Identification and regulation of whole-cell Cl- and Ca(2+)-activated K+ currents in cultured medullary thick ascending limb cells.
The whole-cell patch-clamp technique has been used to study membrane currents in cultured rabbit medullary thick ascending limb (MTAL) epithelial cells. A Ca(2+)-activated K+ current was characterized by its voltage-dependent and Ca(2+)-dependent properties. When the extracellular K+ ion concentration was increased from 2 to 140 mM, the re-reversal potential (Ek) was shifted from -85 to 0 mV with a slope of 46 mV per e-fold change. The Ca(2+)-activated K+ current is blocked by charybdotoxin (CTX) in a manner similar to the apical membrane Ca(2+)-activated K+ channel studied with the single channel patch-clamp technique. The results suggest that the Ca(2+)-activated K+ current is the predominant, large conductance and Ca(2+)-dependent K+ pathway in the cultured MTAL cell apical membrane. The biophysical properties and physiological regulation of a Cl- current were also investigated. This current was activated by stimulation of intracellular cAMP using forskolin and isobutyl-1-methylxanthine (IBMX). The current-voltage (I-V) relationship of the Cl- current showed an outward-rectifying pattern in symmetrical Cl- solution. The Cl- selectivity of the whole-cell current was confirmed by tail current analysis in different Cl- concentration bath solutions. Several Cl- channel blockers were found to be effective in blocking the outward-rectifying Cl- current in MTAL cells. The cAMP-dependent Cl- transport in MTAL cells was further confirmed by measuring changes in the intensity of Cl- sensitive dye using fluorescence microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Physiology and Biophysics, Wright State University School of Medicine, Dayton, Ohio 45435.
Markakis
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Guggino
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D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000493:pharmacokinetics / Q000494:pharmacology*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D018999:Charybdotoxin; D018118:Chloride Channels / Q000502:physiology; D002712:Chlorides / Q000032:analysis / Q000493:pharmacokinetics / Q000494:pharmacology*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D004847:Epithelial Cells; D004848:Epithelium / Q000737:chemistry / Q000502:physiology; D007686:Kidney Tubules, Distal / Q000737:chemistry / Q000166:cytology* / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D011188:Potassium / Q000032:analysis; D015221:Potassium Channels / Q000032:analysis / Q000187:drug effects / Q000502:physiology*; D011817:Rabbits; D012604:Scorpion Venoms / Q000494:pharmacology
D018118:Chloride Channels; D002712:Chlorides; D015221:Potassium Channels; D012604:Scorpion Venoms; D018999:Charybdotoxin; D005576:Colforsin; D000242:Cyclic AMP; D011188:Potassium; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine