title: Anoctamin-1 Cl(-) channels in nociception: activation by an N-aroylaminothiazole and capsaicin and inhibition by T16A[inh]-A01.
abstract: BACKGROUND
  Anoctamin 1 (ANO1 or TMEM16A) Ca(2+)-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 Ca(2+) ([Ca(2+)]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.
  
  
  RESULTS
  ANO1 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-Ca(2+) 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).
  
  
  CONCLUSIONS
  An 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.
authors.affiliation: Department of Pharmaceutical Sciences, I. L. Rangel College of Pharmacy, Texas A&M Health Science Center, 1010 West Avenue B MSC 131, Kingsville, TX, 78363, USA. deba@pharmacy.tamhsc.edu.
authors.forename: Farah
authors.initials: F
authors.lastname: Deba
authors.affiliation: Department of Pharmaceutical Sciences, I. L. Rangel College of Pharmacy, Texas A&M Health Science Center, 1010 West Avenue B MSC 131, Kingsville, TX, 78363, USA. bessac@pharmacy.tamhsc.edu.
authors.forename: Bret F
authors.initials: BF
authors.lastname: Bessac
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D000075369:Anoctamin-1; D002211:Capsaicin / Q000494:pharmacology*; D018118:Chloride Channels / Q000378:metabolism*; D005260:Female; D005727:Ganglia, Spinal / Q000187:drug effects / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008807:Mice, Inbred BALB C; D059225:Nociception / Q000187:drug effects*; D011743:Pyrimidines / Q000494:pharmacology*; D013844:Thiazoles / Q000494:pharmacology*; D014162:Transfection
chemical_list: C531181:ANO1 protein, mouse; D000075369:Anoctamin-1; D018118:Chloride Channels; D011743:Pyrimidines; C578466:T16AInh-A01; D013844:Thiazoles; D002211:Capsaicin