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. 2012 May 25;287(22):18562-72.
doi: 10.1074/jbc.M111.322271. Epub 2012 Apr 4.

Neuromedin U type 1 receptor stimulation of A-type K+ current requires the βγ subunits of Go protein, protein kinase A, and extracellular signal-regulated kinase 1/2 (ERK1/2) in sensory neurons

Yiming Zhang  1 , Dongsheng Jiang, Yuan Zhang, Xinghong Jiang, Fen Wang, Jin Tao
Affiliations

Neuromedin U type 1 receptor stimulation of A-type K+ current requires the βγ subunits of Go protein, protein kinase A, and extracellular signal-regulated kinase 1/2 (ERK1/2) in sensory neurons

Yiming Zhang et al. J Biol Chem. .

Abstract

Although neuromedin U (NMU) has been implicated in analgesia, the detailed mechanisms still remain unclear. In this study, we identify a novel functional role of NMU type 1 receptor (NMUR1) in regulating the transient outward K(+) currents (I(A)) in small dorsal root ganglion (DRG) neurons. We found that NMU reversibly increased I(A) in a dose-dependent manner, instead the sustained delayed rectifier K(+) current (I(DR)) was not affected. This NMU-induced I(A) increase was pertussis toxin-sensitive and was totally reversed by NMUR1 knockdown. Intracellular application of GDPβS (guanosine 5'-O-(2-thiodiphosphate)), QEHA peptide, or a selective antibody raised against the Gα(o) or Gβ blocked the stimulatory effects of NMU. Pretreatment of the cells with the protein kinase A (PKA) inhibitor or ERK inhibitor abolished the NMU-induced I(A) response, whereas inhibition of phosphatidylinositol 3-kinase or PKC had no such effects. Exposure of DRG neurons to NMU markedly induced the phosphorylation of ERK (p-ERK), whereas p-JNK or p-p38 was not affected. Moreover, the NMU-induced p-ERK increase was attenuated by PKA inhibition and activation of PKA by foskolin would mimic the NMU-induced I(A) increase. Functionally, we observed a significant decrease of the firing rate of neuronal action potential induced by NMU and pretreatment of DRG neurons with 4-AP could abolish this effect. In summary, these results suggested that NMU increases I(A) via activation of NMUR1 that couples sequentially to the downstream activities of Gβγ of the G(o) protein, PKA, and ERK, which could contribute to its physiological functions including neuronal hypoexcitability in DRG neurons.

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Figures

FIGURE 1.
FIGURE 1.
NMU selectively increased IA in small DRG neurons. A, isolation of IA. Na+ currents, Ca2+ currents, and Ca2+-activated K+ currents have been eliminated (see “Experimental Procedures”). Left, the membrane voltage was held at −80 mV and IA was isolated by a two-step voltage protocol. Right, the remaining current after off-line subtraction of the noninactivating portion of current remaining after the prepulse to −10 mV. This protocol is used for isolation of IA in all figures. B and C, representative current traces and the time course showed that IA (n = 7, B), but not sustained delayed rectifier K+ current (IDR) (n = 9, C), was significantly increased during exposure to 1 μm NMU. D, summary data of current density showed that 1 μm NMU selectively increased IA. E, dose-response curve for the stimulatory effects of NMU on IA. The line represents the best fit of the data points to the sigmoidal Hill equation. Number of cells tested at each concentration of NMU is indicated in parentheses. F, time course and exemplary traces showed no effect of 1 μm NMU on IA in the presence of 4-AP (n = 5). ***, p < 0.001 versus control.
FIGURE 2.
FIGURE 2.
Effects of NMU on biophysical properties of IA. A, representative current traces of IA recorded before and after exposure to 1 μm NMU. B, current-voltage (I-V) curve in the absence (n = 9) and presence (n = 9) of 1 μm NMU. C, the steady-state activation of IA channels is not altered by 1 μm NMU (n = 9). The voltage-dependent activation of the IA component; as determined by measuring the peak current amplitude elicited by depolarizing test pulses. The conductance at each voltage was calculated normalized to the conductance determined at +40 mV, and plotted versus the test voltage. To determine the voltage dependence of activation, voltage steps of 400 ms were applied at 5-s intervals in +10 mV increase to a maximum of +70 mV. D, NMU at 1 μm rightward shifted the steady-state inactivation curve of IA (n = 7). To determine the steady-state inactivation, conditioning prepulses ranging from −100 to +40 mV were applied at 5-s intervals in +10 mV increments for 150 ms followed by a voltage step to +40 mV for 500 ms. *, p < 0.05 versus control, **, p < 0.01 versus control.
FIGURE 3.
FIGURE 3.
NMU-mediated response is prevented by NMUR1 knockdown. A, membrane expression of NMUR1 determined by confocal microscopy. a, fluorescent signals of NMUR1. b, differential interference contrast images. c, merged picture. Scale bar, 25 μm. B, protein expression of NMUR1 (46 kDa) was measured using Western blot analysis in control siRNA (ctrl siRNA) and NMUR1 siRNA-treated groups. β-Actin (42 kDa) showed the equal loading. C, left, photomicrographs of phase-contrast (a) and fluorescent images (b) of small DRG neurons transfected with the NMUR1 siRNA. Scale bar, 20 μm. Right, representative current traces showed the effects of control siRNA or NMUR1 siRNA on the NMU-induced IA increase. D, summary data showed the effects of control siRNA (n = 8) or NMUR1 siRNA (n = 9) on the 1 μm NMU-induced IA increase in small DRG neurons. **, p < 0.01 versus control.
FIGURE 4.
FIGURE 4.
NMUR1-mediated IA increase requires the Gβγ subunits of Go protein. A and B, representative current traces (A) and summary data (B) showed the effects of 1 μm NMU on IA in the presence of GDPβS (1 mm, intracellular applied, n = 7), CTX (0.5 μg/ml for 24 h pretreatment, n = 9), PTX (0.2 μg/ml for 24 h pretreatment, n = 9), anti-Go (intracellular applied, n = 7), boiled anti-Go (intracellular applied, n = 5), QEHA (200 μm, intracellular applied, n = 9), SKEE (200 μm, intracellular applied, n = 7), or anti-Gβ (intracellular applied, n = 8), respectively. **, p < 0.01 versus control; ***, p < 0.001 versus control.
FIGURE 5.
FIGURE 5.
NMUR1-mediated IA increase was PKA-dependent. A, summary data showed the increase of IA induced by 1 μm NMU in the presence of LY294002 (3 μm for 30 min, n = 9), U73122 (3 μm for 30 min, n = 9), GF109203X (1 μm for 30 min, n = 8), chelerythrine chloride (1 μm for 30 min, n = 7), calphostin C (50 nm, n = 7), H89 (1 μm for 30 min, n = 9), and PKI 6-22 (1 μm, intracellular applied, n = 7), respectively. B, representative current traces (left) and summary data (right) showed the effects of 20 μm forskolin on the peak amplitude of IA in small DRG neurons (n = 9). C, NMU increased PKA activity via NMUR1. Cells were treated with either vehicle (control) or 1 μm NMU for 10 min and assayed for PKA activity as described under “Experimental Procedures.” The PKA activity data are normalized to the vehicle-treated cells as 100%. *, p < 0.05 versus control; **, p < 0.01 versus control; ##, p < 0.001 versus NMU groups.
FIGURE 6.
FIGURE 6.
NMU-induced IA increase involves activation of ERK. A–C, NMU induced increased phosphorylation of ERK (p-ERK, A) in DRG neurons, whereas the protein expression levels of p-JNK (B) or p-38 (C) were not affected. β-Actin showed equal loading. D and E, NMU-induced p-ERK increase was abolished by NMUR1 knockdown and pretreatment of the cells with PD98059 or H89. Exposure of DRG neurons to NMU still increased p-ERK expression in cells transfected with control siRNA. All experiments were performed in triplicate with similar results. Inset, numbers 1–4 represents control siRNA, NMUR1 siRNA, H89, and PD98059, respectively. F, representative current traces (upper) and summary data (lower) showed the effects of 1 μm NMU on IA in the presence of U0126 (n = 9) or PD98059 (n = 7). **, p < 0.01 versus control; ***, p < 0.01 versus control.
FIGURE 7.
FIGURE 7.
NMUR1 induces neuronal hypoexcitability in small DRG neurons. A and B, time course and summary data showed that NMU had no effects on voltage-gated Na+ channel currents (n = 6). Inset, representative examples of Na+ currents recorded before and after application of 1 μm NMU. C and D, exemplary current traces and current-voltage plot of current density of Na+ current versus test voltage recorded before (n = 7) and after (n = 7) 1 μm NMU. E, representative traces of firing at the resting membrane potential under control conditions, during exposure to 1 μm NMU, and washout. F, summary data comparing the average firing rates under the conditions indicated in panel E (n = 14). Cells were given +130 pA current injections. G, representative traces and summary data showed the effects of 4-AP on neuronal firing (n = 11). H, representative traces and summary data showed 4-AP abolished 1 μm NMU-induced hypoexcitability in small DRG neurons (n = 17). *, p < 0.05 versus pre-drug; **, p < 0.01 versus pre-drug.
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References

    1. Brighton P. J., Szekeres P. G., Willars G. B. (2004) Neuromedin U and its receptors. Structure, function, and physiological roles. Pharmacol. Rev. 56, 231–248 - PubMed
    1. Budhiraja S., Chugh A. (2009) Neuromedin U, physiology, pharmacology, and therapeutic potential. Fundam. Clin. Pharmacol. 23, 149–157 - PubMed
    1. Raddatz R., Wilson A. E., Artymyshyn R., Bonini J. A., Borowsky B., Boteju L. W., Zhou S., Kouranova E. V., Nagorny R., Guevarra M. S., Dai M., Lerman G. S., Vaysse P. J., Branchek T. A., Gerald C., Forray C., Adham N. (2000) Identification and characterization of two neuromedin U receptors differentially expressed in peripheral tissues and the central nervous system. J. Biol. Chem. 275, 32452–32459 - PubMed
    1. Shan L., Qiao X., Crona J. H., Behan J., Wang S., Laz T., Bayne M., Gustafson E. L., Monsma F. J., Jr., Hedrick J. A. (2000) Identification of a novel neuromedin U receptor subtype expressed in the central nervous system. J. Biol. Chem. 275, 39482–39486 - PubMed
    1. Mitchell J. D., Maguire J. J., Davenport A. P., (2009) Emerging pharmacology and physiology of neuromedin U and the structurally related peptide neuromedin S. Br. J. Pharmacol. 158, 87–103 - PMC - PubMed

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