Article

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: 3.53). 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.

0 Bookmarks
 · 
185 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In neurons, entry of extracellular calcium (Ca2+) into synaptic terminals through Cav2.1 (P/Q-type) Ca2+ channels is the driving force for exocytosis of neurotransmitter containing synaptic vesicles. This class of Ca2+-channel is, therefore, pivotal during normal neurotransmission in higher organisms. In response to channel opening and Ca2+-influx, specific Ca2+-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 Ca2+-sensing protein Calmodulin (CaM) regulates the activity of all types of mammalian voltage gated Ca2+ channel, including P/Q class, by direct binding to specific regulatory motifs. More recently, experimental evidence has highlighted a role for additional Ca2+-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 man and which 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 Cav2.1 alpha subunit. Using distinct but complimentary approaches including in vitro binding of bacterially expressed recombinant proteins, fluorescence spectrophotometry, ITC, NMR and expression of fluorescently tagged proteins in mammalian cells we show direct binding and demonstrate that it can be competed for by CaM. We speculate about how NCS-1/Cav2.1 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.
    Biochemistry 09/2014; · 3.38 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ca2 + regulates multiple aspects of neuronal function over varying time scales•The specificity of Ca2 + signalling is determined by the properties of Ca2 + sensors•Differing properties of closely-related Ca2 + sensors contributes to specificity•The structure of Ca2 + sensors determines specificity of target protein interactions.•We discuss three case studies involving calmodulin, NCS proteins and CaBP proteins•The examples illustrate the structural-function relationships of Ca2 + sensors
    Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 11/2014; · 5.30 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Neurodegenerative disorders are strongly linked to protein misfolding, and crucial to their explication is a detailed understanding of the underlying structural rearrangements and pathways that govern the formation of misfolded states. Here we use single-molecule optical tweezers to monitor misfolding reactions of the human neuronal calcium sensor-1, a multispecific EF-hand protein involved in neurotransmitter release and linked to severe neurological diseases. We directly observed two misfolding trajectories leading to distinct kinetically trapped misfolded conformations. Both trajectories originate from an on-pathway intermediate state and compete with native folding in a calcium-dependent manner. The relative probability of the different trajectories could be affected by modulating the relaxation rate of applied force, demonstrating an unprecedented real-time control over the free-energy landscape of a protein. Constant-force experiments in combination with hidden Markov analysis revealed the free-energy landscape of the misfolding transitions under both physiological and pathological calcium concentrations. Remarkably for a calcium sensor, we found that higher calcium concentrations increased the lifetimes of the misfolded conformations, slowing productive folding to the native state. We propose a rugged, multidimensional energy landscape for neuronal calcium sensor-1 and speculate on a direct link between protein misfolding and calcium dysregulation that could play a role in neurodegeneration.
    Proceedings of the National Academy of Sciences 08/2014; · 9.81 Impact Factor

Full-text (2 Sources)

Download
39 Downloads
Available from
May 20, 2014