Stable, polarised, functional expression of Kir1.1b channel protein in Madin-Darby canine kidney cell line

• Published in: The Journal of physiology Vol. 528; no. 1; pp. 5 - 13
• Main Authors: Ortega, B., Millar, I. D., Beesley, A. H., Robson, L., White, S. J.
• Format: Journal Article
• Language: English
• Published: Oxford, UK The Physiological Society 01-10-2000; Blackwell Publishing Ltd; Blackwell Science Inc
• Subjects: Animals; Barium - pharmacology; Cell Line; Cell Membrane - metabolism; Cycloheximide - pharmacology; Dogs; Green Fluorescent Proteins; Kidney - cytology; Kidney - drug effects; Kidney - metabolism; Luminescent Proteins - genetics; Original; Patch-Clamp Techniques; Potassium - metabolism; Potassium Channel Blockers; Potassium Channels - biosynthesis; Potassium Channels - genetics; Potassium Channels, Inwardly Rectifying; Protein Synthesis Inhibitors - pharmacology; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Spectrometry, Fluorescence; Temperature; Transfection
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1111/j.1469-7793.2000.00005.x

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.
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 26°C.
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.
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.
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.