CFTR mediates electrogenic chloride secretion in mouse inner medullary collecting duct (mIMCD-K2) cells.
Previously we demonstrated that the inner medullary collecting duct cell line mIMCD-K2 secretes Cl- by an electrogenic mechanism [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995; N. L. Kizer, D. Vandorpe, B. Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F854-F861, 1995]. The goal of the present study was to characterize the Cl- channel responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl- secretion. To this end, using the patch-clamp technique, we measured Cl- currents. In whole cell patch-clamp experiments, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) activated Cl- currents that were time and voltage independent, inhibited by diphenylamine 2-carboxylate (DPC), and had a linear current-voltage (I-V) relation. In cell-attached patches of the apical membrane, we identified 7-pS Cl- channels that were stimulated by CPT-cAMP. In inside-out patches with Cl- in the pipette and bath solutions, Cl- currents had a linear I-V relation. The halide permeability sequence was PCl = PBr > PI. The Cl- channel inhibitors DPC, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and glibenclamide blocked the 7-pS Cl- channel, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was ineffective. By reverse transcriptase polymerase chain reaction, we isolated a partial cDNA clone encoding the cystic fibrosis transmembrane conductance regulator in mIMCD-K2 cells. We conclude that cAMP stimulates electrogenic Cl- secretion in inner medullary collecting duct cells by activating cystic fibrosis transmembrane conductance regulator Cl- channels.
Vandorpe
D
D
Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Kizer
N
N
Ciampollilo
F
F
Moyer
B
B
Karlson
K
K
Guggino
W B
WB
Stanton
B A
BA
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000235:genetics / Q000502:physiology; D002712:Chlorides / Q000378:metabolism*; D003001:Cloning, Molecular; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D004553:Electric Conductivity; D007679:Kidney Medulla; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000378:metabolism
D018118:Chloride Channels; D002712:Chlorides; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator