Spinal bestrophin-1 and anoctamin-1 channels have a pronociceptive role in the tactile allodynia induced by REM sleep deprivation in rats.

Bestrophin-1 and anoctamin-1 are members of the calcium-activated chloride channels (CaCCs) family and are involved in inflammatory and neuropathic pain. However, their role in pain hypersensitivity induced by REM sleep deprivation (REMSD) has not been studied. This study aimed to determine if anoctamin-1 and bestrophin-1 are involved in the pain hypersensitivity induced by REMSD. We used the multiple-platform method to induce REMSD. REM sleep deprivation for 48 h induced tactile allodynia and a transient increase in corticosterone concentration at the beginning of the protocol (12 h) in female and male rats. REMSD enhanced c-Fos and α2δ-1 protein expression but did not change activating transcription factor 3 (ATF3) and KCC2 expression in dorsal root ganglia and dorsal spinal cord. Intrathecal injection of CaCCinh-A01, a non-selective bestrophin-1 blocker, and T16Ainh-A01, a specific anoctamin-1 blocker, reverted REMSD-induced tactile allodynia. However, T16Ainh-A01 had a higher antiallodynic effect in male than female rats. In addition, REMSD increased bestrophin-1 protein expression in DRG but not in DSC in male and female rats. In marked contrast, REMSD decreased anoctamin-1 protein expression in DSC but not in DRG, only in female rats. Bestrophin-1 and anoctamin-1 promote pain and maintain tactile allodynia induced by REM sleep deprivation in both male and female rats, but their expression patterns differ between the sexes.

Blockade of anoctamin-1 in injured and uninjured nerves reduces neuropathic pain.

The aim of this study was to determine the participation of anoctamin-1 in 2 models of neuropathic pain in rats (L5/L6 spinal nerve ligation [SNL] and L5 spinal nerve transection [SNT]). SNL and SNT diminished withdrawal threshold in rats. Moreover, SNL up-regulated anoctamin-1 protein expression in injured L5 and uninjured L4 DRG whereas that it enhanced activating transcription factor 3 (ATF-3) and caspase-3 expression only in injured L5 DRG. In marked contrast, SNT enhanced ATF-3 and caspase-3, but not anoctamin-1, expression in injured L5 DRG but it did not modify anoctamin-1, ATF-3 nor caspase-3 expression in uninjured L4 DRG. Accordingly, repeated (3 times) intrathecal injection of the anoctamin-1 blocker T16Ainh-A01 (0.1-1 µg) or MONNA (1-10 µg) partially reverted SNL-induced mechanical allodynia in a dose-dependent manner. In contrast, anoctamin-1 blockers only produced a modest effect in SNT-induced mechanical allodynia. Interestingly, intrathecal injection of T16Ainh-A01 (1 µg) or MONNA (10 µg) prevented SNL-induced up-regulation of anoctamin-1, ATF-3 and caspase-3 in injured L5 DRG. Repeated intrathecal injection of T16Ainh-A01 or MONNA also reduced SNT-induced up-regulation of ATF-3 in injured L5 DRG. In contrast, T16Ainh-A01 and MONNA did not affect SNT-induced up-regulation of caspase-3 expression in L5 DRG. Likewise, gabapentin (100 µg) diminished SNL-induced up-regulation of anoctamin-1, ATF-3 and caspase-3 expression in injured L5 DRG. These data suggest that spinal anoctamin-1 in injured and uninjured DRG participates in the maintenance of neuropathic pain in rats. Our data also indicate that expression of anoctamin-1 in DRG is differentially regulated depending on the neuropathic pain model.

Phosphatidylinositol 4,5-bisphosphate, cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1).

The TMEM16A-mediated Ca2+-activated Cl- current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca2+. On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-ß-cyclodextrin M-ßCD) or restoration (with M-ßCD+cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-ßCD alone transiently increases TMEM16A activity and dampens rundown whereas M-ßCD+cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-ßCD, M-ßCD+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.

A CLCA regulatory protein present in the chemosensory cilia of olfactory sensory neurons induces a Ca2+-activated Cl- current when transfected into HEK293.

BACKGROUND: CLCA is a family of metalloproteases that regulate Ca2+-activated Cl- fluxes in epithelial tissues. In HEK293 cells, CLCA1 promotes membrane expression of an endogenous Anoctamin 1 (ANO1, also termed TMEM16A)-dependent Ca2+-activated Cl- current. Motif architecture similarity with CLCA2, 3 and 4 suggested that they have similar functions. We previously detected the isoform CLCA4L in rat olfactory sensory neurons, where Anoctamin 2 is the principal chemotransduction Ca2+-activated Cl- channel. We explored the possibility that this protein plays a role in odor transduction. RESULTS: We cloned and expressed CLCA4L from rat olfactory epithelium in HEK293 cells. In the transfected HEK293 cells we measured a Cl--selective Ca2+-activated current, blocked by niflumic acid, not present in the non-transfected cells. Thus, CLCA4L mimics the CLCA1 current on its ability to induce the ANO1-dependent Ca2+-activated Cl- current endogenous to these cells. By immunocytochemistry, a CLCA protein, presumably CLCA4L, was detected in the cilia of olfactory sensory neurons co-expressing with ANO2. CONCLUSION: These findings suggests that a CLCA isoform, namely CLCA4L, expressed in OSN cilia, might have a regulatory function over the ANO2-dependent Ca2+-activated Cl- channel involved in odor transduction.

Expression of anoctamin 1 is associated with advanced tumor stage in patients with non-small cell lung cancer and predicts recurrence after surgery.

PURPOSE: Anoctamin 1 (ANO1), a recently identified calcium-activated chloride channel, has been found to have a critical role in tumorigenesis and tumor progression in several types of cancer. However, its role in non-small cell lung cancer (NSCLC) remains to be elucidated. In this study, we evaluated the utility of ANO1 as a prognostic marker. PATIENTS AND METHODS: ANO1 expression was detected in tumor tissues and paraneoplastic tissues of I-IV stage NSCLC patients who received surgical treatment by using immunohistochemical and quantitative RT-PCR analyses. Epidermal growth factor receptor (EGFR) was investigated using immunohistochemistry. Then the TNM stage of the tumor samples was assessed and patients were followed up for developing recurrence. RESULTS: ANO1 expression was significantly increased in NSCLC tumor tissues compared to the paraneoplastic tissues at both RNA and protein level. In addition, ANO1 overexpression was correlated with the high expression of EGFR and led to an advanced tumor stage. And also high ANO1 expression was significantly correlated with high recurrence rate at 1-year follow-up. CONCLUSIONS: ANO1 overexpression associated with the high expression of EGFR can be a predictive marker of recurrence after surgery in NSCLC patients.

Single Ca(2+)-activated Cl(-) channel currents recorded from toad olfactory cilia.

BACKGROUND: Odor transduction, occurring in the chemosensory cilia of vertebrate olfactory sensory neurons, is triggered by guanosine triphosphate-coupled odor receptors and mediated by a cyclic adenosine monophosphate (cAMP) signaling cascade, where cAMP opens cationic non-selective cyclic nucleotide-gated (CNG) channels. Calcium enters through CNG gates Ca(2+)-activated Cl(-) channels, allowing a Cl(-) inward current that enhances the depolarization initiated by the CNG-dependent inward current. The anoctamin channel 2, ANO2, is considered the main Ca(2+)-activated Cl(-) channel of olfactory transduction. Although Ca(2+)-activated Cl(-) channel-dependent currents in olfactory sensory neurons were reported to be suppressed in ANO2-knockout mice, field potentials from their olfactory epithelium were only modestly diminished and their smell-dependent behavior was unaffected, suggesting the participation of additional Ca(2+)-activated Cl(-) channel types. The Bestrophin channel 2, Best2, was also detected in mouse olfactory cilia and ClCa4l, belonging to the ClCa family of Ca(2+)-activated Cl(-) channels, were found in rat cilia. Best2 knock-out mice present no electrophysiological or behavioral impairment, while the ClCa channels have not been functionally studied; therefore, the overall participation of all these channels in olfactory transduction remains unresolved. RESULTS: We explored the presence of detectable Ca(2+)-activated Cl(-) channels in toad olfactory cilia by recording from inside-out membrane patches excised from individual cilia and detected unitary Cl(-) current events with a pronounced Ca(2+) dependence, corresponding to 12 and 24 pS conductances, over tenfold higher than the aforementioned channels, and a approx. fivefold higher Ca(2+) affinity (K0.5 = 0.38 µM). Remarkably, we observed immunoreactivity to anti-ClCa and anti-ANO2 antibodies in the olfactory cilia, suggesting a possible cooperative function of both channel type in chemotransduction. CONCLUSIONS: These results are consistent with a novel olfactory cilia channel, which might play a role in odor transduction.

Evidence for the participation of Ca(2+)-activated chloride channels in formalin-induced acute and chronic nociception.

In this study we determined the role of Ca(2+)-activated chloride channels (CaCC) in acute and chronic nociceptive responses elicited by 1% formalin. Formalin injection produced a typical pattern of flinching behavior for about 1h. Moreover, it produced secondary allodynia and hyperalgesia in the ipsilateral and contralateral paws for at least 6 days. Local peripheral and intrathecal pre-treatment (-10 min) with the non-selective and selective CaCC blockers niflumic acid and CaCCinh-A01, respectively, prevented formalin-induced flinching behavior mainly during phase 2 of the formalin test. Furthermore, niflumic acid and CaCCinh-A01 also prevented in a dose-dependent manner the long-lasting evoked secondary mechanical allodynia and hyperalgesia in the ipsilateral and contralateral paws. Moreover, local peripheral and intrathecal post-treatment (on day 6) with both CaCC blockers decreased the established formalin-induced secondary mechanical allodynia and hyperalgesia behavior in both paws. CaCC anoctamin-1 and bestrophin-1 were detected in the dorsal root ganglia. Formalin injection increased anoctamin-1, but not bestrophin-1 protein levels at 6 days. Intrathecal injection of the CaCC inhibitor CaCCinh-A01 prevented formalin-induced anoctamin-1 increase. Data suggest that peripheral and spinal CaCC, and particularly anoctamin-1, participates in the acute nociception induced by formalin as well as in the development and maintenance of secondary mechanical allodynia and hyperalgesia. Thus, CaCC activity contributes to neuronal excitability in the process of nociception induced by formalin.

TMEM16A alternative splicing isoforms in Xenopus tropicalis: distribution and functional properties.

Oocytes of Xenopus tropicalis elicit a Ca(2+)-dependent outwardly rectifying, low-activating current (ICl,Ca) that is inhibited by Cl(-) channel blockers. When inactivated, ICl,Ca shows an exponentially decaying tail current that is related to currents generated by TMEM16A ion channels. Accordingly, RT-PCR revealed the expression of five alternatively spliced isoforms of TMEM16A in oocytes, which, after expression in HEK-293 cells, gave rise to fully functional Cl(-) channels. Upon hyperpolarization to -80 mV a transient current was observed only in isoforms that carry the exon 1d, coding for two potentially phosphorylatable Threonine residues. The identified isoforms are differentially expressed in several tissues of the frog. Thus, it appears that X. tropicalis oocytes express TMEM16A that gives rise to a Ca(2+)-dependent Cl(-) current, which is different from the previously reported voltage-dependent outwardly rectifying Cl(-) current.