High Extracellular Ca2+ Stimulates Ca2+-Activated Cl− Currents in Frog Parathyroid Cells through the Mediation of Arachidonic Acid Cascade

Elevation of extracellular Ca2+ concentration induces intracellular Ca2+ signaling in parathyroid cells. The response is due to stimulation of the phospholipase C/Ca2+ pathways, but the direct mechanism responsible for the rise of intracellular Ca2+ concentration has remained elusive. Here, we describe the electrophysiological property associated with intracellular Ca2+ signaling in frog parathyroid cells and show that Ca2+-activated Cl− channels are activated by intracellular Ca2+ increase through an inositol 1,4,5-trisphophate (IP3)-independent pathway. High extracellular Ca2+ induced an outwardly-rectifying conductance in a dose-dependent manner (EC50∼6 mM). The conductance was composed of an instantaneous time-independent component and a slowly activating time-dependent component and displayed a deactivating inward tail current. Extracellular Ca2+-induced and Ca2+ dialysis-induced currents reversed at the equilibrium potential of Cl− and were inhibited by niflumic acid (a specific blocker of Ca2+-activated Cl− channel). Gramicidin-perforated whole-cell recording displayed the shift of the reversal potential in extracellular Ca2+-induced current, suggesting the change of intracellular Cl− concentration in a few minutes. Extracellular Ca2+-induced currents displayed a moderate dependency on guanosine triphosphate (GTP). All blockers for phospholipase C, diacylglycerol (DAG) lipase, monoacylglycerol (MAG) lipase and lipoxygenase inhibited extracellular Ca2+-induced current. IP3 dialysis failed to induce conductance increase, but 2-arachidonoylglycerol (2-AG), arachidonic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HPETE) dialysis increased the conductance identical to extracellular Ca2+-induced conductance. These results indicate that high extracellular Ca2+ raises intracellular Ca2+ concentration through the DAG lipase/lipoxygenase pathway, resulting in the activation of Cl− conductance

Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

KCNQ2/KCNQ3 channels are the molecular correlates of the neuronal M-channels, which play a major role in the control of neuronal excitability. Notably, they differ from homomeric KCNQ2 channels in their distribution pattern within neurons, with unique expression of KCNQ2 in axons and nerve terminals. Here, combined reciprocal coimmunoprecipitation and two-electrode voltage clamp analyses in Xenopus oocytes revealed a strong association of syntaxin 1A, a major component of the exocytotic SNARE complex, with KCNQ2 homomeric channels resulting in a ∼2-fold reduction in macroscopic conductance and ∼2-fold slower activation kinetics. Remarkably, the interaction of KCNQ2/Q3 heteromeric channels with syntaxin 1A was significantly weaker and KCNQ3 homomeric channels were practically resistant to syntaxin 1A. Analysis of different KCNQ2 and KCNQ3 chimeras and deletion mutants combined with in-vitro binding analysis pinpointed a crucial C-terminal syntaxin 1A-association domain in KCNQ2. Pull-down and coimmunoprecipitation analyses in hippocampal and cortical synaptosomes demonstrated a physical interaction of brain KCNQ2 with syntaxin 1A, and confocal immunofluorescence microscopy showed high colocalization of KCNQ2 and syntaxin 1A at presynaptic varicosities. The selective interaction of syntaxin 1A with KCNQ2, combined with a numerical simulation of syntaxin 1A's impact in a firing-neuron model, suggest that syntaxin 1A's interaction is targeted at regulating KCNQ2 channels to fine-tune presynaptic transmitter release, without interfering with the function of KCNQ2/3 channels in neuronal firing frequency adaptation

Tripartite interactions of PKA catalytic subunit and C-terminal domains of cardiac Ca(2+) channel may modulate its β-adrenergic regulation

BACKGROUND: The β-adrenergic augmentation of cardiac contraction, by increasing the conductivity of L-type voltage-gated CaV1.2 channels, is of great physiological and pathophysiological importance. Stimulation of β-adrenergic receptors (βAR) activates protein kinase A (PKA) through separation of regulatory (PKAR) from catalytic (PKAC) subunits. Free PKAC phosphorylates the inhibitory protein Rad, leading to increased Ca2+ influx. In cardiomyocytes, the core subunit of CaV1.2, CaV1.2α1, exists in two forms: full-length or truncated (lacking the distal C-terminus (dCT)). Signaling efficiency is believed to emanate from protein interactions within multimolecular complexes, such as anchoring PKA (via PKAR) to CaV1.2α1 by A-kinase anchoring proteins (AKAPs). However, AKAPs are inessential for βAR regulation of CaV1.2 in heterologous models, and their role in cardiomyocytes also remains unclear. RESULTS: We show that PKAC interacts with CaV1.2α1 in heart and a heterologous model, independently of Rad, PKAR, or AKAPs. Studies with peptide array assays and purified recombinant proteins demonstrate direct binding of PKAC to two domains in CaV1.2α1-CT: the proximal and distal C-terminal regulatory domains (PCRD and DCRD), which also interact with each other. Data indicate both partial competition and possible simultaneous interaction of PCRD and DCRD with PKAC. The βAR regulation of CaV1.2α1 lacking dCT (which harbors DCRD) was preserved, but subtly altered, in a heterologous model, the Xenopus oocyte. CONCLUSIONS: We discover direct interactions between PKAC and two domains in CaV1.2α1. We propose that these tripartite interactions, if present in vivo, may participate in organizing the multimolecular signaling complex and finetuning the βAR effect in cardiomyocytes

G-protein signaling: back to the future

Heterotrimeric G-proteins are intracellular partners of G-protein-coupled receptors (GPCRs). GPCRs act on inactive Gα·GDP/Gβγ heterotrimers to promote GDP release and GTP binding, resulting in liberation of Gα from Gβγ. Gα·GTP and Gβγ target effectors including adenylyl cyclases, phospholipases and ion channels. Signaling is terminated by intrinsic GTPase activity of Gα and heterotrimer reformation — a cycle accelerated by ‘regulators of G-protein signaling’ (RGS proteins). Recent studies have identified several unconventional G-protein signaling pathways that diverge from this standard model. Whereas phospholipase C (PLC) β is activated by Gαq and Gβγ, novel PLC isoforms are regulated by both heterotrimeric and Ras-superfamily G-proteins. An Arabidopsis protein has been discovered containing both GPCR and RGS domains within the same protein. Most surprisingly, a receptor-independent Gα nucleotide cycle that regulates cell division has been delineated in both Caenorhabditis elegans and Drosophila melanogaster. Here, we revisit classical heterotrimeric G-protein signaling and explore these new, non-canonical G-protein signaling pathways

Peculiar Behaviour of the Manicouagan 3 Dam\u27s Core

The presence of some horizontal or subhorizontal zones of weakness (cracks or decompressed layers) led to high pressure development in the downstream part of the relatively wide till core of Manic 3 dam. A short description of the investigation works to determine the location of the weakness zones and an analysis of the causes which produced these zones are presented in this paper

Hydraulic efficiency of the Manicouagan-3 cutoff

The hydraulic efficiency of a double-wall cutoff built through 126 m (420 ft) of pervious alluvial deposit filling a V-shaped canyon and under a 107 m (350 ft) high earthfill dam is discussed. The cutoff walls are made up of cast-in-place concrete interlocking piles and panels and are intended primarily to minimize the danger of piping, by controlling the pore pressures in the downstream zone of the dam. The behaviour of the cutoff, monitored by a comprehensive instrumentation program, indicated after [Formula: see text] years a relatively high hydraulic efficiency (over 92%). During this period the cutoff has exhibited a very slight decrease in its efficiency (less than 1%), probably due to leaching of the grout at the rock-wall contact in the grout curtain, and probably also due to some removal of the bentonite from between the piles and panels. The analysis of the efficiency of each wall considered individually (using Casagrande's latest recommendation) indicates a lower efficiency for the upstream wall as compared to that of the downstream wall, probably because of the migration of bentonite particles downstream under the hydraulic gradient. The double-wall cutoff exhibits an efficiency about 18–30% higher than that for a similar single-wall cutoff. </jats:p

Caractéristiques de compressibilité des argiles du complexe Nottaway – Broadback – Rupert (Baie James)

The geological and geotechnical investigations carried out in the southeast area of James Bay, have revealed the presence of two types of clay, a proglacial clay of lacustrine origin underlying a postglacial marine clay. The two deposits, which are characterized by a high sensitivity, a low shear strength and a large compressibility, belong to the normally consolidated type of clay from a geological point of view. However, the clays exhibit an important 'quasi preconsolidation' due to the delayed consolidation phenomenon.It is why the establishment of the compression characteristics of these clays has a special importance to predict their behavior under the load of the projected structures.Considering the large number of structures, the extent of the area involved and the access difficulties to the sites, the soil investigations had to be limited to the use of light equipment and the realization of simple tests, the undisturbed sampling being restricted to a few boreholes on each site.The aim of the present paper is to establish some correlations between certain, simple and easy to determine, characteristics of the clays and their compression behavior, to allow settlement evaluation.The results obtained show a very good correlation for these clays between the moisture content (W) or the initial void ratio (eo) and the compression ratio (Cc/1+eo). The analysis carried out has disclosed also that these foundations have a higher compressibility index (Cc) than some clays which had been previously tested, confirming at the same time the linear correlation between these two characteristics (W vs. Cc).The analysis carried out to establish a correlation between Pc/Po and the other physical characteristics was less fruitful probably because of the lack of precision in the evaluation of the effective overburden pressure and numerous other factors involved in the determination of the preconsolidation pressure. </jats:p

Behaviour of instrumented Hydro-Qu\ue9bec Dams during the Saguenay Earthquake

Peer reviewed: YesNRC publication: Ye