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      Leak K + channel mRNAs in dorsal root ganglia: Relation to inflammation and spontaneous pain behaviour

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          Abstract

          Two pore domain potassium (K2P) channels (KCNKx.x) cause K + leak currents and are major contributors to resting membrane potential. Their roles in dorsal root ganglion (DRG) neurons normally, and in pathological pain models, are poorly understood. Therefore, we examined mRNA levels for 10 K2P channels in L4 and L5 rat DRGs normally, and 1 day and 4 days after unilateral cutaneous inflammation, induced by intradermal complete Freund's adjuvant (CFA) injections. Spontaneous foot lifting (SFL) duration (spontaneous pain behaviour) was measured in 1 day and 4 day rats < 1 h before DRG harvest. mRNA levels for KCNK channels and Kv1.4 relative to GAPDH (n = 4–6 rats/group) were determined with real-time RT-PCR. This study is the first to demonstrate expression of THIK1, THIK2 and TWIK2 mRNA in DRGs. Abundance in normal DRGs was, in descending order:

          Kv1.4 > TRESK(KCNK18) > TRAAK(KCNK4) > TREK2(KCNK10) = TWIK2(KCNK6) > TREK1 (KCNK2) = THIK2(KCNK12) > TASK1(KCNK3) > TASK2(KCNK5) > THIK1(KCNK13) = TASK3(KCNK9).

          During inflammation, the main differences from normal in DRG mRNA levels were bilateral, suggesting systemic regulation, although some channels showed evidence of ipsilateral modulation. By 1 day, bilateral K2P mRNA levels had decreased (THIK1) or increased (TASK1, THIK2) but by 4 days they were consistently decreased (TASK2, TASK3) or tended to decrease (excluding TRAAK). The decreased TASK2 mRNA was mirrored by decreased protein (TASK2-immunoreactivity) at 4 days. Ipsilateral mRNA levels at 4 days compared with 1 day were lower (TRESK, TASK1, TASK3, TASK2 and THIK2) or higher (THIK1). Ipsilateral SFL duration during inflammation was positively correlated with ipsilateral TASK1 and TASK3 mRNAs, and contralateral TASK1, TRESK and TASK2 mRNAs. Thus changes in K2P mRNA levels occurred during inflammation and for 4 K2P channels were associated with spontaneous pain behaviour (SFL). K2P channels and their altered expression are therefore associated with inflammation-induced pain.

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          Molecular background of leak K+ currents: two-pore domain potassium channels.

          Péter Enyedi, Gábor Árpád Czirják (2010)
          Two-pore domain K(+) (K(2P)) channels give rise to leak (also called background) K(+) currents. The well-known role of background K(+) currents is to stabilize the negative resting membrane potential and counterbalance depolarization. However, it has become apparent in the past decade (during the detailed examination of the cloned and corresponding native K(2P) channel types) that this primary hyperpolarizing action is not performed passively. The K(2P) channels are regulated by a wide variety of voltage-independent factors. Basic physicochemical parameters (e.g., pH, temperature, membrane stretch) and also several intracellular signaling pathways substantially and specifically modulate the different members of the six K(2P) channel subfamilies (TWIK, TREK, TASK, TALK, THIK, and TRESK). The deep implication in diverse physiological processes, the circumscribed expression pattern of the different channels, and the interesting pharmacological profile brought the K(2P) channel family into the spotlight. In this review, we focus on the physiological roles of K(2P) channels in the most extensively investigated cell types, with special emphasis on the molecular mechanisms of channel regulation.
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            An overview of real-time quantitative PCR: applications to quantify cytokine gene expression.

            A Giulietti, L Overbergh, D Valckx … (2001)
            The analysis of cytokine profiles helps to clarify functional properties of immune cells, both for research and for clinical diagnosis. The real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantify cytokines from cells, body fluids, tissues, or tissue biopsies. Being a very powerful and sensitive method it can be used to quantify mRNA expression levels of cytokines, which are often very low in the tissues under investigation. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in one single step. In this review we discuss the principle of real-time RT-PCR, the different methodologies and chemistries available, the assets, and some of the pitfalls. With the TaqMan chemistry and the 7700 Sequence Detection System (Applied Biosystems), validation for a large panel of murine and human cytokines and other factors playing a role in the immune system is discussed in detail. In summary, the real-time RT-PCR technique is very accurate and sensitive, allows a high throughput, and can be performed on very small samples; therefore it is the method of choice for quantification of cytokine profiles in immune cells or inflamed tissues. Copyright 2001 Elsevier Science (USA).
            Article 0 comments Cited 239 times – based on 0 reviews     Review now
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              Is Open Access

              MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments

              Stephen A. Bustin, Jean-François Beaulieu, Jim Huggett … (2010)
              The conclusions of thousands of peer-reviewed publications rely on data obtained using fluorescence-based quantitative real-time PCR technology. However, the inadequate reporting of experimental detail, combined with the frequent use of flawed protocols is leading to the publication of papers that may not be technically appropriate. We take the view that this problem requires the delineation of a more transparent and comprehensive reporting policy from scientific journals. This editorial aims to provide practical guidance for the incorporation of absolute minimum standards encompassing the key assay parameters for accurate design, documentation and reporting of qPCR experiments (MIQE précis) and guidance on the publication of pure 'reference gene' articles.
              Article 0 comments Cited 227 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): Mol Cell Neurosci
                Journal ID (iso-abbrev): Mol. Cell. Neurosci
                Title: Molecular and Cellular Neurosciences
                Publisher: Academic Press
                ISSN (Print): 1044-7431
                ISSN (Electronic): 1095-9327
                Publication date PMC-release: March 2012
                Publication date (Print): March 2012
                Volume: 49-531
                Issue: 3
                Pages: 375-386
                Affiliations
                School of Physiology and Pharmacology, University of Bristol, BS8 1TD, UK
                Author notes
                [* ]Corresponding author. c.acosta@ 123456bristol.ac.uk
                [1]

                Present address: Institute of Membrane and Systems Biology, University of Leeds LS2 9JT, UK.

                [2]

                Present address: Department of Molecular & Clinical Pharmacology, Sherrington Building, University of Liverpool, Ashton Street, Liverpool L69 3GE, UK.

                Article
                Publisher ID: YMCNE2691
                DOI: 10.1016/j.mcn.2012.01.002
                PMC ID: 3334831
                PubMed ID: 22273507
                SO-VID: 5bdb4d1b-d95d-48f2-be5a-36307607da3c
                Copyright © © 2012 Elsevier Inc.
                License:

                This document may be redistributed and reused, subject to certain conditions.

                History
                Date received : 23 August 2011
                Date revision received : 2 January 2012
                Date accepted : 4 January 2012
                Categories
                Subject: Article

                ScienceOpen disciplines: Neurosciences
                Keywords: task,potassium channel,k2p mrna,kcnkx.x,tresk,drg
                Data availability:
                ScienceOpen disciplines: Neurosciences
                Keywords: task, potassium channel, k2p mrna, kcnkx.x, tresk, drg

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