Ca2+-activated-Cl–channels encoded by ANO1 are exclusively expressed in specialized interstitial cells of Cajal (ICC) in the gastrointestinal (GI) tract muscularis, and serves as the ubiquitous excitatory conductance that generates electrical pacemaker activity and excitatory post-junctional neuronal responses in these cells (Sanders et al., 2026). Activation of ANO1 in ICC leads to depolarization, which is transduced to electrically coupled smooth muscle cells to coordinate regular contractions that underlie GI motility. The importance of ANO1 to normal gut function is demonstrated by the perturbation of normal electrical and contractile behaviours of GI tissues by genetic ablation of ANO1 or selective pharmacological inhibitors of ANO1 (such as Ani9). However, in many GI tract pathophysiological disorders, increasing regular contractions would be desirable (such as delayed gastric emptying or slow transit constipation), however, to date the effects of increasing ANO1 activity on GI tissues to accomplish this has not been characterised. Recently, a small molecule that acts as a positive allosteric modulator of ANO1, PAM_16A, was identified and characterized to enhance ANO1-dependent changes in vascular smooth muscle tone (Al-Hosni et al., 2025). In our study, we sought to evaluate the functional effects of PAM_16A on GI contractility and electrical activity.
We performed isometric tension recordings of circularly orientated strips of mouse gastric antrum and proximal colon (C57/wild-type, 10-16 weeks old), both of which generated spontaneous phasic contractions after an initial application of 5 mN of stretch. In gastric antrum, application of PAM_16A (300 nM – 1 mM) lead to an enhancement in the amplitude of spontaneous contractile amplitude by 42 % (n=5). Similarly, in strips of proximal colon, 300 nM PAM_16A enhanced contractile amplitude by 45% and this was further potentiated to an 80% increase when the concentration of PAM_16A was increased to 1 mM (n=3). In both gastric antrum and proximal colon, application of the ANO1 inhibitor Ani9 (1 mM) significantly reduced contractile activity in the continued presence of PAM_16A (abolishing it in the proximal colon (n=3) and reduced gastric antrum contraction amplitude to 4 % of control (n=x)). In antrum and colon, application of the muscarinic agonist carbachol (CCh, 300 nM – 1 mM) evoked a large amplitude, sustained contraction with superimposed phasic contractions over the length of agonist exposure. In gastric antrum and proximal colon, PAM_16A (1 mM) enhanced the amplitude of the CCh-evoked superimposed phasic contractions by 27 % and 30 % respectively, and these effects were reversed by Ani9, with neither drug affecting the sustained component of CCh responses. In intracellular recordings, spontaneous electrical slow waves were recorded from mouse gastric antrum. Application of PAM 16_A (300 nM) accelerated the upstroke component, increasing the initial amplitude of slow waves and caused a slight depolarization of resting membrane potential.
Our preliminary data suggests that the small molecule PAM_16A enhances electrical and contractile activity of mouse gastric antrum and proximal colon, resulting in increased contractile amplitude. PAM_16A may be a potentially effective pharmacological tool to probe the effects of ANO1 channel modulation in GI tissues.