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.