IDENTIFICATION AND REGULATION OF WHOLE-CELL CL- AND CA2+-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 Ca2+-activated K+ current was characterized by its voltage-dependent and Ca2+-dependent properties. When the extracellular K+ ion concentration was increased from 2 to 140 mm, the rereversal potential (Ek) was shifted from -85 to 0 mV with a slope of 46 mV per e-fold change. The Ca2+-activated K+ current is blocked by charybdotoxin (CTX) in a manner similar to the apical membrane Ca2+-activated K+ channel studied with the single channel patch-clamp technique. The results suggest that the Ca2+-activated K+ current is the predominant, large conductance and Ca2+-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. These results suggest that the Cl- channel in the apical or basolateral membrane of MTAL cells may be regulated by cAMP-dependent protein-kinase-induced phosphorylation.