Anoctamin-1 Cl⁻ Channels in Nociception: Activation by an N-Aroylaminothiazole and Capsaicin and Inhibition by T16A[inh]-A01

Abstract

BackgroundAnoctamin 1 (ANO1 or TMEM16A) Ca2+-gated Cl− channels of nociceptor neurons are emerging as important molecular components of peripheral pain transduction. At physiological intracellular Cl− concentrations ([Cl−]i) sensory neuronal Cl− channels are excitatory. The ability of sensory neuronal ANO1 to trigger action potentials and subsequent nocifensive (pain) responses were examined by direct activation with an N-aroylaminothiazole. ANO1 channels are also activated by intracellular Ca2+ ([Ca2+]i) from sensory neuronal TRPV1 (transient-receptor-potential vallinoid 1) ion channels and other noxicant receptors. Thus, sensory neuronal ANO1 can facilitate TRPV1 triggering of action potentials, resulting in enhanced nociception. This was investigated by reducing ANO1 facilitation of TRPV1 effects with: (1) T16A[inh]-A01 ANO1-inhibitor reagent at physiological [Cl−]i and (2) by lowering sensory neuronal [Cl−]i to switch ANO1 to be inhibitory.ResultsANO1 effects on action potential firing of mouse dorsal root ganglia (DRG) neurons in vitro and mouse nocifensive behaviors in vivo were examined with an N-aroylaminothiazole ANO1-activator (E-act), a TRPV1-activator (capsaicin) and an ANO1-inhibitor (T16A[inh]-A01). At physiological [Cl−]i (40 mM), E-act (10 µM) increased current sizes (in voltage-clamp) and action potential firing (in current-clamp) recorded in DRG neurons using whole-cell electrophysiology. To not disrupt TRPV1 carried-Ca2+ activation of ANO1 in DRG neurons, ANO1 modulation of capsaicin-induced action potentials was measured by perforated-patch (Amphotericin–B) current-clamp technique. Subsequently, at physiological [Cl−]i, capsaicin (15 µM)-induced action potential firing was diminished by co-application with T16A[inh]-A01 (20 µM). Under conditions of low [Cl−]i (10 mM), ANO1 actions were reversed. Specifically, E-act did not trigger action potentials; however, capsaicin-induced action potential firing was inhibited by co-application of E-act, but was unaffected by co-application of T16A[inh]-A01. Nocifensive responses of mice hind paws were dramatically induced by subcutaneous injections of E-act (5 mM) or capsaicin (50 µM). The nocifensive responses were attenuated by co-injection with T16A[inh]-A01 (1.3 mM).ConclusionsAn ANO1-activator (E-act) induced [Cl−]i-dependent sensory neuronal action potentials and mouse nocifensive behaviors; thus, direct ANO1 activation can induce pain perception. ANO1-inhibition attenuated capsaicin-triggering of action potentials and capsaicin-induced nocifensive behaviors. These results indicate ANO1 channels are involved with TRPV1 actions in sensory neurons and inhibition of ANO1 could be a novel means of inducing analgesia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12990-015-0061-y) contains supplementary material, which is available to authorized users.

Ca2+ activated Cl− channels as targets for analgesics

“…This confirmed previous reports demonstrating that TRPV1 and ANO1 channels can directly interact with each other [5]. Furthermore, blockers of ANO1 channels were shown to reduce the excitatory actions of the TRPV1 channel activator capsaicin on sensory neurons, on the one hand, and the nocifensive behavior of mice in response to TRPV1 activation, on the other hand [5, 6]. The presumed underlying process is the gating of CaCCs by Ca 2+ which enters the neurons via TRPV1 channels (Figure 1).…”

Eact, a small molecule activator of TMEM16A, activates TRPV1 and elicits pain‐ and itch‐related behaviours

“…RNAi knockdown or genetic deletion of TMEM16A from DRG neurons also substantially attenuated thermal hyperalgesia in both inflammatory and neuropathic pain models (Lee et al, ; Liu et al, ). Moreover, a recent study also showed that Eact activated DRG neurons and induced pain response, both of which were suppressed by the TMEM16A inhibitor A01 (Deba and Bessac, ). Indeed, we also found that co‐expression of TMEM16A potentiated capsaicin‐activated currents in mTRPV1‐expressing HEK293T cells, suggesting that Eact activation of TMEM16A might also contribute to Eact‐elicited sensory hypersensitivity.…”

“…Although different experimental settings, cell culture conditions, and differences between HEK293T cells and DRG neurons might partly account for the discrepancy of Eact responses in DRG and heterologous cells between these two studies (Deba and Bessac, ), our results using TRPV1 knockout mice clearly demonstrated that Eact‐induced excitation and sensory hypersensitivity require functional TRPV1 channels, suggesting that Eact is not an ideal tool to dissect the function of TMEM16A in the primary sensory neurons because it could directly activate TRPV1 in the same cells. Because TRPV1 is a dominant initiator of pain and itch in the primary sensory neurons, Eact activation of TRPV1 could override TMEM16A‐mediated Eact responses.…”

Use of Capsaicin to Treat Pain: Mechanistic and Therapeutic Considerations

“…TRPV1 mediated Ca 2+ influx activates anoctamine 1 (ANO1), Ca 2+ -activated Cl − channels in nociceptive terminals, which leads to further depolarization [78]. Indeed, pharmacological inhibition of ANO1 attenuates capsaicin-mediated acute nocifensive behaviors in rodents [78,79]. …”