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Actin Filaments Interact with the Cytosolic Tails and Regulate Hypotonic Activation of ClC‐3
Author(s) -
McCloskey Diana Teresa,
Doherty Lynda,
Dai YanPing,
Miller Lisa,
Hume Joseph R.,
Yamboliev Ilia A.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a963-a
Subject(s) - actin , cytosol , pipette , cytoskeleton , actin cytoskeleton , chemistry , microbiology and biotechnology , biophysics , actin binding protein , amino acid , biochemistry , cell , biology , enzyme
Previous studies have shown that remodeling of the actin cytoskeleton modulates hypotonic activation of the volume‐sensitive outwardly rectifying anion current (VSOAC). Human short ClC‐3 has been shown to contribute to VSOAC in various cell types. We determined the amino acid domains of the cytosolic termini of ClC‐3 that interact with actin. We further examined how inhibition of this actin‐ClC‐3 binding affected VSOAC activation. Association between the cytosolic tails of ClC‐3 and actin was assessed by cosedimentation assays in vitro. Synthetic peptides (CTP1, CTP2, CTP3), mapping basic clusters of the cytosolic C‐terminus of ClC‐3, were dialyzed into NIH/3T3 cells via the patch pipette under hypotonic conditions. The C‐terminus exhibited significantly higher binding affinity for both G‐ and F‐actin compared to the N‐terminus of ClC‐3. Cosedimentation assays using progressively truncated C‐termini indicated that the F‐actin binding region of ClC‐3 is located between amino acids 690‐760. Dialysis through the patch pipette of CTP3 (aa 688–709) and CTP2 (aa 716–734), but not CTP1 (aa 737–748), reduced the maximal VSOAC current density in NIH/3T3 cells by 60% and 38%, respectively. We conclude that by direct interaction with subcortical actin filaments, ClC‐3 contributes to the hypotonic stress‐induced VSOAC in NIH/3T3 cells. This study was supported by NIH grants P20RR 15581 and HL49254.