Mining recent brain proteomic databases for ion channel phosphosite nuggets

Cerda, Oscar; Baek, Je‐Hyun; Trimmer, James S.

doi:10.1085/jgp.201010555

Cited by 22 publications

(29 citation statements)

References 58 publications

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“…Phosphorylation and dephosphorylation are common processes that regulate diverse protein functions, including the gating of plasma membrane channels; a number of reviews have summarized the effects of phosphorylation in voltage-gated channels as well as in NMDA, GABA, TRP and ACh receptors [10; 37; 45; 50]. P2XRs gating is also modulated by phosphorylation and dephosphorylation.…”

Section: Discussionmentioning

confidence: 99%

Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372

Coddou

Sandoval

Castro

et al. 2017

PAIN

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The purinergic P2X2 receptor (P2X2R) is an ATP-gated ion channel widely expressed in the nervous system. Here, we identified a putative Cdk5 phosphorylation site in the full-size variant P2X2aR (372TPKH375), which is absent in the splice variant P2X2bR. We therefore investigated the effects of Cdk5 and its neuronal activator, p35, on P2X2aR function. We found an interaction between P2X2aR and Cdk5/p35 by co-immunofluorescence and co-immunoprecipitation in HEK293 cells. We also found that threonine-phosphorylation was significantly increased in HEK293 cells co-expressing P2X2aR and p35 as compared to cells expressing only P2X2aR. Moreover, P2X2aR-derived peptides encompassing the Cdk5 consensus motif were phosphorylated by Cdk5/p35. Whole-cell patch-clamp recordings indicated a delay in development of use-dependent desensitization (UDD) of P2X2aR but not of P2X2bR in HEK293 cells co-expressing P2X2aR and p35. In Xenopus oocytes, P2X2aRs showed a slower UDD than in HEK293 cells and Cdk5-activation prevented this effect. A similar effect was found in P2X2a/3R heteromeric currents in HEK293 cells. The P2X2aR-T372A mutant was resistant to UDD. In endogenous cells, we observed similar distribution between P2X2R and Cdk5/p35 by co-localization using immunofluorescence in primary culture of nociceptive neurons. Moreover, co-immunoprecipitation experiments showed an interaction between Cdk5 and P2X2R in mouse trigeminal ganglia. Finally, endogenous P2X2aR-mediated currents in PC12 cells, and P2X2/3R mediated increases of intracellular Ca2+ in trigeminal neurons were Cdk5-dependent, since inhibition with roscovitine accelerated the desensitization kinetics of these responses. These results indicate that the P2X2aR is a novel target for Cdk5-mediated phosphorylation, which might play important physiological roles including pain signaling.

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Section: Discussionmentioning

confidence: 99%

Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372

Coddou

Sandoval

Castro

et al. 2017

PAIN

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“…There are several mechanisms by which the expression of ion channel proteins in the human CNS is regulated. First, there is a multitude of discrete isoforms and molecular subunit combinations within each ion channel subclass, each of which has distributional specificity that is controlled developmentally, temporally and adaptively (Abernethy and Soldatov 2002; Ader et al 2008; Angelino and Brenner 2007; Anselmi et al 2007; Catterall and Few 2008; Cerda et al 2011; Chahine et al 2008; Dai et al 2009; Gaudet 2007; Goetz et al 2007; Gotti et al 2009; Hashimoto and Panchenko 2010; Jegla et al 2009; Jensen et al 2008; Kurokawa et al 2009; Levitan 2006; Marionneau et al 2011; Milstein et al 2007; Molina et al 2006; Olsen and Sieghart 2009; Paoletti 2011; Perrais et al 2010; Picton and Fisher 2007; Tseng et al 2007; Vanoye et al 2010; Waxman et al 2000; Yang et al 2009). Second, most ion channels have known alternatively spliced isoforms, offering additional modulatory capacity (Chahine et al 2008; Daniel and Ohman 2009; Honore 2008; Jenkinson 2006; Kurokawa et al 2009; Lin et al 2009; Mohapatra et al 2007; Vazquez and Valverde 2006; Yan et al 2008).…”

Section: Background and Rationale For Our Hypothesis Aims And Methodsmentioning

confidence: 99%

Ion channels and schizophrenia: a gene set-based analytic approach to GWAS data for biological hypothesis testing

et al. 2011

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Schizophrenia is a complex genetic disorder. Gene set-based analytic (GSA) methods have been widely applied for exploratory analyses of large, high-throughput datasets, but less commonly employed for biological hypothesis testing. Our primary hypothesis is that variation in ion channel genes contribute to the genetic susceptibility to schizophrenia. We applied Exploratory Visual Analysis (EVA), one GSA application, to analyze European-American (EA) and African-American (AA) schizophrenia genome-wide association study datasets for statistical enrichment of ion channel gene sets, comparing GSA results derived under three SNP-to-gene mapping strategies: (1) GENIC; (2) 500-Kb; (3) 2.5-Mb and three complimentary SNP-to-gene statistical reduction methods: (1) minimum p value (pMIN); (2) a novel method, proportion of SNPs per Gene with p-values below a pre-defined α-threshold (PROP); and (3) the truncated product method (TPM). In the EA analyses, ion channel gene set(s) were enriched under all mapping and statistical approaches. In the AA analysis, ion channel gene set(s) were significantly enriched under pMIN for all mapping strategies and under PROP for broader mapping strategies. Less extensive enrichment in the AA sample may reflect true ethnic differences in susceptibility, sampling or case ascertainment differences, or higher dimensionality relative to sample size of the AA data. More consistent findings under broader mapping strategies may reflect enhanced power due to increased SNP inclusion, enhanced capture of effects over extended haplotypes or significant contributions from regulatory regions. While extensive pMIN findings may reflect gene size bias, the extent and significance of PROP and TPM findings suggest that common variation at ion channel genes may capture some of the heritability of schizophrenia.

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“…This work has been recently reviewed [ 23 ]. The pore-forming α-subunits of the voltage-gated Na + and Ca 2+ channels were both found to possess between 20 and 25 phosphorylation sites each.…”

Section: Phosphorylationmentioning

confidence: 99%

Applications for Mass Spectrometry in the Study of Ion Channel Structure and Function

Samways

2014

Advances in Experimental Medicine and Biology

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Ion channels are intrinsic membrane proteins that form gated ion-permeable pores across biological membranes. Depending on the type, ion channels exhibit sensitivities to a diverse range of stimuli including changes in membrane potential, binding by diffusible ligands, changes in temperature and direct mechanical force. The purpose of these proteins is to facilitate the passive diffusion of ions down their respective electrochemical gradients into and out of the cell, and between intracellular compartments. In doing so, ion channels can affect transmembrane potentials and regulate the intracellular homeostasis of the important second messenger, Ca(2+). The ion channels of the plasma membrane are of particular clinical interest due to their regulation of cell excitability and cytosolic Ca(2+) levels, and the fact that they are most amenable to manipulation by exogenously applied drugs and toxins. A critical step in improving the pharmacopeia of chemicals available that influence the activity of ion channels is understanding how their three-dimensional structure imparts function. Here, progress has been slow relative to that for soluble protein structures in large part due to the limitations of applying conventional structure determination methods, such as X-ray crystallography, nuclear magnetic resonance imaging, and mass spectrometry, to membrane proteins. Although still an underutilized technique in the assessment of membrane protein structure, recent advances have pushed mass spectrometry to the fore as an important complementary approach to studying the structure and function of ion channels. In addition to revealing the subtle conformational changes in ion channel structure that accompany gating and permeation, mass spectrometry is already being used effectively for identifying tissue-specific posttranslational modifications and mRNA splice variants. Furthermore, the use of mass spectrometry for high-throughput proteomics analysis, which has proven so successful for soluble proteins, is already providing valuable insight into the functional interactions of ion channels within the context of the macromolecular-signaling complexes that they inhabit in vivo. In this chapter, the potential for mass spectrometry as a complementary approach to the study of ion channel structure and function will be reviewed with examples of its application.

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Mining recent brain proteomic databases for ion channel phosphosite nuggets

Cited by 22 publications

References 58 publications

Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372

Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372

Ion channels and schizophrenia: a gene set-based analytic approach to GWAS data for biological hypothesis testing

Applications for Mass Spectrometry in the Study of Ion Channel Structure and Function

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