Korean J Physiol Pharmacol.  2012 Aug;16(4):237-241. 10.4196/kjpp.2012.16.4.237.

A Novel Carbamoyloxy Arylalkanoyl Arylpiperazine Compound (SKL-NP) Inhibits Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channel Currents in Rat Dorsal Root Ganglion Neurons

• Chung G
• Kim TH
• Shin H
• Chae E
• Yi H
• Moon H
• Kim HJ
• Kim JS
• Jung SJ
• Oh SB

• KMID: 2011157
• DOI: http://doi.org/10.4196/kjpp.2012.16.4.237

Abstract

In this study, we determined mode of action of a novel carbamoyloxy arylalkanoyl arylpiperazine compound (SKL-NP) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents (Ih) that plays important roles in neuropathic pain. In small or medium-sized dorsal root ganglion (DRG) neurons (<40 microm in diameter) exhibiting tonic firing and prominent Ih, SKL-NP inhibited Ih and spike firings in a concentration dependent manner (IC50=7.85 microM). SKL-NP-induced inhibition of Ih was blocked by pretreatment of pertussis toxin (PTX) and N-ethylmaleimide (NEM) as well as 8-Br-cAMP, a membrane permeable cAMP analogue. These results suggest that SKL-NP modulates Ih in indirect manner by the activation of a Gi-protein coupled receptor that decreases intracellular cAMP concentration. Taken together, SKL-NP has the inhibitory effect on HCN channel currents (I h) in DRG neurons of rats.

Keyword

cAMP; Gi-protein; Hyperpolarization-activated cyclic nucleotide-gated channel; Ih; Neuropathic pain

MeSH Terms

Animals
Diagnosis-Related Groups
Ethylmaleimide
Fires
Ganglia, Spinal
Membranes
Neuralgia
Neurons
Pertussis Toxin
Rats
Spinal Nerve Roots
Ethylmaleimide
Pertussis Toxin

Figure

• 

  Fig. 1 Structure of a novel carbamoyloxy arylalkanoyl arylpiperazine compound (R-Carbamic acid 3-[4-(3,4-dimethoxy-phenyl)-piperazin-1-yl]-3-oxo-1-phenyl-propyl ester, molecular weight 388.27, SKL-NP).

• 

  Fig. 2 The effects of SKL-NP on Ih in rat DRG neurons. (A) (a) Representative trace of firing pattern elicited by a 2 s current injection (500 pA) before and during exposure to SKL-NP (10 µM). SKL-NP abolished tonic firing. Injection of a hyperpolarizing current (-100 pA) elicited a sag in the voltage trace (bottom trace), which was abolished by application of SKL-NP (10 µM). (b) Representative trace of Ih currents, which was inhibited by SKL-NP (10 µM). (c) Summary of firing rates (white columns) and amplitude of Ih (black columns) under influence of SKL-NP (n=7, *p<0.05). (B) Tonic firing of neurons, which did not have discernable Ih, was not modified by application of SKL-NP (n=4, p>0.05).

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  Fig. 3 (A) Concentration-response curve of SKL-NP effects on Ih. SKL-NP inhibited Ih, and spike firings in concentration dependent manner. (B) An irreversible action of SKL-NP effects on Ih. Inhibitory effect of SKL-NP showed a delayed response in a irreversible manner. After washout of SKL-NP, Ih did not recover to control level (SKL-NP 10 and 50 µM, *p<0.05).

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  Fig. 4 Signaling pathways involved in SKL-NP-induced Ih inhibition. (A) PTX abolished the inhibitory effects of SKL-NP on Ih (n=7). Superimposed Ih traces before and after application of SKL-NP following PTX pretreatment. (B) Another Gi-protein inhibitor, NEM, also blocked the inhibitory effects of SKL-NP on Ih during and after SKL-NP application (n=4, p>0.05). Superimposed Ih traces before and after application of SKL-NP following NEM pretreatment.

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  Fig. 5 8Br-cAMP (200 µM) was perfused extracelluarly before (1), during (2) and after (3) application of SKL-NP. Superimposed Ih traces before, during and after application of SKL-NP as indicated by appropriate numbers in the time course trace shown in lower panel (n=4, p> 0.05).

Reference

1. Momin A, Cadiou H, Mason A, McNaughton PA. Role of the hyperpolarization-activated current Ih in somatosensory neurons. J Physiol. 2008. 586:5911–5929.

2. Wahl-Schott C, Biel M. HCN channels: structure, cellular regulation and physiological function. Cell Mol Life Sci. 2009. 66:470–494.

3. Wickenden AD, Maher MP, Chaplan SR. HCN pacemaker channels and pain: a drug discovery perspective. Curr Pharm Des. 2009. 15:2149–2168.

4. Campbell JN, Meyer RA. Mechanisms of neuropathic pain. Neuron. 2006. 52:77–92.

5. Yeon KY, Chung G, Kim YH, Hwang JH, Davies AJ, Park MK, Ahn DK, Kim JS, Jung SJ, Oh SB. Eugenol reverses mechanical allodynia after peripheral nerve injury by inhibiting hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Pain. 2011. 152:2108–2116.

6. Chaplan SR, Guo HQ, Lee DH, Luo L, Liu C, Kuei C, Velumian AA, Butler MP, Brown SM, Dubin AE. Neuronal hyperpolarization-activated pacemaker channels drive neuropathic pain. J Neurosci. 2003. 23:1169–1178.

7. Binshtok AM, Bean BP, Woolf CJ. Inhibition of nociceptors by TRPV1-mediated entry of impermeant sodium channel blockers. Nature. 2007. 449:607–610.

8. Shapiro MS, Wollmuth LP, Hille B. Modulation of Ca²⁺ channels by PTX-sensitive G-proteins is blocked by N-ethylmaleimide in rat sympathetic neurons. J Neurosci. 1994. 14:7109–7116.

9. Dunlop J, Vasilyev D, Lu P, Cummons T, Bowlby MR. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and pain. Curr Pharm Des. 2009. 15:1767–1772.

10. Jiang YQ, Sun Q, Tu HY, Wan Y. Characteristics of HCN channels and their participation in neuropathic pain. Neurochem Res. 2008. 33:1979–1989.

11. Kouranova EV, Strassle BW, Ring RH, Bowlby MR, Vasilyev DV. Hyperpolarization-activated cyclic nucleotide-gated channel mRNA and protein expression in large versus small diameter dorsal root ganglion neurons: correlation with hyperpolarization-activated current gating. Neuroscience. 2008. 153:1008–1019.

12. Emery EC, Young GT, Berrocoso EM, Chen L, McNaughton PA. HCN2 ion channels play a central role in inflammatory and neuropathic pain. Science. 2011. 333:1462–1466.

13. Ingram SL, Williams JT. Opioid inhibition of Ih via adenylyl cyclase. Neuron. 1994. 13:179–186.