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

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 (I(h)) that plays important roles in neuropathic pain. In small or medium-sized dorsal root ganglion (DRG) neurons (<40 µm in diameter) exhibiting tonic firing and prominent I(h), SKL-NP inhibited I(h) and spike firings in a concentration dependent manner (IC(50)=7.85 µM). SKL-NP-induced inhibition of I(h) 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 I(h) 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.

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

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 I_h 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 I_h currents, which was inhibited by SKL-NP (10 µM). (c) Summary of firing rates (white columns) and amplitude of I_h (black columns) under influence of SKL-NP (n=7, ^*p<0.05). (B) Tonic firing of neurons, which did not have discernable I_h, was not modified by application of SKL-NP (n=4, p>0.05).

Fig. 3

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

Fig. 4

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

Fig. 5

8Br-cAMP (200 µM) was perfused extracelluarly before (1), during (2) and after (3) application of SKL-NP. Superimposed I_h 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).