Abstract
Anoctamins (ANOs) are multifunctional membrane proteins that consist of 10 homologs. ANO1 (TMEM16A) and ANO2 (TMEM16B) are anion channels activated by intracellular calcium that meditate numerous physiological functions. ANO6 is a scramblase that redistributes phospholipids across the cell membrane. The other homologs are not well characterized. We found ANO9/TMEM16J is a cation channel activated by a cAMP-dependent protein kinase A (PKA). Intracellular cAMP-activated robust currents in whole cells expressing ANO9, which were inhibited by a PKA blocker. A cholera toxin that persistently stimulated adenylate cyclase activated ANO9 as did the application of PKA. The cAMP-induced ANO9 currents were permeable to cations. The cAMP-dependent ANO9 currents were augmented by intracellular Ca2+. Ano9 transcripts were predominant in the intestines. Human intestinal SW480 cells expressed high levels of Ano9 transcripts and showed PKA inhibitor-reversible cAMP-dependent currents. We conclude that ANO9 is a cation channel activated by a cAMP/PKA pathway and could play a role in intestine function.
Highlights
The Anoctamin/TMEM16 family consists of transmembrane proteins in 10 isoforms, ranging from ANO1/TMEM16A to ANO10/TMEM16K
CAMP activates ANO9 To determine if ANO9 is a channel, we expressed mouse ANO9 tagged on the C terminus with enhanced green fluorescence protein for visual identification in HEK 293T cells
Upon whole-cell formation in the HEK 293T cells transfected with Ano9-enhanced green fluorescence protein (eGFP) (ANO9/HEK cells), robust inward currents (65.9 ± 6.99 pA/pF, n = 36) were observed with Ehold = −60 mV when the pipette solution contained 100 M cAMP
Summary
The Anoctamin/TMEM16 family consists of transmembrane proteins in 10 isoforms, ranging from ANO1/TMEM16A to ANO10/TMEM16K. The best known anoctamin gene is Ano, which is a Cl− channel activated by Ca2+ (Caputo et al, 2008; Schroeder et al, 2008; Yang et al, 2008). ANO1 is known to mediate transepithelial fluid movements such as salivation in the salivary glands, mucin secretion in the airway and Cl− and fluid secretion in the intestine (Huang et al, 2012a; Jang & Oh, 2014; Namkung et al, 2011; Ousingsawat et al, 2009; Romanenko et al, 2010). ANO1 is highly expressed in small-diameter dorsal-root ganglion neurons, implicating its role in nociception as a heat sensor (Cho et al, 2012). ANO1 has been implicated in tumorigenesis (Britschgi et al, 2013; Duvvuri et al, 2012; Jia et al, 2015) and benign prostate hyperplasia (Cha et al, 2015)
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