Stable, polarised, functional expression of Kir1.1b channel protein in Madin-Darby canine kidney cell line.
1. The family of Kir1.1 (ROMK) channel proteins constitute a secretory pathway for potassium in principal cells of cortical collecting duct and thick ascending limb of Henle's loop. Mutations in Kir1.1 account for some types of Bartter's syndrome. 2. Here we report that stable transfection of Kir1.1b (ROMK2) in Madin-Darby canine kidney (MDCK) cell line results in expression of inwardly rectifying K+ currents and transmonolayer electrical and transport properties appropriate to Kir1.1 function. When grown on permeable supports, transfected monolayers secreted K+ into the apical solution. This secretion was inhibited by application of barium to the apical membrane, or by reduction in expression temperature from 37 to 26 C. However, whole-cell voltage clamp electrophysiology showed that K+ conductance was higher in cells expressing Kir1.1b at 26C. 3. To investigate this further, Kir1.1b was tagged with (EGFP), a modification that did not affect channel activity. Protein synthesis was inhibited with cycloheximide. Spectrofluorimetry was used to compare protein degradation at 37 and 26 C. The increased level of Kir1.1b at the plasma membrane at 26 C was due to an increase in protein stability. 4. Confocal microscopic investigation of EGFP-Kir1. 1b fluorescence in transfected cells showed that the channel protein was targeted to the apical domain of the cell. 5. These results demonstrate that Kir1.1b is capable of appropriate trafficking and function in MDCK cell lines at physiological temperatures. In addition, expression of Kir1.1b in MDCK cell lines provides a useful and convenient tool for the study of functional activity and targeting of secretory K+ channels.
Ortega
B
B
Laboratory for Membrane Protein Function, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
Millar
I D
ID
Beesley
A H
AH
Robson
L
L
White
S J
SJ
D000818:Animals; D001464:Barium / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004285:Dogs; D049452:Green Fluorescent Proteins; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000096:biosynthesis* / Q000235:genetics; D024661:Potassium Channels, Inwardly Rectifying; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014162:Transfection
D008164:Luminescent Proteins; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001464:Barium; D003513:Cycloheximide; D011188:Potassium