Background

Colonic motility is regulated by coordinated interactions between electrically coupled smooth muscle cells (SMC) and non-contractile ICC – interstitial cells of Cajal (Sanders et al., 2014). In the gut, ICC exclusively express the Ca²⁺-activated-Cl^– channel, ANO1, activated by intracellular Ca²⁺ transients from endoplasmic reticulum (ER) stores (Zhu et al., 2015). ANO1 activation leads to depolarisation in ICC, and increased excitability of electrically coupled SMC, increasing opening of voltage-dependent Ca²⁺ channels and resulting excitation-contraction coupling (Sanders et al., 2024). Ca²⁺ transient firing in ICC must be continually sustained by regular ER Ca²⁺ refilling to prevent store depletion. This is accomplished by store-operated Ca²⁺ entry (SOCE), mediated by interactions between ER-bound STIM proteins and plasmalemmal Orai channels to allow Ca²⁺ influx for ER refilling (Zheng et al., 2018). Much of the evidence for the role of SOCE in ICC derives from pharmacological Orai channel inhibitors such as GSK 7975A, however effects of potentiating Orai channel activity on colonic contractility or behaviours of ICC are unknown.

 

Results

This study investigated the newly characterised Orai1 modulator IA65 (Azimi et al., 2020) on colonic smooth muscle contractile behaviour in isolated circular muscle strips from mouse proximal colon. In isometric tension recordings, spontaneous contractions developed following equilibration at a frequency of 3.3 ± 0.8 min^-1, with an average amplitude of 1.1 ± 0.9 mN and duration of 5.0 ± 2.5 sec (n=5, N=9). Following incubation with the Orai1 enhancer IA65 (1 – 3 micromolar), the average amplitude of contractions increased from 1.1 ± 0.9 mN to 1.9 ± 1.2 mN, while frequency decreased by 30.7 % (3 micromolar; n=5, N=9). Potentiated contractile amplitude in the presence of IA65 was reversed by the specific ANO1 antagonist Ani9 (1 µM, n=5) and the Orai channel inhibitor GSK 7975A (10 micromolar; n=4). Application of the muscarinic agonist carbachol (CCh; 1 micromolar) for 5 mins evoked repeatable responses consisting of an initial transient contraction, following by oscillatory contractions superimposed on a decaying baseline (n=5). While incubation with IA65 had no effect on the initial transient component of CCh responses, the second component was dramatically altered, with the baseline more sustained and oscillatory contractions converted to a tonic response, increasing the sustained contraction from 16.75 to 35.5 % (n=5). Using a mouse expressing a genetically encoded Ca²⁺ reporter exclusively in ICC (Kit-iCre-GCaMP6f), we performed whole tissue recordings of Ca²⁺ transient firing in two populations of ICC in proximal colon, intramuscular ICC (ICC-IM) and ICC at the myenteric plexus (ICC-MY). Both ICC were spontaneous active, generating Ca²⁺ transients in situ. 3 micromolar IA65 significantly increased Ca²⁺ transient frequency of ICC-IM (P=0.005, n=6, cells=19) and ICC-MY (P=0.005, n=5, cells=10) by 45%.

 

Conclusion

IA65, a pharmacological enhancer of Orai1 channels, increased Ca²⁺ transient firing in ICC-IM and ICC-MY in mouse proximal colon, leading to increased ANO1 activation in these cells. Enhanced activation of ANO1 increases the excitability of the colonic syncytium, increasing open probability of voltage-dependent Ca²⁺ channels in SMC, resulting in increased contractile amplitude.