title: Na+-K+-Cl- cotransporter (NKCC) maintains the chloride gradient to sustain pacemaker activity in interstitial cells of Cajal.
abstract: Interstitial cells of Cajal (ICC) generate electrical slow waves by coordinated openings of ANO1 channels, a Ca2+-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 (ESTICs) 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 ESTICsto more negative potentials within a few minutes and reduced pacemaker activity. Bumetanide had no direct effects on ANO1 or CaV3.2 channels expressed in HEK293 cells or L-type Ca2+currents. Reducing extracellular Cl-to 10 mM shifted ESTICsto positive potentials as predicted by the Nernst equation. The relatively rapid shift in ESTICswhen 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.
authors.affiliation: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada.
authors.forename: Mei Hong
authors.initials: MH
authors.lastname: Zhu
authors.affiliation: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada.
authors.forename: Tae Sik
authors.initials: TS
authors.lastname: Sung
authors.affiliation: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kurahashi
authors.affiliation: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada.
authors.forename: Lauren E
authors.initials: LE
authors.lastname: O'Kane
authors.affiliation: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada.
authors.forename: Kate
authors.initials: K
authors.lastname: O'Driscoll
authors.affiliation: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada.
authors.forename: Sang Don
authors.initials: SD
authors.lastname: Koh
authors.affiliation: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada ksanders@medicine.nevada.edu.
authors.forename: Kenton M
authors.initials: KM
authors.lastname: Sanders
mesh_terms: D000200:Action Potentials; D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D020747:Calcium Channels, T-Type / Q000378:metabolism; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D056885:Interstitial Cells of Cajal / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D010507:Periodicity; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D064506:Solute Carrier Family 12, Member 2 / Q000235:genetics / Q000378:metabolism*
chemical_list: C504883:Cacna1h protein, mouse; D020747:Calcium Channels, T-Type; D002712:Chlorides; C578878:Slc12a2 protein, mouse; D049994:Sodium Potassium Chloride Symporter Inhibitors; D064506:Solute Carrier Family 12, Member 2; D002034:Bumetanide
keywords: ANO1; Ca2+-activated Cl− current; electrical slow waves; gastrointestinal motility; smooth muscle