Structural and Functional Deficits in a Neuronal Calcium Sensor-1 Mutant Identified in a Case of Autistic Spectrum Disorder

The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Liverpool, United Kingdom.
PLoS ONE (Impact Factor: 3.23). 05/2010; 5(5):e10534. DOI: 10.1371/journal.pone.0010534
Source: PubMed

ABSTRACT Neuronal calcium sensor-1 (NCS-1) is a Ca(2+) sensor protein that has been implicated in the regulation of various aspects of neuronal development and neurotransmission. It exerts its effects through interactions with a range of target proteins one of which is interleukin receptor accessory protein like-1 (IL1RAPL1) protein. Mutations in IL1RAPL1 have recently been associated with autism spectrum disorders and a missense mutation (R102Q) on NCS-1 has been found in one individual with autism. We have examined the effect of this mutation on the structure and function of NCS-1. From use of NMR spectroscopy, it appeared that the R102Q affected the structure of the protein particularly with an increase in the extent of conformational exchange in the C-terminus of the protein. Despite this change NCS-1(R102Q) did not show changes in its affinity for Ca(2+) or binding to IL1RAPL1 and its intracellular localisation was unaffected. Assessment of NCS-1 dynamics indicated that it could rapidly cycle between cytosolic and membrane pools and that the cycling onto the plasma membrane was specifically changed in NCS-1(R102Q) with the loss of a Ca(2+) -dependent component. From these data we speculate that impairment of the normal cycling of NCS-1 by the R102Q mutation could have subtle effects on neuronal signalling and physiology in the developing and adult brain.

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Available from: Lee Philip Haynes, Sep 28, 2015
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    • "In humans , the interaction is conserved , suggesting a potential role in pathology . IL1RAPL1 and NCS - 1 are implicated in X - linked mental retardation and autism ( Handley et al . , 2010 ; Pavlowsky et al . , 2010 ) . The autism - related missense ( R102Q ) mutation in NCS - 1 abolishes Ca 2+ dependence , owing to a weakened conformational stability of its C - terminus that affects the cytosolic - to - membrane cycling of NCS - 1 ( Heidarsson et al . , 2012 ) . Because Frq2 – NCS - 1 is a negative regulator of Ric8 , it "
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    ABSTRACT: TThe conserved Ca(2+)-binding protein Frequenin (homolog of the mammalian NCS-1, neural calcium sensor) is involved in pathologies that result from abnormal synapse number and probability of neurotransmitter release per synapse. Both synaptic features are likely to be co-regulated but the intervening mechanisms remain poorly understood. We show here that Drosophila Ric8a (a homolog of mammalian synembryn, which is also known as Ric8a), a receptor-independent activator of G protein complexes, binds to Frq2 but not to the virtually identical homolog Frq1. Based on crystallographic data on Frq2 and site-directed mutagenesis on Frq1, the differential amino acids R94 and T138 account for this specificity. Human NCS-1 and Ric8a reproduce the binding and maintain the structural requirements at these key positions. Drosophila Ric8a and Gαs regulate synapse number and neurotransmitter release, and both are functionally linked to Frq2. Frq2 negatively regulates Ric8a to control synapse number. However, the regulation of neurotransmitter release by Ric8a is independent of Frq2 binding. Thus, the antagonistic regulation of these two synaptic properties shares a common pathway, Frq2-Ric8a-Gαs, which diverges downstream. These mechanisms expose the Frq2-Ric8a interacting surface as a potential pharmacological target for NCS-1-related diseases and provide key data towards the corresponding drug design.
    Journal of Cell Science 07/2014; 127(19)::4246-59. DOI:10.1242/jcs.152603 · 5.43 Impact Factor
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    • "NCS-1, the primordial member of the NCS family, has orthologues from Saccharomyces cerevisiae (Frq1) [11] to man and has been implicated in several neuronal functions including regulation of neurotransmitter release [12,13], membrane traffic [14], voltage gated Ca2+ channels [15-17], neuronal development [18,19], synaptic plasticity [20,21] and learning [22,23]. NCS-1 is N-terminally myristoylated which allows its association with the plasma membrane and the trans-Golgi network [7] and it cycles between membrane and cytosolic pools [24]. NCS-1 is known to interact with a wide range of potential target proteins [25,26] including phosphatidylinositol-4-kinase (PI4K) IIIβ [14,27] and its orthologue Pik1 in yeast [11], ARF1 [14,28], interleukin receptor accessory protein like-1 (IL1RAPL1) [29], TRPC5 channels [18], InsP(3) receptors [30] and dopamine D2 and D3 receptors [31]. "
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    ABSTRACT: Intracellular Ca2+ regulates many aspects of neuronal function through Ca2+ binding to EF hand-containing Ca2+ sensors that in turn bind target proteins to regulate their function. Amongst the sensors are the neuronal calcium sensor (NCS) family of proteins that are involved in multiple neuronal signalling pathways. Each NCS protein has specific and overlapping targets and physiological functions and specificity is likely to be determined by structural features within the proteins. Common to the NCS proteins is the exposure of a hydrophobic groove, allowing target binding in the Ca2+-loaded form. Structural analysis of NCS protein complexes with target peptides has indicated common and distinct aspects of target protein interaction. Two key differences between NCS proteins are the size of the hydrophobic groove that is exposed for interaction and the role of their non-conserved C-terminal tails. We characterised the role of NCS-1 in a temperature-dependent locomotion assay in C. elegans and identified a distinct phenotype in the ncs-1 null in which the worms do not show reduced locomotion at actually elevated temperature. Using rescue of this phenotype we showed that NCS-1 functions in AIY neurons. Structure/function analysis introducing single or double mutations within the hydrophobic groove based on information from characterised target complexes established that both N- and C-terminal pockets of the groove are functionally important and that deletion of the C-terminal tail of NCS-1 did not impair its ability to rescue. The current work has allowed physiological assessment of suggestions from structural studies on the key structural features that underlie the interaction of NCS-1 with its target proteins. The results are consistent with the notion that full length of the hydrophobic groove is required for the regulatory interactions underlying NCS-1 function whereas the C-terminal tail of NCS-1 is not essential. This has allowed discrimination between two potential modes of interaction of NCS-1 with its targets.
    Molecular Brain 08/2013; 6(1):39. DOI:10.1186/1756-6606-6-39 · 4.90 Impact Factor
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    • "Since the LAMP1–YCaM construct responded to TG treatment, this demonstrates its capacity to detect global increases in cytosolic Ca2+. That these responses exhibited a lag not observed when using cytosolic Ca2+ indicators [50] is consistent with the time taken for diffusion of Ca2+ from release sites at the ER to the lysosome. Our TG-histamine sequential-treatment experiments on intact cells suggests that the cameleon can probably register endolysosomal-specific release events. "
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    ABSTRACT: Distinct spatio-temporal calcium (Ca2+) signalling events regulate fundamental aspects of eukaryotic cell physiology. Complex Ca2+-signals can be driven by release of Ca2+ from intracellular organelles that sequester Ca2+ such as the endoplasmic reticulum (ER) or through the opening of Ca2+-permeable channels in the plasma membrane and influx of extracellular Ca2+. Late endocytic pathway compartments including late-endosomes and lysosomes have recently been observed to sequester Ca2+ to levels comparable with those found within the ER lumen. These organelles harbour ligand gated Ca2+-release channels and evidence indicates that they can operate as Ca2+-signalling platforms. Lysosomes sequester Ca2+ to a greater extent than any other endocytic compartment and signalling from this organelle has been postulated to provide 'trigger' release events that can subsequently elicit more extensive Ca2+-signals from stores including the ER. In order to investigate lysosomal specific Ca2+-signalling a simple method for measuring lysosomal Ca2+-release is essential. In this study we describe the generation and characterisation of a genetically encoded, lysosomally targeted, cameleon sensor which is capable of registering specific Ca2+ release in response to extracellular agonists and intracellular second messengers. This probe represents a novel tool that will permit detailed investigations examining the impact of lysosomal Ca2+-handling on cellular physiology.
    Biochemical Journal 10/2012; 449(Pt 2). DOI:10.1042/BJ20120898 · 4.40 Impact Factor
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