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

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.