyl-D-aspartate) receptors (NMDARs). AMPARs in Purkinje neurons are characterized by a low permeability for Ca2+ ions. AMPAR-mediated synaptic depolarization may activate voltage-gated Ca2+ channels, mostly of the P/Q-type. The resulting intracellular Ca2+ signals are shaped by the Ca2+ buffers calbindin and parvalbumin. Ca2+ clearance from the cytosol is brought about by Ca2+-ATPases in the plasma membrane and the endoplasmic reticulum, as well as the Na+-Ca2+-exchanger. Binding of glutamate to mGluRs induces postsynaptic Ca2+-transients through two G protein-dependent pathways: involving (1) the release of Ca2+ ions from intracellular Ca2+ stores and (2) the opening of the cation channel TRPC1. Homer proteins appear to play an important role in postsynaptic Ca2+ signaling by providing a direct link between the plasma membrane-resident elements (mGluRs and TRPC1) and their intracellular partners, including the IP3Rs.     

Ca2+ buffering and action potential-evoked Ca2+ signaling in dendrites of pyramidal neurons.

The effect of the fluorescent Ca2+ indicator dye Fura-2 on Ca2+ dynamics was studied in proximal apical dendrites of neocortical layer V and hippocampal CA1 pyramidal neurons in rat brain slices using somatic whole-cell recording and a charge-coupled device camera. A single action potential evoked a transient increase of intradendritic calcium concentration ([Ca2+]i) that was reduced in size and prolonged when the Fura-2 concentration was increased from 20 to 250 microM. Extrapolation to zero Fura-2 concentration suggests that "physiological" transients at 37 degrees C have large amplitudes (150-300 nM) and fast decays (time constant < 100 ms). Assuming a homogeneous compartment model for the dendrite, 0.5-1% of the total Ca2+ entering during an action potential was estimated to remain free. Washout of cytoplasmic Ca2+ buffers was not detectable, suggesting that they are relatively immobile. During trains of action potentials, [Ca2+]i increased and rapidly reached a steady state (time constant < 200 ms), fluctuating around a plateau level which depended linearly on the action potential frequency. Thus, the mean dendritic [Ca2+]i encodes the action potential frequency during physiological patterns of electrical activity and may regulate Ca(2+)-dependent dendritic functions in an activity-dependent way.     

Heterogeneity of microsomal Ca2+ stores in chicken Purkinje neurons.

Chicken cerebellum microsomes were subfractionated on isopycnic, linear sucrose (15-50%) density gradients. The distribution of four markers of intracellular, rapidly-exchanging Ca2+ stores, i.e. the Ca2+ pump, the receptors for inositol 1,4,5-trisphosphate (IP3) and ryanodine (Ry), and calsequestrin (CS, an intralumenal, high capacity Ca2+ binding protein) was investigated biochemically and immunologically. In the cerebellum, high levels of these markers are expressed by one of the cell types, the Purkinje neuron. Heavy subfractions were enriched in both CS and Ry receptor, intermediate subfractions in the IP3 receptor, while the Ca2+ pump was present in both intermediate and heavy subfractions. Intact cells and pelleted subfractions were examined by conventional and immuno-electron microscopy (immunogold labeling of ultrathin cryosections with anti-CS and anti-IP3 receptor antibodies). Of the strongly CS-labeled, moderately dense-cored vacuoles (calciosomes) recently described in chicken Purkinje neurons only partly exhibited labeling for the IP3 receptor as well, and the rest appeared negative. The latter were enriched in a heavy subfraction of the gradient where Ry receptors were also concentrated, whereas the CS-rich vacuoles in an intermediate subfraction were almost always IP3 receptor-positive. The population of CS-rich calciosomes of chicken Purkinje neurons appears therefore to be molecularly heterogeneous, with a part responsive to IP3 and the rest possibly sensitive to Ry.     

Dynamic buffering of mitochondrial Ca2+ during Ca2+ uptake and Na+-induced Ca2+ release

In cardiac mitochondria, matrix free Ca²⁺ ([Ca²⁺]_m) is primarily regulated by Ca²⁺ uptake and release via the Ca²⁺ uniporter (CU) and Na⁺/Ca²⁺ exchanger (NCE) as well as by Ca²⁺ buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca²⁺ buffering affects these dynamics under various Ca²⁺ uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca²⁺ buffering on the uptake and release of Ca²⁺, we monitored Ca²⁺ dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca²⁺] ([Ca²⁺]_e) and [Ca²⁺]_m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl₂] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca²⁺]_e and [Ca²⁺]_m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca²⁺-induced changes in mitochondrial bioenergetics. Our [Ca²⁺]_e and [Ca²⁺]_m measurements demonstrate that Ca²⁺ uptake and release do not show reciprocal Ca²⁺ dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca²⁺ buffering system in the matrix compartment. The Na⁺- induced Ca²⁺ release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca²⁺ uptake in the presence of large amounts of CaCl₂, but not by Na⁺- induced Ca²⁺ release.     

Modulation of L-type Ca2+ current by fast and slow Ca2+ buffering in guinea pig ventricular cardiomyocytes.

Free Ca2+ near Ca2+ channel pores is expected to be lower in cardiomyocytes dialyzed with bis-(o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) than with ethyleneglycol-bis-(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) because BAPTA chelates incoming Ca2+ more rapidly. The consequences of intracellular Ca2+ buffering by BAPTA (0.2-60 mM) and by EGTA (0.2-67 mM) on whole-cell L-type Ca2+ current (ICa,L) were investigated in voltage-clamped guinea pig ventricular cardiomyocytes; bulk cytoplasmic free Ca2+ (Cac2+) was monitored using the fluorescent Ca2+ indicator indo-1. ICa,L was augmented by approximately 12-fold when BAPTA in the cell dialysate was increased from 0.2 to 50 mM (half-maximal stimulation at 31 mM), whereas elevating internal EGTA from 0.2 to 67 mM increased ICa,L only by approximately 2-fold. Cac2+ was < 20 nM with internal BAPTA or EGTA > or = 20 mM. While EGTA up to 67 mM had only an insignificant inhibitory effect on the stimulation of ICa,L by 3 microM forskolin, ICa,L in 50 mM BAPTA-dialyzed myocytes was insensitive to forskolin-induced elevation of adenosine 3',5'-cyclic monophosphate (cAMP); conversely, ICa,L in cAMP-loaded cells was unresponsive to BAPTA dialysis. Cell dialysis with BAPTA, but not with EGTA, accelerated the slow component of ICa,L inactivation (tau S) without affecting its fast component (tau F), resembling the effects of cAMP-dependent phosphorylation. BAPTA-stimulated ICa,L was inhibited by acetylcholine and by the cAMP-dependent protein kinase (PKA) blocker H-89. These results suggest that BAPTA-induced lowering of peri-channel Ca2+ stimulates cAMP synthesis and channel phosphorylation by disinhibiting Ca(2+)-sensitive adenylyl cyclase.     

Inositol trisphosphate and ryanodine receptors share a common functional Ca2+ pool in cerebellar Purkinje neurons

Changes in the intracellular free calcium concentration ([Ca2+]i) control many important processes in excitable and nonexcitable cells. In cerebellar Purkinje neurons, increases in [Ca2+]i modulate excitability by turning on calcium-activated potassium and chloride conductances, and modifying the synaptic efficacy of inhibitory and excitatory inputs to the cell. Calcium release from the intracellular stores plays an important role in the regulation of [Ca2+]i. Purkinje neurons contain both inositol trisphosphate (InsP3) and ryanodine (Ry) receptors. With the exception of the dendritic spines, where only InsP3 receptors are found, InsP3 and Ry receptors are present in the entire cell. The distribution of the two calcium release channels, however, is not uniform, and it has been suggested that InsP3 and Ry receptors use separate Ca2+ pools. The functional properties of InsP3 and Ry Ca2+ pools were investigated by flash photolysis and single-cell microspectrofluorimetry. It was found that depletion of ryanodine-sensitive Ca2+ stores renders InsP3 incapable of releasing more Ca2+ from the stores. Abolishing calcium-induced calcium release by blocking ryanodine receptors with ruthenium red did not have a significant effect on InsP3-evoked Ca2+ release. It is concluded that InsP3 receptors use the same functional Ca2+ pool as that utilized by Ry receptors in Purkinje neurons.     

Ca2+ signaling in prokaryotes

The role of Ca²⁺ ions in the regulation of motility, cell cycle, and division of prokaryotes is discussed, as well as their involvement in the pathogenesis of some infectious diseases. The structural and functional organization of the prokaryotic Ca²⁺ signaling system and the mechanisms of Ca²⁺ membrane transport and homeostasis are described. Special attention is paid to the role of Ca²⁺ cation channels, Ca²⁺ transporters, and Ca²⁺-binding proteins in the regulation of the intercellular Ca²⁺ concentration.     

Sustained Ca2+ transfer across mitochondria is Essential for mitochondrial Ca2+ buffering,sore-operated Ca2+ entry,and Ca2+ store refilling

Mitochondria have been found to sequester and release Ca2+ during cell stimulation with inositol 1,4,5-triphosphate-generating agonists, thereby generating subplasmalemmal microdomains of low Ca2+ that sustain activity of capacitative Ca2+ entry (CCE). Procedures that prevent mitochondrial Ca2+ uptake inhibit local Ca2+ buffering and CCE, but it is not clear whether Ca2+ has to transit through or remains trapped in the mitochondria. Thus, we analyzed the contribution of mitochondrial Ca2+ efflux on the ability of mitochondria to buffer subplasmalemmal Ca2+, to maintain CCE, and to facilitate endoplasmic reticulum (ER) refilling in endothelial cells. Upon the addition of histamine, the initial mitochondrial Ca2+ transient, monitored with ratio-metric-pericam-mitochondria, was largely independent of extracellular Ca2+. However, subsequent removal of extracellular Ca2+ produced a reversible decrease in [Ca2+]mito, indicating that Ca2+ was continuously taken up and released by mitochondria, although [Ca2+]mito had returned to basal levels. Accordingly, inhibition of the mitochondrial Na+/Ca2+ exchanger with CGP 37157 increased [Ca2+]mito and abolished the ability of mitochondria to buffer subplasmalemmal Ca2+, resulting in an increased activity of BKCa channels and a decrease in CCE. Hence, CGP 37157 also reversibly inhibited ER refilling during cell stimulation. These effects of CGP 37157 were mimicked if mitochondrial Ca2+ uptake was prevented with oligomycin/antimycin A. Thus, during cell stimulation a continuous Ca2+ flux through mitochondria underlies the ability of mitochondria to generate subplasmalemmal microdomains of low Ca2+, to facilitate CCE, and to relay Ca2+ from the plasma membrane to the ER.     

Ca2+]i signaling between mitochondria and endoplasmic reticulum in neurons is regulated by microtubules. From mitochondrial permeability transition pore to Ca2+-induced Ca2+ release

The positioning and dynamics of organelles depend on membrane-cytoskeleton interactions. Mitochondria relocate along microtubules (MT), but it is not clear whether MT have direct effects on mitochondrial function. Using two-photon microscopy and the mitochondrial fluorescent dyes rhodamine 123 and Rhod-2, we showed that Taxol and nocodazole, which correspondingly stabilize and disrupt MT, decreased potential and Ca(2+) in the mitochondria of brain stem pre-Botzinger complex neurons. Without changing basal cytoplasmic Ca(2+) ([Ca(2+)](i)), Taxol promoted the generation of [Ca(2+)](i) spikes in dendrites. These spikes were abolished after blockade of Ca(2+) influx and after depletion of internal Ca(2+) stores, indicating the involvement of Ca(2+)-induced Ca(2+) release. Nocodazole decreased mitochondrial potential and [Ca(2+)](m) and produced a long lasting increase in [Ca(2+)](i). MT-acting drugs depolarized single immobilized mitochondria and released previously stored Ca(2+). All of these effects were inhibited by pretreatment with blockers of mitochondrial permeability transition pore (mPTP), cyclosporin A, and 2-aminoethoxydiphenyl borate. Induction of mPTP by Taxol and nocodazole was confirmed by using a calcein/Co(2+) imaging technique. Electron and optical microscopy revealed tubulin bound to mitochondria. Mitochondria, MT, and endoplasmic reticulum (ER) showed strong co-localization, the degree of which decreased after MT were disrupted. We propose that changes in the structure of MT by Taxol and nocodazole promote the induction of mPTP. Subsequent Ca(2+) efflux stimulates the Ca(2+) release from the ER that drives spontaneous [Ca(2+)](i) transients. Thus, close positioning of mitochondria to the ER as determined by MT can be essential for the local [Ca](i) signaling in neurons.     

Bile acids induce Ca2+ signaling and membrane permeabilizations in vagal nodose ganglion neurons

Bile acids (BAs) play an important role in the digestion of dietary fats and act as signaling molecules. However, due to their solubilizing properties, high concentrations in the gut may negatively affect gut epithelium and possibly afferent fibers innervating the gastrointestinal tract (GI). To determine the effect of BAs on intracellular Ca²⁺ and membrane permeabilization we tested a range of concentrations of two BAs on vagal nodose ganglion (NG) neurons, Chinese Hamster Ovary (CHO), and PC12 cell lines. NG explants from mice were drop-transduced with the genetically encoded Ca²⁺ indicator AAV9-Syn-jGCaMP7s and used to measure Ca²⁺ changes upon application of deoxycholic acid (DCA) and taurocholic acid (TCA). We found that both BAs induced a Ca²⁺ increase in NG neurons in a dose-dependent manner. The DCA-induced Ca²⁺ increase was dependent on intracellular Ca²⁺ stores. NG explants, with an intact peripheral part of the vagus nerve, showed excitation of NG neurons in nerve field recordings upon exposure to DCA. The viability of NG neurons at different BA concentrations was determined, and compared to CHO and PC12 cells lines using propidium iodide labeling, showing threshold concentrations of BA-induced cell death at 400–500 μM. These observations suggest that BAs act as Ca²⁺-inducing signaling molecules in vagal sensory neurons at low concentrations, but induce cell death at higher concentrations, which may occur during inflammatory bowel diseases.     

Synchronized Ca2+ signaling by intercellular propagation of Ca2+ action potentials in NRK fibroblasts

Theintercellular propagation of Ca²⁺waves by diffusion of inositol trisphosphate has been shown to be ageneral mechanism by which nonexcitable cells communicate. Here, weshow that monolayers of normal rat kidney (NRK) fibroblasts behave likea typical excitable tissue. In confluent monolayers of these cells,Ca²⁺ action potentials can begenerated by local depolarization of the monolayer on treatment witheither bradykinin or an elevation of the extracellularK⁺ concentration. Theseelectrotonically propagating action potentials travel intercellularlyover long distances in an all-or-none fashion at a speed of ~6.1 mm/sand can be blocked by L-typeCa²⁺ channel blockers. The actionpotentials are generated by depolarizations beyond the threshold valuefor L-type Ca²⁺ channels of about15 mV. The result of these locally induced, propagatingCa²⁺ action potentials is analmost synchronous, transient increase in the intracellularCa²⁺ concentration in largenumbers of cells. These data show that electrically coupled fibroblastscan form an excitable syncytium, and they elucidate a novel mechanismof intercellular Ca²⁺ signaling inthese cells that may coordinate synchronized multicellular responses tolocal stimuli.

     

Activation of Ca2+-dependent signaling by TLR2

Upon contact with airway epithelial cells, bacterial products activate Ca(2+) fluxes that are required for induction of NF-kappaB-dependent gene expression. TLR2 is apically displayed on airway cells, making it a likely transducer linking bacterial stimuli and kinases that affect Ca(2+) release. Using biochemical and genetic approaches, we demonstrate that TLR2 ligands stimulate release of Ca(2+) from intracellular stores by activating TLR2 phosphorylation by c-Src, and recruiting PI3K and phospholipase Cgamma to affect Ca(2+) release through inositol (1,4,5) trisphosphate receptors. In the absence of TLR2, murine macrophages as well as airway cells do not generate Ca(2+) fluxes or induce proinflammatory signaling. Thus, Ca(2+) participates as a second messenger in TLR2-dependent signaling and provides another target to modulate proinflammatory responses to bacterial infection.