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Intrinsic dynamics study identifies two amino acids of TIMP-1 critical for its LRP-1-mediated endocytosis in neurons.

ABSTRACT: The tissue inhibitor of metalloproteinases-1 (TIMP-1) exerts inhibitory activity against matrix metalloproteinases and cytokine-like effects. We previously showed that TIMP-1 reduces neurite outgrowth in mouse cortical neurons and that this cytokine-like effect depends on TIMP-1 endocytosis mediated by the low-density lipoprotein receptor-related protein-1 (LRP-1). To gain insight into the interaction between TIMP-1 and LRP-1, we considered conformational changes that occur when a ligand binds to its receptor. TIMP-1 conformational changes have been studied using biomolecular simulations, and our results provide evidence for a hinge region that is critical for the protein movement between the N- and C-terminal TIMP-1 domains. In silico mutants have been proposed on residues F12 and K47, which are located in the hinge region. Biological analyses of these mutants show that F12A or K47A mutation does not alter MMP inhibitory activity but impairs the effect of TIMP-1 on neurite outgrowth. Interestingly, these mutants bind to LRP-1 but are not endocytosed. We conclude that the intrinsic dynamics of TIMP-1 are not involved in its binding to LRP-1 but rather in the initiation of endocytosis and associated biological effects.

SUBMITTER: Verzeaux L

PROVIDER: S-EPMC5511134 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Intrinsic dynamics study identifies two amino acids of TIMP-1 critical for its LRP-1-mediated endocytosis in neurons.

Verzeaux Laurie L Belloy Nicolas N Thevenard-Devy Jessica J Devy Jérôme J Ferracci Géraldine G Martiny Laurent L Dedieu Stéphane S Dauchez Manuel M Emonard Hervé H Etique Nicolas N Devarenne-Charpentier Emmanuelle E

Scientific reports 20170714 1

The tissue inhibitor of metalloproteinases-1 (TIMP-1) exerts inhibitory activity against matrix metalloproteinases and cytokine-like effects. We previously showed that TIMP-1 reduces neurite outgrowth in mouse cortical neurons and that this cytokine-like effect depends on TIMP-1 endocytosis mediated by the low-density lipoprotein receptor-related protein-1 (LRP-1). To gain insight into the interaction between TIMP-1 and LRP-1, we considered conformational changes that occur when a ligand binds t ...[more]

PMID: 28710453

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Project description:KEY POINTS:Neurons in the hypothalamus of the brain which secrete the peptide kisspeptin are important regulators of reproduction, and normal reproductive development. Electrical activity, in the form of action potentials, or spikes, leads to secretion of peptides and neurotransmitters, influencing the activity of downstream neurons; in kisspeptin neurons, this activity is highly irregular, but the mechanism of this is not known. In this study, we show that irregularity depends on the presence of a particular type of potassium ion channel in the membrane, which opens transiently in response to electrical excitation. The results contribute to understanding how kisspeptin neurons generate and time their membrane potential spikes, and how reliable this process is. Improved understanding of the activity of kisspeptin neurons, and how it shapes their secretion of peptides, is expected to lead to better treatment for reproductive dysfunction and disorders of reproductive development. ABSTRACT:Kisspeptin neurons in the hypothalamus are critically involved in reproductive function, via their effect on GnRH neuron activity and consequent gonadotropin release. Kisspeptin neurons show an intrinsic irregularity of firing, but the mechanism of this remains unclear. To address this, we carried out targeted whole-cell patch-clamp recordings of kisspeptin neurons in the arcuate nucleus (Kiss1Arc ), in brain slices isolated from adult male Kiss-Cre:tdTomato mice. Cells fired irregularly in response to constant current stimuli, with a wide range of spike time variability, and prominent subthreshold voltage fluctuations. In voltage clamp, both a persistent sodium (NaP) current and a fast transient (A-type) potassium current were apparent, activating at potentials just below the threshold for spiking. These currents have also previously been described in irregular-spiking cortical interneurons, in which the A-type current, mediated by Kv4 channels, interacts with NaP current to generate complex dynamics of the membrane potential, and irregular firing. In Kiss1Arc neurons, A-type current was blocked by phrixotoxin, a specific blocker of Kv4.2/4.3 channels, and consistent expression of Kv4.2 transcripts was detected by single-cell RT-PCR. In addition, firing irregularity was correlated to the density of A-type current in the membrane. Using conductance injection, we demonstrated that adding Kv4-like potassium conductance (gKv4 ) to a cell produces a striking increase in firing irregularity, and excitability is reduced, while subtracting gKv4 has the opposite effects. Thus, we propose that Kv4 interacting dynamically with NaP is a key determinant of the irregular firing behaviour of Kiss1Arc neurons, shaping their physiological function in gonadotropin release.

Dataset Information

Intrinsic dynamics study identifies two amino acids of TIMP-1 critical for its LRP-1-mediated endocytosis in neurons.

Publications

Intrinsic dynamics study identifies two amino acids of TIMP-1 critical for its LRP-1-mediated endocytosis in neurons.

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