Our initial work was focused on exploration of the structurally novel arylpiperazine as analgesics, because its elements are most frequently served as pharmacophores in central nervous system field. During the structural modification, we identified SKL-NP as having an analgesic effect of pain models, but its analgesic mechanism was not elucidated yet. Chemical structure of SKL-NP is shown in Fig. 1. Family patent applications of this compound were filed in several countries including Korea (PCT/KR2008/002470), US (US2011/0195963 A1), EU, Brazil, Canada, China, Japan and so on.

All surgical and experimental procedures complied with guidelines from the Institutional Animal Care and Use Committee (IACUC) at the School of Dentistry, Seoul National University. DRG neurons from 2- to 7-day-old neonatal rats (OrientBio, Sungnam, Korea) were prepared as previously described [7]. Briefly, DRG neurons taken from L3-5 DRG were prepared in 4℃ HBSS (Life Technologies, Carlsbad CA, USA) and incubated in 3 ml DMEM (Invitrogen, Carlsbad CA, USA) containing collagenase (5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) and dispase (1 mg/ml; Sigma-Aldrich) then in 2 ml DMEM of 0.25% trypsin (Sigma-Aldrich) at 37℃ for 8 min. The cells were washed in DMEM and triturated with a flame-polished Pasteur pipette to separate cells and remove processes, and subsequently centrifuged (750 RPM, 5 min), re-suspended and placed on poly-D-lysine (Sigma-Aldrich)-coated glass cover-slips (12 mm in diameter). The cells were maintained at 37℃ in 95% O₂/5% CO₂ incubator.

Whole-cell recording were made using patch electrodes (4~6 MΩ) pulled from borosilicate glass on a Brown-Flaming P-97 horizontal micropipette puller (Sutter Instrument, USA). Voltage- and current-clamp experiments were performed using an HEKA EPC9 amplifier and digitized using ITC16 and Pulse v8.54 software (both from HEKA, Germany). Signals were filtered at 1 kHz and sampled at 3 kHz. Series resistance was typically less than 20 MΩ and was compensated by about 70~80%. Data were analyzed using Origin6.0 software (Microcal Software Inc., USA). All recordings were performed at room temperature (21~23℃).

Extracellular solution contained (in mM): 140 NaCl, 2 CaCl₂, 1 MgCl₂, 5 KCl, 10 HEPES, 10 D-Glucose, adjusted to pH 7.4 with NaOH, 290~310 mOsm. In experiments, BaCl₂ (500 µM/l) was used to eliminate inward rectifier K⁺ currents. Pipettes were filled with an intracellular solution containing (in mM): 136 K-gluconate, 10 NaCl, 1 MgCl₂, 10 EGTA, 2 Mg-ATP, 0.1 Na-GTP adjusted to pH 7.4 with KOH, 290~310 mOsm.

I_h were elicited every 15 sec by a 1 s pulse from a holding potential of -50 mV to a command potential of -100 mV. SKL-NP was bath-applied after obtaining stable I_h for 5 min. Recorded I_h was normalized for pulled data analysis. IC₅₀ was determined as concentration of SKL-NP that would inhibit 50% of the maximum amplitude of I_h based on curve drawn by Origin software. In current-clamp experiments, potential change or action potential firing was elicited by either hyperpolarizing or depolarizing current pulses.

All chemicals except SKL-NP were purchased from Sigma-Aldrich (USA). N-ethylmaleimide (NEM) and 8-bromoadenosine 3',5'-cyclic monophosphate (sodium salt; 8-Bromo-cAMP) was added to the extracellular solutions.

All data are presented as mean±S.E.M; the numbers of cells tested are indicated in parentheses where applicable. Unpaired or paired Student's t-test was used for statistical analysis using Origin6.0 software. Differences were considered to be significant when p-value was less than 0.05.

In our previous study, we found that the mechanical allodynia in neuropathic pain could be associated with I_h that contributes ectopic action potential firings in sensory neurons [5]. Thus, we investigated whether SKL-NP affects the firing pattern in small or medium-sized DRG neurons (<40 µm in diameter), which were characterized by tonic discharge of action potentials and prominent I_h. As shown in Fig. 2Aa, tonic firing and I_h-induced sag in DRG neuron were mostly decreased by 10 µM SKL-NP (58.2±10.4%, n=7, p<0.05). In addition, I_h was blocked by SKL-NP (51.6±3.5%, n=7, p<0.05, Fig. 2Ab). However, tonic firing pattern in DRG neurons with no discernible I_h was not altered by SKL-NP (n=4, p>0.05, Fig. 2B).

Next, we determined the concentration-dependent effect of SKL-NP on I_h. Maximal inhibitory effect of SKL-NP was 52.7% inhibition of I_h at 50 µM, and IC₅₀ was 7.85 µM (Fig. 3A). These results suggest that SKL-NP reduced the excitability of small and medium-sized DRG neurons by inhibiting I_h in a cell-specific and dose-dependent manner. In addition, inhibitory effect of SKL-NP on I_h showed a delayed response in an irreversible manner (Fig. 3B).

Because HCN channels are regulated by cyclic nucleotide in addition to hyperpolarization, we next examined whether the modulating mechanism of SKL-NP effect on I_h is associated with G_i-protein. The pretreatment of pertussis toxin (PTX, 4 µg/ml) and NEM (50 µM) [8] abolished the inhibitory effect of SKL-NP on I_h during application of SKL-NP and at 5 min after application of SKL-NP (96.4±5.3% and 94.5±0.5%, n=7, p>0.05, Fig. 4A). Thus, SKL-NP inhibits I_h in DRG neurons via PTX-sensitive G_i-protein activation. Another G_i-protein inhibitor, NEM, which slightly decreased the amplitude of I_h, (88.7±5.7%, n=4, p>0.05, Fig. 4B), also blocked the SKL-NP effect on I_h (86.0±9.8%, n=4, p>0.05). These results indicate that DRG neurons expressing a G_i-protein coupled receptor (GPCR) may be sensitive to SKL-NP, and their excitability is inhibited by SKL-NP via indirect pathway.

Next, we examined the G_i-protein downstream pathway (i.e. the decrease of cAMP via inhibition of adenylyl cyclase) with the pretreatment of 8-Br-cAMP (200 µM), a membrane permeable cAMP analog, for 10 min before application of SKL-NP. In DRG neurons tested (n=4), 8-Br-cAMP application induced the fast activation of I_h, without affecting the amplitude of I_h (100.3±7.2%, n=4, p>0.05). SKL-NP-induced I_h inhibition was not observed by the pretreatment of 8-Br-cAMP (91.8±18.7%, n=4, p>0.05), indicating that SKL-NP effect was associated with decrease of intracellular cAMP concentration (Fig. 5).