Identification and regulation of whole-cell Cl⁻ and Ca²⁺-activated K⁺ currents in cultured medullary thick ascending limb cells

Abstract

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 (E_k) 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.