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

Luo Lu, Diane Markakis, William B. Guggino

Research output: Contribution to journalArticle

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 m m, 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.

Original languageEnglish (US)
Pages (from-to)181-189
Number of pages9
JournalThe Journal of Membrane Biology
Volume135
Issue number2
DOIs
StatePublished - Aug 1 1993

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Keywords

  • Ca-activated K current
  • Cl current
  • MTAL epithelial cells
  • Patch clamp
  • SPQ fluorescence

ASJC Scopus subject areas

  • Biophysics
  • Physiology
  • Cell Biology

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