Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles

Specialized cells, referred to as interstitial cells of Cajal (ICC), generate and actively propagate the spontaneous electrical activity known as slow waves in the gastrointestinal (GI) tract (Ward et al. 1994, Huizinga et al. 1995; Sanders, 1996). Slow waves time the phasic contractions of GI muscles and provide the underlying organization of excitability for gastric peristalsis and intestinal segmentation. ICC are also interposed between nerve terminals and smooth muscle cells and serve as sites of post-junctional transduction of responses to enteric motor neurotransmitters (see Burns et al. 1996; Ward et al. 2000a). There are at least two classes of ICC, those intermingled within muscle bundles of the circular (CM) and longitudinal muscle (LM) layers (ICC-IM), and those arranged in a dense network between the CM and LM (ICC-MY), at the submucosal surface of the CM (ICC-SM) and in the septa between bundles of smooth muscle cells (ICC-SEP). There have been many studies regarding the mechanisms of pacemaker activity and slow wave propagation, but the precise mechanisms of this behaviour remain controversial. Studies of pacemaker activity in intact muscle strips and bundles have suggested the involvement of a Ca2+-dependent inward current because activity was reduced when the muscles were treated with membrane-permeable Ca2+ buffers (Edwards et al. 1999; Hirst et al. 2002; Kito & Suzuki, 2003). The Ca2+-dependent conductance has been thought to be a Cl− conductance, since a variety of Cl− channel blocking drugs reduced pacemaker activity in guinea-pig and murine muscles (see Hirst et al. 2002; Kito et al. 2002a; Kito & Suzuki, 2003). Similar conclusions were reached from studies of slow waves recorded directly from ICC-MY of the small intestine (e.g. Kito & Suzuki, 2003). In contrast, studies of isolated and cultured ICC have suggested that spontaneous activity is generated by activation of a non-selective cation conductance (Ward et al. 2000; Koh et al. 2002; Sanders et al. 2006), and the putative conductance was found to be inhibited by Ca2+ (Koh et al. 2002). Thus, pacemaker current may be initiated by a transient reduction in [Ca2+]i in a sub-compartment under the plasma membrane containing the non-selective cation conductance (Sanders et al. 2006). No Ca2+-activated inward currents were observed in cultured ICC, and the non-selective cation channels activated by reduced Ca2+ were inhibited by niflumic acid (Koh et al. 2002). Thus, use of Cl− channel antagonists does not necessarily indicate a role for Ca2+- activated Cl− channels in pacemaker activity. A microarray genetic screen recently revealed that Tmem16a is expressed at far greater levels in ICC than in the rest of the muscularis (Chen et al. 2007). Tmem16a encodes ANO1, a Ca2+-activated Cl− channel (Caputo et al. 2008; Schroeder et al. 2008; Yang et al. 2008), and immunohistochemical studies have documented expression of ANO1 (also known as DOG1) protein by ICC (Espinosa et al. 2008; Gomez-Pinilla et al. 2009). Taken together these data suggest the hypothesis that expression and function of these channels may be important in pacemaker activity in the GI tract. Therefore, we have characterized expression of Tmem16a transcripts and ANO1 protein in the tunica muscularis of mouse, monkey (Macaca fascicularis) and human GI tracts using RT-PCR, amplicon sequencing and immunohistochemical techniques. We also evaluated the electrical activity of murine gastric and small intestine muscles, and tested the effects of Cl− channel-blocking drugs. Finally, we tested whether slow wave activity is affected in mice homozygous with null Tmem16a alleles (Tmem16atm1Bdh/tm1Bdh, see Rock et al. 2008). Our data show ubiquitous expression of ANO1 in ICC throughout the GI tract and inhibitory effects of Cl− channel blocking drugs on slow waves. Tmem16a−/− animals failed to generate slow waves, and pacemaker activity did not develop in organ culture after birth, as occurs in wildtype muscles. Together with voltage-clamp studies of isolated ICC (Zhu et al. 2009), our findings strongly support a role for ANO1 in the generation of slow wave currents of GI ICC and electrical slow waves in intact muscles. The model of pacemaker activity deduced from previous studies of cultured ICC (e.g. as detailed in Sanders et al. 2006) will require reconsideration in light of these new findings.