Capsazepine concentration dependently inhibits currents in HEK 293 cells mediated by human hyperpolarization-activated cyclic nucleotide-gated 2 and 4 channels

Abstract

Recent studies indicate that blockade of currents (Ih) mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (particularly HCN1) may partly account for the antinociceptive effects of capsazepine (CPZ). Unfortunately, determining whether capsazepine is a selective HCN channel blocker and determining its adverse effects when it is used for the treatment of neuropathic pain, have been thus far understudied. In this study, we aimed to elucidate the effects of capsazepine on human HCN2 (hHCN2) and HCN4 (hHCN4) channels in HEK293 cells. The vectors that expressed hHCN2 and hHCN4 cDNA were constructed and transfected into HEK293 cells. Enhanced green fluorescent protein (EGFP) fluorescence and the reverse transcription polymerase chain reaction (RT-PCR) were used to confirm the successful transfection of the vectors. After G418 (neomycin) screening, cell lines that expressed hHCN2 and hHCN4 were obtained. The whole-cell voltage-clamp technique was used to determine the currents from hHCN2 and hHCN4 channels, which were perfused with five concentrations (0.1 µM, 1 µM, 5 µM, 10 µM and 50 µM) of capsazepine. The results showed that capsazepine at the range from 0.1 to 50 µM markedly inhibited hHCN2 and hHCN4 currents in a concentration-dependent manner, with most inhibition achieved at a concentration of 10 µM of capsazepine. When compared with the control group, a V0.5 for the hHCN2 and hHCN4 channel showed that 10 µM capsazepine significantly shifted the membrane potential towards hyperpolarization. The present results indicate that capsazepine is not a selective HCN1 channel blocker and that it may have adverse effects when used to treat neuropathic pain.

Peer Review #2 of "Capsazepine prolongation of the duration of lidocaine block of sensory transmission in mice may be mediated by modulation of HCN channel currents (v0.1)"

“…In the present study, CPZ inhibits transfected mice HCN channel currents without selectivity between HCN1 and HCN2. The IC 50 of CPZ measured in this study (~10 µM) is similar to the potency on human I h (Gill et al, 2004;Zuo et al, 2013), indicating the enhancement of CPZ for regional anesthesia might be conserved between species.…”

“…Fifty-six mice were randomly divided into groups (n = 8/group): respectively receiving lidocaine alone, CPZ alone, ZD7288 (HCN channel blocker) alone, CPZ + lidocaine, ZD7288 + lidocaine, ZD7288 + CPZ + lidocaine, forskolin (an activator of adenylyl cyclase) + CPZ + lidocaine. The concentrations of lidocaine, ZD7288, CPZ and forskolin were 1% (w/v, ~35 mM), 5 mM, 53 µM and 153 µM, respectively (Kroin et al, 2004;Zuo et al, 2013). For the groups that received two or three drugs, ZD7288 and/or forskolin were injected 20 min before lidocaine and CPZ was injected 10 min before lidocaine (Ando et al, 2004;Kroin et al, 2004).…”

“…CPZ can inhibit transfected human HCN channel currents (Zuo et al, 2013). HCN channel is widely expressed in peripheral nervous system (Acosta et al, 2012;Cao et al, 2016).…”

Systemic desensitization through TRPA1 channels by capsazepine and mustard oil - a novel strategy against inflammation and pain

“…This high percentage may account for the efficiency of chronic CPZ to induce general nociceptor desensitization. Of note, all previously reported off-target effects of CPZ were related to blocking other ion channels than TRPV1 at higher (>50 μM) concentrations: nicotinic acetylcholine receptors, voltage-gated calcium channels, transient receptor potential melastatin 8 (TRPM8), and the hyperpolarization-activated HCN 1 2 3 4 channels 28 29 30 31 . Only the block of TRPM8 by CPZ was reported to mediate an anti-nociceptive effect in a behavioral model of neuropathic hypersensitivity to cold stimulation 32 .…”

HCN Channels Modulators: The Need for Selectivity

“…Capsazepine is however not selective: when tested on recombinant hHCN2 and hHCN4 isoforms expressed in HEK293 cells, its IC 50 values were comparable, being 6.1±0.8 and 5.8±0.5 μM, respectively [182]. …”