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Efflux of Cl<jats:sup>\u2212<\/jats:sup>during slow waves must be significant, as there is high current density during slow-wave currents and slow waves are of sufficient magnitude to depolarize the syncytium of smooth muscle cells and PDGFR\u03b1<jats:sup>+<\/jats:sup>cells to which they are electrically coupled. We investigated how the driving force for Cl<jats:sup>\u2212<\/jats:sup>current is maintained in ICC. We found robust expression of Slc12a2 (which encodes an Na<jats:sup>+<\/jats:sup>-K<jats:sup>+<\/jats:sup>-Cl<jats:sup>\u2212<\/jats:sup>cotransporter, NKCC1) and immunohistochemical confirmation that NKCC1 is expressed in ICC. With the use of the gramicidin permeabilized-patch technique, which is reported to not disturb [Cl<jats:sup>\u2212<\/jats:sup>]<jats:sub>i<\/jats:sub>, the reversal potential for spontaneous transient inward currents ( E<jats:sub>STICs<\/jats:sub>) was \u221210.5 mV. This value corresponds to the peak of slow waves when they are recorded directly from ICC in situ. Inhibition of NKCC1 with bumetanide shifted E<jats:sub>STICs<\/jats:sub>to more negative potentials within a few minutes and reduced pacemaker activity. Bumetanide had no direct effects on ANO1 or Ca<jats:sub>V<\/jats:sub>3.2 channels expressed in HEK293 cells or L-type Ca<jats:sup>2+<\/jats:sup>currents. Reducing extracellular Cl<jats:sup>\u2212<\/jats:sup>to 10 mM shifted E<jats:sub>STICs<\/jats:sub>to positive potentials as predicted by the Nernst equation. The relatively rapid shift in E<jats:sub>STICs<\/jats:sub>when NKCC1 was blocked suggests that significant changes in the transmembrane Cl<jats:sup>\u2212<\/jats:sup>gradient occur during the slow-wave cycle, possibly within microdomains formed between endoplasmic reticulum and the plasma membrane in ICC. Recovery of Cl<jats:sup>\u2212<\/jats:sup>via NKCC1 might have additional consequences on shaping the waveforms of slow waves via Na<jats:sup>+<\/jats:sup>entry into microdomains.<\/jats:p>","DOI":"10.1152\/ajpgi.00277.2016","type":"journal-article","created":{"date-parts":[[2016,10,15]],"date-time":"2016-10-15T02:59:53Z","timestamp":1476500393000},"page":"G1037-G1046","source":"Crossref","is-referenced-by-count":34,"title":["Na<sup>+<\/sup>-K<sup>+<\/sup>-Cl<sup>\u2212<\/sup>cotransporter (NKCC) maintains the chloride gradient to sustain pacemaker activity in interstitial cells of Cajal"],"prefix":"10.1152","volume":"311","author":[{"given":"Mei Hong","family":"Zhu","sequence":"first","affiliation":[{"name":"Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tae Sik","family":"Sung","sequence":"additional","affiliation":[{"name":"Department of Physiology and Cell Biology, 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