Page 43 - Q. Neuroscience

Anoctamin 8/TMEM16H is a cAMP/PKA activated cation channel
in the nociceptive pathway

Huan-Jun Lu, Thien-Luan Nguyen, Hyungsup Kim, Hyesu Kim and Uhtaek Oh *
Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea.
Abstract Intracellular calcium amplifies ANO8 currents.
The Anoctamin (ANO, or TMEM16) gene family consists of ten
isoforms. ANO1 and ANO2 are identified as Ca2+-activated anion cha
nnel ANO6 is a lipid scramblase disrupting polarized phospholipids in
the membrane. However, the function of ANO8 is not known. Here we
found that ANO8/TMEM16H is a cAMP-activated cation channel.
Intracellular cAMP-induced robust current when heterologously
expressed in mammalian cells. The cAMP-dependent currents were
inhibited by a protein kinase-A inhibitor, suggesting the active role of
protein kinase A in its activation mechanism. A cholera toxin, an
activator of adenylate cyclase also activated ANO8. The cAMP-induce
currents in ANO8-expressing cells were cationic, which did not
discriminate among cations. ANO8 is highly expressed in neurons in
the brain regions as well as dorsal root ganglion neurons. The
knockdown of Ano8 causes a decrease in cAMP-dependent currents in
dorsal root ganglion neurons as well as nociceptive behaviors. These
results now suggest that ANO8 is a cation channel activated by the cA
MP/pathway and involved in nociception in the pain pathway. (a) Representative traces of cAMP activated currents in ANO8-HEK cells with different concentrations of Ca2+.
(b) Summary of the cAMP-induced whole -cell currents of ANO8-HEK cells in 1 or 10um Ca2+ .
Results
Ano8 is activated by intracellular c-AMP ANO8 is highly expressed in nociceptive neurons.

(a) The image of overexpression of mAno8-pEGFP-N1 in the HEK293T cells with Hoechst 33342 and f-actin
staining. Scale bar represents 50 μm. (b) Representative traces of whole-cell currents in HEK cells transfected with
GFP and Ano8(up left and right) and Ano8 overexpression cells treatment with MONNA, Ano1 antagonist, and
H89 (below left and right). (c) Summary of the cAMP-induced whole -cell currents in GFP and ANO8-HEK cells a
nd H89, MONNA pre-treated ANO8 transfected HEK cells. (d) Representative traces of cholera toxin (CTX)
activated currents in ANO8- HEK cells. (e) Summary of the CTX-induced whole -cell currents in GFP and ANO8-
HEK cells.
Ano8 is cation channel and not sensitive to voltage.

(a) mRNA transcript levels of ANO8 in various mouse tissue organs. Note that ANO8 is highly expressed central
nerve system and DRGs. (b) Immunofluorescent images of ANO8 in DRGs. The DRG neurons were stained with
IB4, CGRP, neurofilament H (NFH) and TRPV1. Scale bar represents 50 μm. (c) The mRNA expression of ANO8
in DRG neurons assayed by RT-PCR. (d) Proportion of IB4+, CGRP+, NFH+ and TRPV1+ neurons among
ANO8+ DRG neurons. (e) Representative traces of cAMP induced whole currents of scr treatment or siANO8
treatment DRG neurons. (f) The average current amplitude of scr treatment or siANO8 treatment DRG neurons a
ctivated by intracellular cAMP. (g) The knock-down effeciency of ANO8 siRNA.
Conclusion

 Anoctamin 8/TMEM16H is a newly discovered
cAMP/PKA activated cation channel.
 Anoctamin 8/TMEM16H highly expresses in nociceptive
DRG neurons.
(a) The cAMP induced whole-cell currents were observed in NaCl (left) but not in NMDG-Cl (right) bath solution.  Anoctamin 8/TMEM16H may mediate pain sensation in
(b) Summary of the cAMP-induced whole-cell currents of ANO8-HEK cells in NaCl and NMDG-Cl bath solution.
(c) Reversal potentials in KCl concentration gradient between pipette and bath solution. (d) I-V curves of ANO8 the nociceptive pathway.
currents obtained at different salt conditions in ANO8 overexpressed HEK cells. The pipette solution contained
140mM KCl. (e) Cation permeability ratios(Px/PCs). (f) Representative traces of whole currents of ANO8 transfected
HEK cells or GFP transfected HEK cells. activated by voltage. Voltage steps from -120mV to +120mV in 10 mV
increments. (g) I-V curves of ANO8-HEK cells activated by voltage.