Purification and characterization of epithelial Ca(2+)-activated K+ channels.
Reabsorption of NaCl in the thick ascending limb of Henle's loop in the kidney and in the surface cells in the distal colon involves the integrated function of several membrane transport systems including ion channels, the Na,K,Cl-cotransport system and the Na,K-pump. To determine if their properties are consistent with a role in regulation of transepithelial transport, Ca(2+)-activated K+ channels from the luminal membrane of the TAL cells and from the basolateral membrane of the distal colon cells have been characterized by flux studies in plasma membrane vesicle preparations and by single channel measurements in lipid bilayers. The channels are found to be activated by Ca2+ in the physiological range of concentration with a strong dependence on intracellular pH and the membrane potential. The Ca(2+)-sensitivity of the K+ channels is modulated by phosphorylation and dephosphorylation and the K+ channel protein must be in a phosphorylated state to respond to intracellular concentrations of Ca2+. As a step towards purification of the K+ channel proteins, procedures for solubilization and reconstitution of the K+ channels have been developed. The observation that the epithelial Ca(2+)-activated K+ channels bind calmodulin in the presence of Ca2+ have allowed for partial purification of the K+ channel proteins by calmodulin affinity chromatography. In the sequences for the two cloned Ca(2+)-activated K+ channels, the mSlo channel and the slowpoke channel, putative calmodulin binding regions can be identified.
Klaerke
D A
DA
Biomembrane Research Centre, August Krogh Institute, University of Copenhagen, Denmark.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003106:Colon / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008051:Lipid Bilayers / Q000378:metabolism; D008138:Loop of Henle / Q000378:metabolism; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D024681:Potassium Channels, Calcium-Activated
D002147:Calmodulin; C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008051:Lipid Bilayers; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D002118:Calcium