Demonstration of Binding of Neuronal Calcium Sensor-1 to the Ca(v)2.1 P/Q-Type Calcium Channel

[Image: see text] In neurons, entry of extracellular calcium (Ca(2+)) into synaptic terminals through Ca(v)2.1 (P/Q-type) Ca(2+) channels is the driving force for exocytosis of neurotransmitter-containing synaptic vesicles. This class of Ca(2+) channel is, therefore, pivotal during normal neurotransmission in higher organisms. In response to channel opening and Ca(2+) influx, specific Ca(2+)-binding proteins associate with cytoplasmic regulatory domains of the P/Q channel to modulate subsequent channel opening. Channel modulation in this way influences synaptic plasticity with consequences for higher-level processes such as learning and memory acquisition. The ubiquitous Ca(2+)-sensing protein calmodulin (CaM) regulates the activity of all types of mammalian voltage-gated Ca(2+) channels, including the P/Q class, by direct binding to specific regulatory motifs. More recently, experimental evidence has highlighted a role for additional Ca(2+)-binding proteins, particularly of the CaBP and NCS families in the regulation of P/Q channels. NCS-1 is a protein found from yeast to humans and that regulates a diverse number of cellular functions. Physiological and genetic evidence indicates that NCS-1 regulates P/Q channel activity, including calcium-dependent facilitation, although a direct physical association between the proteins has yet to be demonstrated. In this study, we aimed to determine if there is a direct interaction between NCS-1 and the C-terminal cytoplasmic tail of the Ca(v)2.1 α-subunit. Using distinct but complementary approaches, including in vitro binding of bacterially expressed recombinant proteins, fluorescence spectrophotometry, isothermal titration calorimetry, nuclear magnetic resonance, and expression of fluorescently tagged proteins in mammalian cells, we show direct binding and demonstrate that CaM can compete for it. We speculate about how NCS-1/Ca(v)2.1 association might add to the complexity of calcium channel regulation mediated by other known calcium-sensing proteins and how this might help to fine-tune neurotransmission in the mammalian central nervous system

Chronic and acute alterations in the functional levels of Frequenins 1 and 2 reveal their roles in synaptic transmission and axon terminal morphology

Frequenin (Frq) and its mammalian homologue, neuronal calcium sensor 1 (NCS-1), are important calcium-binding proteins which enhance neurotransmitter release and facilitation. Here, we report the discovery of a second Frq-encoding gene (frq2) in Drosophila. The temporal and spatial expression patterns of the two genes are very similar, and the proteins they encode, Frq1 and Frq2, are 95% identical in amino acid sequence. Frq1 is more abundant than Frq2, and is most highly expressed in larva. Loss-of-function phenotypes were studied using dominant negative peptides to prevent Frq target binding, RNAi to reduce gene transcription, or both methods. To discriminate chronic from acute loss-of-function effects, we compared the effects of transgenic expression and forward-filling the dominant-negative peptide into presynaptic terminals. In both cases, a 70% reduction in quantal content per bouton occurred, demonstrating that this trait does not result from homeostatic adaptations of the synapse during development. The chronic treatment also produced more synaptic boutons from MNSNb/d-Is motorneurons, but fewer active zones per bouton. By contrast, excess-of-function conditions yielded a 1.4- to 2-fold increase in quantal content and fewer boutons in the same motorneuron. These synaptic effects resulted in behavioural changes in the Buridan locomotion assay, showing that walking speed is dependent on Frq activity in the nervous system. All the effects were identical for both Frqs, and consistent with excess- and loss-of-function genotypes. We conclude that Frqs have two distinct functions: one in neurotransmission, regulating the probability of release per synapse, and another in axonal growth and bouton formation. © The Authors (2007).Peer Reviewe

Multiple roles for frequenin/NCS-1 in synaptic function and development

The calcium-binding protein frequenin (Frq), discovered in the fruit fly Drosophila, and its mammalian homologue neuronal calcium sensor 1 (NCS-1) have been reported to affect several aspects of synaptic transmission, including basal levels of neurotransmission and short- and long-term synaptic plasticities. However, discrepant reports leave doubts about the functional roles of these conserved proteins. In this review, we attempt to resolve some of these seemingly contradictory reports. We discuss how stimulation protocols, sources of calcium (voltage-gated channels versus internal stores), and expression patterns (presynaptic versus postsynaptic) of Frq may result in the activation of various protein targets, leading to different synaptic effects. In addition, the potential interactions of Frq's C-terminal and N-terminal domains with other proteins are discussed. Frq also has a role in regulating neurite outgrowth, axonal regeneration, and synaptic development. We examine whether the effects of Frq on neurotransmitter release and neurite outgrowth are distinct or interrelated through homeostatic mechanisms. Learning and memory are affected by manipulations of Frq probably through changes in synaptic transmission and neurite outgrowth, raising the possibility that Frq may be implicated in human pathological conditions, including schizophrenia, bipolar disorder, and X-linked mental retardation. © Springer Science+Business Media, LLC 2012.Peer Reviewe

Frequenin/NCS-1 and the Ca2+-channel α1-subunit co-regulate synaptic transmission and nerve-terminal growth

Drosophila Frequenin (Frq) and its mammalian and worm homologue, NCS-1, are Ca2+-binding proteins involved in neurotransmission. Using site-specific recombination in Drosophila, we created two deletions that removed the entire frq1 gene and part of the frq2 gene, resulting in no detectable Frq protein. Frq-null mutants were viable, but had defects in larval locomotion, deficient synaptic transmission, impaired Ca2+ entry and enhanced nerve-terminal growth. The impaired Ca2+ entry was sufficient to account for reduced neurotransmitter release. We hypothesized that Frq either modulates Ca2+ channels, or that it regulates the PI4Kβ pathway as described in other organisms. To determine whether Frq interacts with PI4K. with consequent effects on Ca2+ channels, we first characterized a PI4Kβ-null mutant and found that PI4Kβ was dispensable for synaptic transmission and nerve-terminal growth. Frq gain-of-function phenotypes remained present in a PI4Kβ-null background. We conclude that the effects of Frq are not due to an interaction with PI4Kβ. Using flies that were trans-heterozygous for a null frq allele and a null cacophony (encoding the α1-subunit of voltage-gated Ca2+ channels) allele, we show a synergistic effect between these proteins in neurotransmitter release. Gain-of-function Frq phenotypes were rescued by a hypomorphic cacophony mutation. Overall, Frq modulates Ca2+ entry through a functional interaction with the voltage-gated Ca2+-channel subunit; this interaction regulates neurotransmission and nerveterminal growth.Peer Reviewe

Quantitative proteomic analysis of amniocytes reveals potentially dysregulated molecular networks in Down syndrome

Abstract Background Down syndrome (DS), caused by an extra copy of chromosome 21, affects 1 in 750 live births and is characterized by cognitive impairment and a constellation of congenital defects. Currently, little is known about the molecular pathogenesis and no direct genotype-phenotype relationship has yet been confirmed. Since DS amniocytes are expected to have a distinct biological behaviour compared to normal amniocytes, we hypothesize that relative quantification of proteins produced from trisomy and euploid (chromosomally normal) amniocytes will reveal dysregulated molecular pathways. Results Chromosomally normal- and Trisomy 21-amniocytes were quantitatively analyzed by using Stable Isotope Labeling of Amino acids in Cell culture and tandem mass spectrometry. A total of 4919 unique proteins were identified from the supernatant and cell lysate proteome. More specifically, 4548 unique proteins were identified from the lysate, and 91% of these proteins were quantified based on MS/MS spectra ratios of peptides containing isotope-labeled amino acids. A total of 904 proteins showed significant differential expression and were involved in 25 molecular pathways, each containing a minimum of 16 proteins. Sixty of these proteins consistently showed aberrant expression from trisomy 21 affected amniocytes, indicating their potential role in DS pathogenesis. Nine proteins were analyzed with a multiplex selected reaction monitoring assay in an independent set of Trisomy 21-amniocyte samples and two of them (SOD1 and NES) showed a consistent differential expression. Conclusions The most extensive proteome of amniocytes and amniotic fluid has been generated and differentially expressed proteins from amniocytes with Trisomy 21 revealed molecular pathways that seem to be most significantly affected by the presence of an extra copy of chromosome 21

Mars Global Surveyor Thermal Emission Spectrometer (TES) Observations: Atmospheric Temperatures During Aerobraking and Science Phasing

Between September 1997, when the Mars Global Surveyor spacecraft arrived at Mars, and September 1998 when the final aerobraking phase of the mission began, the Thermal Emission Spectrometer (TES) has acquired an extensive data set spanning approximately half of a Martian year. Nadir-viewing spectral measurements from this data set within the 15-micrometers CO2 absorption band are inverted to obtain atmospheric temperature profiles from the surface up to about the 0.1 mbar level. The computational procedure used to retrieve the temperatures is presented. Mean meridional cross sections of thermal structure are calculated for periods of time near northern hemisphere fall equinox, winter solstice, and spring equinox, as well as for a time interval immediately following the onset of the Noachis Terra dust storm. Gradient thermal wind cross sections are calculated from the thermal structure. Regions of possible wave activity are identified using cross sections of rms temperature deviations from the mean. Results from both near-equinox periods show some hemispheric asymmetry with peak eastward thermal winds in the north about twice the magnitude of those in the south. The results near solstice show an intense circumpolar vortex at high northern latitudes and waves associated with the vortex jet core. Warming of the atmosphere aloft at mid-northern latitudes suggests the presence of a strong cross-equatorial Hadley circulation. Although the Noachis dust storm did not become global in scale, strong perturbations to the atmospheric structure are found, including an enhanced temperature maximum aloft at high northern latitudes resulting from intensification of the Hadley circulation. TES results for the various seasonal conditions are compared with published results from Mars general circulation models, and generally good qualitative agreement is found