Hypotonic activation of short ClC3 isoform is modulated by direct interaction between its cytosolic C-terminal tail and subcortical actin filaments.
Short ClC3 isoform (sClC3) functions as a volume-sensitive outwardly rectifying anion channel (VSOAC) in some cell types. In previous studies, we have shown that the hypotonic activation of sClC3 is linked to cell swelling-mediated remodeling of the actin cytoskeleton. In the present study, we have tested the hypothesis that the cytosolic tails of sClC3 bind to actin directly and that binding modulates the hypotonic activation of the channel. Co-sedimentation assays in vitro demonstrated a strong binding between the glutathione S-transferase-fused cytosolic C terminus of sClC3 (GST-sClC3-CT) to filamentous actin (F-actin) but not to globular monomeric actin (G-actin). The GST-fused N terminus (GST-sClC3-NT) exhibited low binding affinity to both G- and F-actin. Co-sedimentation experiments with progressively truncated GST-sClC3-CT indicated that the F-actin binding region is located between amino acids 690 and 760 of sClC3. Two synthetic peptides mapping basic clusters of the cytosolic sClC3-CT (CTP2, isoleucine 716 to leucine 734; and CTP3, proline 688 to proline 709) prevented binding of GST-sClC3-CT to F-actin in vitro. Dialysis into NIH/3T3 cells of these two peptides (but not of synthetic peptide CTP1 (isoleucine 737 to glutamine 748)) reduced the maximal current density by 60 and 38%, respectively. Based on these results, we have concluded that, by direct interaction with subcortical actin filaments, sClC3 contributes to the hypotonic stress-induced VSOACs in NIH/3T3 cells.
McCloskey
Diana T
DT
Department of Pharmacology and Center of Biomedical Research Excellence, University of Nevada School of Medicine, Reno, Nevada 89557, USA.
Doherty
Lynda
L
Dai
Yan-Ping
YP
Miller
Lisa
L
Hume
Joseph R
JR
Yamboliev
Ilia A
IA
D000199:Actins / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D018118:Chloride Channels / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009994:Osmolar Concentration; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000199:Actins; D018118:Chloride Channels; C115376:ClC-3 channel; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase