Descripción: Calcium channels of the P/Q subtype mediate transmitter release at the neuromuscular junction and at many central synapses, such as the calyx of Held. Transgenic mice in which α1A channels are ablated provide a powerful tool with which to test compensatory mechanisms at the synapse and to explore mechanisms of presynaptic regulation associated with expression of P/Q channels. Using the calyx of Held preparation from the knock-out (KO) mice, we show here that N-type channels functionally compensate for the absence of P/Q subunits at the calyx and evoke giant synaptic currents [approximately two-thirds of the magnitude of wild-type (WT) responses]. However, although evoked paired-pulse facilitation is prominent in WT, this facilitation is greatly diminished in the KO. In addition, direct recording of presynaptic calcium currents revealed that the major functional difference was the absence of calcium-dependent facilitation at the calyx in the P/Q KO animals. We conclude that one physiological function of P/Q channels is to provide additional facilitatory drive, so contributing to maintenance of transmission as vesicles are depleted during high throughput synaptic transmission.

Descripción: Calcium signals are involved in a large variety of physiological processes. Their versatility relies on the diversity of spatiotemporal behaviors that the calcium concentration can display. Calcium entry through inositol 1,4,5-trisphosphate (IP3) receptors (IP3R's) is a key component that participates in both local signals such as "puffs" and in global waves. IP3R's are usually organized in clusters on the membrane of the endoplasmic reticulum and their spatial distribution has important effects on the resulting signal. Recent high resolution observations [1] of Ca2+ puffs offer a window to study intra-cluster organization. The experiments give the distribution of the number of IP3R's that open during each puff without much processing. Here we present a simple model with which we interpret the experimental distribution in terms of two stochastic processes: IP3 binding and unbinding and Ca2+-mediated inter-channel coupling. Depending on the parameters of the system, the distribution may be dominated by one or the other process. The transition between both extreme cases is similar to a percolation process. We show how, from an analysis of the experimental distribution, information can be obtained on the relative weight of the two processes. The largest distance over which Ca2+mediated coupling acts and the density of IP3-bound IP3R's of the cluster can also be estimated. The approach allows us to infer properties of the interactions among the channels of the cluster from statistical information on their emergent collective behavior. © 2010 Solovey, Dawson.

Descripción: Mean field models are often useful approximations to biological systems, but sometimes, they can yield misleading results. In this work, we compare mean field approaches with stochastic models of intracellular calcium release. In particular, we concentrate on calcium signals generated by the concerted opening of several clustered channels (calcium puffs). To this end we simulate calcium puffs numerically and then try to reproduce features of the resulting calcium distribution using mean field models were all the channels open and close simultaneously. We show that an unrealistic non-linear relationship between the current and the number of open channels is needed to reproduce the simulated puffs. Furthermore, a single channel current which is five times smaller than the one of the stochastic simulations is also needed. Our study sheds light on the importance of the stochastic kinetics of the calcium release channel activity to estimate the release fluxes. © 2011 Solovey, Fraiman and Dawson.

Descripción: Sporadic amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects particularly motoneurons. Several pieces of evidence suggested the involvement of autoimmune mechanisms mediated by antibodies in ALS. However, the significance of those antibodies in the disease and the underlying mechanisms are unknown. Here we showed that IgG purified from a group of sporadic ALS patients, but not familial ALS patients, specifically interact with the presynaptic membrane of motoneurons through an antigen-antibody interaction and modulated synaptic transmission. Immunoreactivity against nerve terminals showed strong correlation with synaptic modulation ability. In addition, several controls have ruled out the possibility for this synaptic modulation to be mediated through proteases or nonspecific effects. Effective IgG potentiated both spontaneous and asynchronous transmitter release. Application of pharmacological inhibitors suggested that activation of this increased release required a nonconstitutive Ca2+ influx through N-type (Ca v2.2) channels and phospholipase C activity and that activation of IP3 and ryanodine receptors were necessary to both activate and sustain the increased release. Consistent with the notion that ALS is heterogeneous disorder, our results reveal that, in ∼50% of ALS patients, motor nerve terminals constitutes a target for autoimmune response. Copyright © 2006 Society for Neuroscience.

Descripción: In the mammalian auditory system, the synapse between efferent olivocochlear (OC) neurons and sensory cochlear hair cells is cholinergic, fast, and inhibitory. This efferent synapse is mediated by the nicotinic α9α10 receptor coupled to the activation of SK2 Ca 2+-activated K+ channels that hyperpolarize the cell. So far, the ion channels that support and/or modulate neurotransmitter release from the OC terminals remain unknown. To identify these channels, we used an isolated mouse cochlear preparation and monitored transmitter release from the efferent synaptic terminals in inner hair cells (IHCs) voltage clamped in the whole-cell recording configuration. Acetylcholine (ACh) release was evoked by electrically stimulating the efferent fibers that make axosomatic contacts with IHCs before the onset of hearing. Using the specific antagonists for P/Q- and N-type voltage-gated calcium channels (VGCCs), ω-agatoxin IVA and ω-conotoxin GVIA, respectively, we show that Ca2+ entering through both types of VGCCs support the release process at this synapse. Interestingly, we found that Ca2+ entering through the dihydropiridine-sensitive L-type VGCCs exerts a negative control on transmitter release. Moreover, using immunostaining techniques combined with electrophysiology and pharmacology, we show that BK Ca2+-activated K+ channels are transiently expressed at the OC efferent terminals contacting IHCs and that their activity modulates the release process at this synapse. The effects of dihydropiridines combined with iberiotoxin, a specific BK channel antagonist, strongly suggest that L-type VGCCs negatively regulate the release of ACh by fueling BK channels that are known to curtail the duration of the terminal action potential in several types of neurons. Copyright © 2010 the authors.

Descripción: In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long-term survival of cerebellar granule cells. The effect of voltage-gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase (MEK1), to the effects of high [K+]e under serum-free conditions. We found that 25 mM KCI (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24-48 h under low-density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin-dependent protein kinase II. This also produced an increase in long-term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin-dependent protein kinase II and of the extracellular signal-regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin-dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.

Descripción: Local intracellular Ca2+ signals result from Ca2+ flux into the cytosol through individual channels or clusters of channels. To gain a mechanistic understanding of these events we need to know the magnitude and spatial distribution of the underlying Ca2+ flux. However, this is difficult to infer from fluorescence Ca2+ images because the distribution of Ca2+-bound dye is affected by poorly characterized processes including diffusion of Ca2+ ions, their binding to mobile and immobile buffers, and sequestration by Ca2+ pumps. Several methods have previously been proposed to derive Ca2+ flux from fluorescence images, but all require explicit knowledge or assumptions regarding these processes. We now present a novel algorithm that requires few assumptions and is largely model-independent. By testing the algorithm with both numerically generated image data and experimental images of sparklets resulting from Ca2+ flux through individual voltage-gated channels, we show that it satisfactorily reconstructs the magnitude and time course of the underlying Ca2+ currents. © 2005 by the Biophysical Society.

Descripción: When Ca2+ is released from internal stores in living cells, the resulting wave of increased concentration can travel without deformation (continuous propagation) or with burst-like behavior (saltatory propagation). We analyze the 'fire-diffuse-fire' model in order to illuminate the differences between these two modes of propagation. We show that the Ca2+ release wave in immature Xenopus oocytes and cardiac myocytes is saltatory, whereas the fertilization wave in the mature oocyte is continuous.

Descripción: Nur factors are critical for proopiomelanocortin (POMC) induction by CRH in corticotrophs, but the pathways linking CRH to Nur are unknown. In this study we show that in AtT-20 corticotrophs CRH and cAMP induce Nur77 and Nurr1 expression and transcription at the NurRE site by protein kinase A (PKA) and calcium-dependent and -independent mechanisms. Calcium pathways depend on calmodulin kinase II (CAMKII) activity, and calcium-independent pathways are accounted for in part by MAPK activation (Rap1/B-Raf/MAPK-ERK kinase/ERK1/2), demonstrated by the use of molecular and pharmacological tools. ATT-20 corticotrophs express B-Raf, as do other cells in which cAMP stimulates MAPK. CRH/cAMP stimulated ERK2 activity and increased transcriptional activity of a Gal4-Elk1 protein, which was blocked by overexpression of dominant negative mutants and kinase inhibitors and stimulated by expression of B-Raf. The MAPK kinase inhibitors did not affect Nur77 and Nurr1 mRNA induction but blocked CRH or cAMP-stimulated Nur transcriptional activity. Moreover, MAPK stimulated phosphorylation and transactivation of Nur77. The functional impact of these pathways was confirmed at the POMC promoter. In conclusion, in AtT-20 corticotrophs the CRH/cAMP signaling that leads to Nur77/Nurr1 mRNA induction and transcriptional activation, and thus POMC expression, is dependent on protein kinase A and involves calcium/calmodulin kinase II (Nur induction/activation) and MAPK calcium-dependent and -independent (Nur phosphorylation-activation) pathways.

Descripción: Calcium signals participate in a large variety of physiological processes. In many instances, they involve calcium entry through inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), which are usually organized in clusters. Recent high-resolution optical experiments by Smith & Parker have provided new information on Ca2+ release from clustered IP3Rs. In the present paper, we use the model recently introduced by Solovey & Ponce Dawson to determine how the distribution of the number of IP3Rs that become open during a localized release event may change by the presence of Ca2+ buffers, substances that react with Ca2+, altering its concentration and transport properties. We then discuss how buffer properties could be extracted from the observation of local signals. © 2010 The Royal Society.

Descripción: In silico analyses have revealed a conserved protein domain (CHDL) widely present in bacteria that has significant structural similarity to eukaryotic cadherins. A CHDL domain was shown to be present in RapA, a protein that is involved in autoaggregation of Rhizobium cells, biofilm formation, and adhesion to plant roots as shown by us and others. Structural similarity to cadherins suggested calcium-dependent oligomerization of CHDL domains as a mechanistic basis for RapA action. Here we show by circular dichroism spectroscopy, light scattering, isothermal titration calorimetry, and other methods that RapA2 from Rhizobium leguminosarum indeed exhibits a cadherin-like β-sheet conformation and that its proper folding and stability are dependent on the binding of one calcium ion per protein molecule. By further in silico analysis we also reveal that RapA2 consists of two CHDL domains and expand the range of CHDLcontaining proteins in bacteria and archaea. However, light scattering assays at various concentrations of added calcium revealed that RapA2 formed neither homo-oligomers nor hetero-oligomers with RapB (a distinct CHDL protein), indicating that RapA2 does not mediate cellular interactions through a cadherin-like mechanism. Instead, we demonstrate that RapA2 interacts specifically with the acidic exopolysaccharides (EPSs) produced by R. leguminosarum in a calcium-dependent manner, sustaining a role of these proteins in the development of the biofilm matrix made of EPS. Because EPS binding by RapA2 can only be attributed to its two CHDL domains, we propose that RapA2 is a calcium-dependent lectin and thatCHDLdomains in various bacterial and archaeal proteins confer carbohydrate binding activity to these proteins. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: In the mature cochlea, inner hair cells (IHCs) transduce acoustic signals into receptor potentials, communicating to the brain by synaptic contacts with afferent fibers. Before the onset of hearing, a transient efferent innervation is found on IHCs, mediated by a nicotinic cholinergic receptor that may contain both α9 and α10 subunits. Calcium influx through that receptor activates calcium-dependent (SK2-containing) potassium channels. This inhibitory synapse is thought to disappear after the onset of hearing [after postnatal day 12 (P12)]. We documented this developmental transition using whole-cell recordings from IHCs in apical turns of the rat organ of Corti. Acetylcholine elicited ionic currents in 88-100% of IHCs between P3 and P14, but in only 1 of 11 IHCs at P16-P22. Potassium depolarization of efferent terminals caused IPSCs in 67% of IHCs at P3, in 100% at P7-P9, in 93% at P10-P12, but in only 40% at P13-P14 and in none of the IHCs tested between P16 and P22. Earlier work had shown by in situ hybridization that α9 mRNA is expressed in adult IHCs but that α10 mRNA disappears after the onset of hearing. In the present study, antibodies to α10 and to the associated calcium-dependent (SK2) potassium channel showed a similar developmental loss. The correlated expression of these gene products with functional innervation suggests that Alpha10 and SK2, but not Alpha9, are regulated by synaptic activity. Furthermore, this developmental knock-out of α10, but not α9, supports the hypothesis that functional nicotinic acetylcholine receptors in hair cells are heteromers containing both these subunits.

Descripción: Transmission at the mouse neuromuscular junction normally relies on P/Q-type channels, but became jointly dependent on both N-and R-type Ca2+ channels when the P/Q-type channel α1A subunit was deleted. R-type channels lay close to Ca2+ sensors for exocytosis and IK(Ca) channel activation, like the P/Q-type channels they replaced. In contrast, N-type channels were less well localized, but abundant enough to influence secretion strongly, particularly when action potentials were prolonged. Our data suggested that active zone structures may select among multiple Ca2+ channels in the hierarchy P/Q>R>N. The α1A-/- neuromuscular junction displayed several other differences from wild-type: lowered quantal content but greater ability to withstand reductions in the Ca2+/Mg2+ ratio, and little or no paired-pulse facilitation, the latter findings possibly reflecting compensatory mechanisms at individual release sites. Changes in presynaptic function were also associated with a significant reduction in the size of postsynaptic acetylcholine receptor clusters.

Descripción: 1. The effects of the voltage-dependent calcium channel (VDCC) blockers ω-agatoxin IVA (ω-AgaIVA), ω-conotoxin GVIA (ω-CgTx), ω-conotoxin MVIIC (ω-MVIIC) and ω-conotoxin MVIID (ω-MVIID) were evaluated on transmitter release in the mouse diaphragm preparation. The effects of ω-AgaIVA and ω-MVIIC were also evaluated on the perineurial calcium and calcium-dependent potassium currents, I(ca), and I(K(Ca)), respectively, in the mouse levator auris preparation. 2. The P- and Q-type VDCC blocker ω-AgaIVA (100 nM) and P- Q- and N-type channel blockers ω-MVIIC (1 μM) and ω-MVIID (3 μM) strongly reduced transmitter release (> 80-90% blockade) whereas the selective N-type channel blocker ω-CgTx (5 μM) was ineffective. 3. The process of release was much more sensitive to ω-MVIIC (IC50 = 39 nM) than to ω-MVIID (IC50 = 1.4 μM). After almost completely blocking transmitter release (quantal content ~0.3% of its control value) with 3 μM ω-MVIIC, elevating the external [Ca2+] from 2 to 10 mM induced an increase of ~20 fold on the quantal content of the endplate potential (e.p.p.) (from 0.2 ± 0.04 to 4.8 ± 1.4). 4. Nerve-evoked transmitter release in a low Ca2+-high Mg2+ medium (low release probability, quantal content = 2 ± 0.1) had the same sensitivity to ω-AgaIVA (IC50 = 16.8 nM) as that in normal saline solutions. In addition, K+-evoked transmitter release was also highly sensitive to the action of this toxin (IC50 = 11.5 nM; 100 nM > 95% blockade). The action of ω-AgaIVA on transmitter release could be reversed by toxin washout if the experiments were carried out at 31-33°C. Conversely, the effect of ω-AgaIVA persisted even after two hours of toxin washout at room temperature. 5. Both the calcium and calcium-dependent potassium presynaptic currents, I(ca), and I(K(Ca)), respectively, were highly sensitive to low concentrations (10-30 nM) of ω-AgaIVA. The I(ca), and the I(K(Ca)) were also strongly reduced by 1 μM ω-MVIIC. The most marked difference between the action of these two toxins was the long incubation times required to achieve maximal effects with ω-MVIIC. 6. In summary these results provide more evidence that synaptic transmission at the mammalian neuromuscular junction is mediated by Ca2+ entry through P- and/or Q-type calcium channels.

Descripción: The efferent synaptic specialization of hair cells includes a near-membrane synaptic cistern, whose presence suggests a role for internal calcium stores in cholinergic inhibition. Calcium release channels from internal stores include 'ryanodine receptors', whose participation is usually demonstrated by sensitivity to the eponymous plant alkaloid, ryanodine. However, use of this and other store-active compounds on hair cells could be confounded by the unusual pharmacology of the α9α10-containing hair cell nicotinic cholinergic receptor (nAChR), which has been shown to be antagonized by a broad spectrum of compounds. Surprisingly, we found that ryanodine, rather than antagonizing, is a positive modulator of the α9α10 nAChR expressed in Xenopus oocytes, the first such compound to be found. The effect of ryanodine was to increase the apparent affinity and efficacy for acetylcholine (ACh). Correspondingly, ACh-evoked currents through the isolated cholinergic receptors of inner hair cells in excised mouse cochleas were approximately doubled by 200 μM ryanodine, a concentration that inhibits gating of the ryanodine receptor itself. This unusual positive modulation was not unique to the mammalian receptor. The response to ACh of chicken 'short' hair cells likewise was enhanced in the presence of 100 μM ryanodine. This facilitatory effect on current through the AChR could enhance brief (∼1 s) activation of associated calcium-dependent K+ (SK) channels in both chicken short hair cells and rat outer hair cells. This novel effect of ryanodine provides new opportunities for the design of compounds that potentiate α9α10- mediated responses and for potential inner ear therapeutics based on this interaction. © 2007 Association for Research in Otolaryngology.

Descripción: Pineal hormone melatonin (N-acetyl-5-methoxytryptamine) is thought to modulate the calcium/calmodulin signaling pathway either by changing intracellular Ca2+ concentration via activation of its G-protein-coupled membrane receptors, or through a direct interaction with calmodulin (CaM). The present work studies the direct interaction of melatonin with intact calcium-saturated CaM both experimentally, by fluorescence and nuclear magnetic resonance spectroscopies, and theoretically, by molecular dynamics simulations. The analysis of the experimental data shows that the interaction is calcium-dependent. The affinity, as obtained from monitoring 15N and 1H chemical shift changes for a melatonin titration, is weak (in the millimolar range) and comparable for the N- and C-terminal domains. Partial replacement of diamagnetic Ca2+ by paramagnetic Tb3+ allowed the measurement of interdomain NMR pseudocontact shifts and residual dipolar couplings, indicating that each domain movement in the complex is not correlated with the other one. Molecular dynamics simulations allow us to follow the dynamics of melatonin in the binding pocket of CaM. Overall, this study provides an example of how a combination of experimental and theoretical approaches can shed light on a weakly interacting system of biological and pharmacological significance.

Descripción: A new species, Geastrum episcopale, is described from Argentina. Detailed descriptions and illustrations of fresh and dry basidiomata are provided along with photographs of microscopic elements, such as spores, capillitium, calcium oxalate crystals, and exo- and endoperidium surfaces. This fungus is typical of the genus in its morphology and ecology but clearly differs from every known species. This species can be distinguished easily by the red-violet (to magenta) exoperidium and the lilac endoperidium. Specimens were collected on wet soil composed of a superficial layer of ca. 5 cm leaf litter and 15 cm of particulate and melanized lignocellulosic material (pH 6.5) under Eugenia uruguayensis and Nectandra sp. © 2009 by The Mycological Society of American.

Descripción: Accurate characterization of the molecular mechanisms of the action of ligands is an extremely important issue for their appropriate research, pharmacological, and therapeutic uses. In view of this fact, the aim of the present work was to investigate the mechanisms involved in the actions of mepyramine at the guinea pig H1 receptor stably expressed in Chinese hamster ovary cells. We found that mepyramine is able to decrease the basal constitutive activity of the guinea pig H1 receptor, to bind with high affinity to a Gq/11 protein-coupled form of the receptor and to promote a G protein-coupled inactive state of the H1 receptor that interferes with the Gq/11-mediated signaling of the endogenously expressed ATP receptor, as predicted by the Cubic Ternary Complex Model of receptor occupancy. The effect of mepyramine on ATP-induced signaling was specifically neutralized by Gα11 overexpression, indicating that mepyramine is able to reduce G protein availability for other non-related receptors associated with the same signaling pathway. Finally, we found a loss of mepyramine efficacy in decreasing basal levels of intracellular calcium at high Gα11 expression levels, which can be theoretically explained in terms of high H1 receptor constitutive activity. The whole of the present work sheds new light on H1 receptor pharmacology and the mechanisms H1 receptor inverse agonists could use to exert their observed negative efficacy.

Descripción: It is generally accepted that the immediately releasable pool is a group of readily releasable vesicles that are closely associated with voltage dependent Ca2+ channels. We have previously shown that exocytosis of this pool is specifically coupled to P/Q Ca2+ current. Accordingly, in the present work we found that the Ca2+ current flowing through P/Q-type Ca2+ channels is 8 times more effective at inducing exocytosis in response to short stimuli than the current carried by L-type channels. To investigate the mechanism that underlies the coupling between the immediately releasable pool and P/Q-type channels we transiently expressed in mouse chromaffin cells peptides corresponding to the synaptic protein interaction site of Cav2.2 to competitively uncouple P/Q-type channels from the secretory vesicle release complex. This treatment reduced the efficiency of Ca2+ current to induce exocytosis to similar values as direct inhibition of P/Q-type channels via ω-agatoxin-IVA. In addition, the same treatment markedly reduced immediately releasable pool exocytosis, but did not affect the exocytosis provoked by sustained electric or high K+ stimulation. Together, our results indicate that the synaptic protein interaction site is a crucial factor for the establishment of the functional coupling between immediately releasable pool vesicles and P/Q-type Ca2+ channels. © 2013 Álvarez et al.