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Sci Rep
2015 Jan 12;5:12548. doi: 10.1038/srep12548.
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Activation of TRESK channels by the inflammatory mediator lysophosphatidic acid balances nociceptive signalling.
Kollert S
,
Dombert B
,
Döring F
,
Wischmeyer E
.
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In dorsal root ganglia (DRG) neurons TRESK channels constitute a major current component of the standing outward current IKSO. A prominent physiological role of TRESK has been attributed to pain sensation. During inflammation mediators of pain e.g. lysophosphatidic acid (LPA) are released and modulate nociception. We demonstrate co-expression of TRESK and LPA receptors in DRG neurons. Heterologous expression of TRESK and LPA receptors in Xenopus oocytes revealed augmentation of basal K(+) currents upon LPA application. In DRG neurons nociception can result from TRPV1 activation by capsaicin or LPA. Upon co-expression in Xenopus oocytes LPA simultaneously increased both depolarising TRPV1 and hyperpolarising TRESK currents. Patch-clamp recordings in cultured DRG neurons from TRESK[wt] mice displayed increased IKSO after application of LPA whereas under these conditions IKSO in neurons from TRESK[ko] mice remained unaltered. Under current-clamp conditions LPA application differentially modulated excitability in these genotypes upon depolarising pulses. Spike frequency was attenuated in TRESK[wt] neurons and, in contrast, augmented in TRESK[ko] neurons. Accordingly, excitation of nociceptive neurons by LPA is balanced by co-activation of TRESK channels. Hence excitation of sensory neurons is strongly controlled by the activity of TRESK channels, which therefore are good candidates for the treatment of pain disorders.
Figure 1. Expression profile of LPA receptors and immunodetection of TRESK channel in DRG neurons.(A) Whole cell extracts of HEK-293 cells transfected with pcDNA3 (mock), mTRESK, hTRESK and myc-mTRESK plasmid DNA (as indicated) were immunoblotted and analysed with polyclonal TRESK antibody (upper panel) or anti-Actin antibody as loading control (lower panel). Extracts of cells transfected with mTRESK or myc-mTRESK displayed double bands with the expected molecular weight indicating high specificity for mouse TRESK of the novel antibody. (B) HEK-293 cells were transfected with mTRESK-eGFP fusion construct and analysed by confocal microscopy. Only transfected cells (GFP in upper panel) were found to have membrane reactivity with TRESK antibodies (middle panel). Lower panel, merge displayed co-reactivity of the antibody with GFP signal. DAPI staining in blue revealed also non-transfected cells. Scale bar 2 μm. (C) Expression of mTRESK, and a selection of Gq-coupled receptors (as indicated) was monitored in adult DRGs by RT-PCR. Primers for GAPDH were used as a positive control and H2O was applied as a negative control in reverse transcription. (D) Immunocytochemistry of cultured DRG neurons from E13.5 mouse embryos probed with antibodies against TRESK (c, g), NFH (b), TRPV1 (f) and DAPI (a, e). Images a-c and e-g are merged in panel d and h, respectively. Merge in d documented that only NFH-stained neurons (grey) displayed TRESK reactivity (magenta); DAPI-stained, non-neuronal cells (a, upper and lower edge) displayed hardly any TRESK signal (c). Neuronal co-expression of TRESK (magenta) and TRPV1 (green) was documented in merge of panel h (out of 29 TRESK-positive cells 18 were also positive for TRPV1). Fused z-stacks of confocal images are shown. Scale bar 10 μm.
Figure 2. Regulation of TRESK channels by Gq-coupled LPA receptors heterologously expressed in Xenopus oocytes.(A) Co-expression of 5-HT2c, bradykinin B1, bradykinin B2, histamine H1 and LPA2 receptors together with TRESK augmented outward currents upon activation with the respective selective agonist. Inset displays representative trace with co-expressed 5-HT2c receptors. (B) Ramp recordings from −100 to +60 mV elicited outwardly rectifying currents amplified after application of LPA and blocked by lamotrigine (100 μM) (C) Dose-response curve of TRESK activation by LPA recorded at a holding potential of +30 mV with a half-maximal activating concentration of 0.2 μM. (D) LPA-induced augmentation of outward currents (upper panel) was abolished either upon incubation with the phospholipase C blocker U73122 (middle trace) or by a mutant lacking the calcineurin binding motif (lower trace).
Figure 3. TRPV1 and TRESK are simultaneously activated by LPA.(A) LPA augmented capsaicin activated currents in TRPV1 injected oocytes. (B) TRPV1 and TRESK co-expressing oocytes displayed an augmented inward and outward current upon application of LPA. (C) Pulse protocol to the respective reversal potentials demonstrated time-locked co-activation of TRESK and TRPV1 with LPA expressed in the same oocyte. (D) LPA-induced augmentation of TRESK and TRPV1 currents were depicted on a larger time scale. Note the fast deactivation of TRPV1 currents upon sole activation with LPA.
Figure 4. Transfection of F-11 cells with TRESK reduces cellular excitability.(A) Expression profile of K2P channels in F-11 cells. PCR-primers specific for transcripts of mouse and rat (as indicated) were used to cover the hybrid genome of F-11 cells. Note that TRESK channels are not expressed. (B) Untransfected F-11 cells displayed typical voltage-gated potassium and sodium channels in ramp recordings and in depolarising step recordings (inset, left panel) whereas TRESK transfected F-11 cells displayed a shift of reversal potential to more negative values in ramp recordings and large outward currents upon depolarising steps (inset, right panel). (C) Current injection (40 pA) leads to a single action potential in non-transfected F-11 cells (left panel) whereas even high amplitude current injection (340 pA) failed to elicit an action potential in TRESK-transfected cells (right panel). Insets display current steps from −60 to 300 pA.
Figure 5. LPA receptors activate IKSO currents in DRG neurons.(A) Patch-clamp recordings from TRESK[wt] neurons displayed an increase in current amplitude of IKSO standing-outward currents after application of 10 μM LPA (upper panel, black trace) reversed by lamotrigine (upper panel, grey trace). In neurons from TRESK[ko] mice LPA application had no effect on IKSO (lower panel). Bar graph summarises data from left panel and from neurons 5 days in culture as indicated. (B) Recordings from capsaicin positive TRESK[wt] neurons display augmentation of either outward and inward currents upon LPA application, respectively whereas TRESK[ko] neurons display only augmentation of inward currents.
Figure 6. Enhanced excitability of DRG neurons by LPA.(A) Current-clamp recordings displayed spike trains from TRESK[wt] and TRESK[ko] neurons upon LPA application. LPA application after depolarising pulses (100 pA) reduced spike frequency in TRESK[wt] neurons (upper trace) and increased spike frequency in TRESK[ko] neurons. (B) Bar graph quantifies data shown in (A).
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