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LES smooth muscle cells (SMCs) are relatively depolarised, facilitating activation of Cav<\/jats:sub>1.2 channels to sustain contractile tone. We hypothesised that intramuscular interstitial cells of Cajal (ICC\u2010IM), through activation of Ca2+<\/jats:sup>\u2010activated Cl\u2212<\/jats:sup> channels (ANO1), set membrane potentials of SMCs favourable for activation of Cav<\/jats:sub>1.2 channels. In some gastrointestinal muscles, ANO1 channels in ICC\u2010IM are activated by Ca2+<\/jats:sup> transients, but no studies have examined Ca2+<\/jats:sup> dynamics in ICC\u2010IM within the LES. Immunohistochemistry and qPCR were used to determine expression of key proteins and genes in ICC\u2010IM and SMCs. These studies revealed that Ano1<\/jats:italic> and its gene product, ANO1, are expressed in c\u2010Kit+<\/jats:sup> cells (ICC\u2010IM) in mouse and monkey LES clasp muscles. Ca2+<\/jats:sup> signalling was imaged in situ<\/jats:italic>, using mice expressing GCaMP6f specifically in ICC (Kit\u2010KI\u2010GCaMP6f). ICC\u2010IM exhibited spontaneous Ca2+<\/jats:sup> transients from multiple firing sites. Ca2+<\/jats:sup> transients were abolished by cyclopiazonic acid or caffeine but were unaffected by tetracaine or nifedipine. Maintenance of Ca2+<\/jats:sup> transients depended on Ca2+<\/jats:sup> influx and store reloading, as Ca2+<\/jats:sup> transient frequency was reduced in Ca2+<\/jats:sup> free solution or by Orai antagonist. Spontaneous tone of LES muscles from mouse and monkey was reduced \u223c80% either by Ani9, an ANO1 antagonist or by the Cav<\/jats:sub>1.2 channel antagonist nifedipine. Membrane hyperpolarisation occurred in the presence of Ani9. These data suggest that intracellular Ca2+<\/jats:sup> activates ANO1 channels in ICC\u2010IM in the LES. Coupling of ICC\u2010IM to SMCs drives depolarisation, activation of Cav<\/jats:sub>1.2 channels, Ca2+<\/jats:sup> entry and contractile tone.<\/jats:p><\/jats:sec>Key points<\/jats:title>\nThe lower oesophageal sphincter (LES) generates contractile tone preventing reflux of gastric contents into the oesophagus. LES smooth muscle cells (SMCs) display depolarised membrane potentials facilitating activation of L\u2010type Ca2+<\/jats:sup> channels.<\/jats:p><\/jats:list-item>\nInterstitial cells of Cajal (ICC) express Ca2+<\/jats:sup>\u2010activated Cl\u2212<\/jats:sup> channels encoded by Ano1<\/jats:italic> in mouse and monkey LES. Ca2+<\/jats:sup> signalling in ICC activates ANO1 currents in ICC.<\/jats:p><\/jats:list-item>\nICC displayed spontaneous Ca2+<\/jats:sup> transients in mice from multiple firing sites in each cell and no entrainment of Ca2+<\/jats:sup> firing between sites or between cells.<\/jats:p>\nInhibition of ANO1 channels with a specific antagonist caused hyperpolarisation of mouse LES and inhibition of tone in monkey and mouse LES muscles.<\/jats:p>\n<\/jats:list-item>\nOur data suggest a novel mechanism for LES tone in which Ca2+<\/jats:sup> transient activation of ANO1 channels in ICC generates depolarising inward currents that conduct to SMCs to activate L\u2010type Ca2+<\/jats:sup> currents, Ca2+<\/jats:sup> entry and contractile tone.<\/jats:p><\/jats:list-item>\n<\/jats:list><\/jats:p><\/jats:sec>","DOI":"10.1113\/jp282570","type":"journal-article","created":{"date-parts":[[2022,3,1]],"date-time":"2022-03-01T11:39:15Z","timestamp":1646134755000},"page":"2613-2636","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":26,"title":["Ca2+<\/sup> signalling in interstitial cells of Cajal contributes to generation and maintenance of tone in mouse and monkey lower oesophageal sphincters"],"prefix":"10.1113","volume":"600","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4762-844X","authenticated-orcid":false,"given":"Bernard T.","family":"Drumm","sequence":"first","affiliation":[{"name":"Department of Physiology and Cell Biology University of Nevada, Reno School of Medicine Reno NV USA"},{"name":"Smooth Muscle Research Centre Dundalk Institute of Technology Dundalk Co. 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