Cell swelling stimulates cytosol to membrane transposition of ICln.
ICln is a multifunctional protein that is essential for cell volume regulation. It can be found in the cytosol and is associated with the cell membrane. Besides its role in the splicing process, ICln is critically involved in the generation of ion currents activated during regulatory volume decrease after cell swelling (RVDC). If reconstituted in artificial bilayers, ICln can form ion channels with biophysical properties related to RVDC. We investigated (i) the cytosol versus cell membrane distribution of ICln in rat kidney tubules, NIH 3T3 fibroblasts, Madin-Darby canine kidney (MDCK) cells, and LLC-PK1 epithelial cells, (ii) fluorescence resonance energy transfer (FRET) in living fibroblasts between fluorescently tagged ICln and fluorochromes in the cell membrane, and (iii) possible functional consequences of an enhanced ICln presence at the cell membrane. We demonstrate that ICln distribution in rat kidneys depends on the parenchymal localization and functional state of the tubules and that cell swelling causes ICln redistribution from the cytosol to the cell membrane in NIH 3T3 fibroblasts and LLC-PK1 cells. The addition of purified ICln protein to the extracellular solution or overexpression of farnesylated ICln leads to an increased anion permeability in NIH 3T3 fibroblasts. The swelling-induced redistribution of ICln correlates to altered kinetics of RVDC in NIH 3T3 fibroblasts, LLC-PK1 cells, and MDCK cells. In these cells, RVDC develops more rapidly, and in MDCK cells the rate of swelling-induced depolarization is accelerated if cells are swollen for a second time. This coincides with an enhanced ICln association with the cell membrane.
Ritter
Markus
M
Department of Physiology, University of Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria. markus.ritter@uibk.ac.at
Ravasio
Andrea
A
Jakab
Martin
M
Chwatal
Sabine
S
Fürst
Johannes
J
Laich
Andreas
A
Gschwentner
Martin
M
Signorelli
Sara
S
Burtscher
Carmen
C
Eichmüller
Sonja
S
Paulmichl
Markus
M
D000818:Animals; D000838:Anions; D055592:Biophysical Phenomena; D001703:Biophysics; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007150:Immunohistochemistry; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism*; D007477:Ions; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D018374:LLC-PK1 Cells; D008051:Lipid Bilayers; D008297:Male; D051379:Mice; D041681:NIH 3T3 Cells; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016622:Silver Staining; D013552:Swine; D013997:Time Factors; D014162:Transfection
D000838:Anions; C498728:Clns1a protein, mouse; C498729:Clns1a protein, rat; D007473:Ion Channels; D007477:Ions; D008051:Lipid Bilayers; D011993:Recombinant Fusion Proteins