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Interstitial cells of Cajal (ICC) generate electrical slow waves by coordinated openings of ANO1 channels, a Ca 2+ -activated Cl −  (CaCC) conductance. Efflux of Cl −  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 PDGFRα +  cells to which they are electrically coupled. We investigated how the driving force for Cl −  current is maintained in ICC. We found robust expression of Slc12a2 (which encodes an Na + -K + -Cl −  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 − ] i , the reversal potential for spontaneous transient inward currents ( E STICs ) was −10.5 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 STICs  to more negative potentials within a few minutes and reduced pacemaker activity. Bumetanide had no direct effects on ANO1 or Ca V 3.2 channels expressed in HEK293 cells or L-type Ca 2+  currents. Reducing extracellular Cl −  to 10 mM shifted E STICs  to positive potentials as predicted by the Nernst equation. The relatively rapid shift in E STICs  when NKCC1 was blocked suggests that significant changes in the transmembrane Cl −  gradient occur during the slow-wave cycle, possibly within microdomains formed between endoplasmic reticulum and the plasma membrane in ICC. Recovery of Cl −  via NKCC1 might have additional consequences on shaping the waveforms of slow waves via Na +  entry into microdomains.

american journal of physiology. gastrointestinal and liver physiology/american journal of physiology: gastrointestinal and liver physiology

Na+-K+-Cl−cotransporter (NKCC) maintains the chloride gradient to sustain pacemaker activity in interstitial cells of Cajal