CFTR surface expression and chloride currents are decreased by inhibitors of N-WASP and actin polymerization

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid turnover at the plasma membrane in various cell types. The ubiquitously expressed N-WASP promotes actin polymerization and regulates endocytic trafficking of other proteins in response to signaling molecules such as Rho-GTPases. In the present study we investigated the effects of wiskostatin, an N-WASP inhibitor, on the surface expression and activity of CFTR. We demonstrate, using surface biotinylation methods, that the steady-state surface CFTR pool in stably transfected BHK cells was dramatically decreased following wiskostatin treatment with a corresponding increase in the amount of intracellular CFTR. Similar effects were observed for latrunculin B, a specific actin-disrupting reagent. Both reagents strongly inhibited macroscopic CFTR-mediated Cl(-) currents in two cell types including HT29-Cl19A colonic epithelial cells. As previously reported, CFTR internalization from the cell surface was strongly inhibited by a cyclic-AMP cocktail. This effect of cyclic-AMP was only partially blunted in the presence of wiskostatin, which raises the possibility that these two factors modulate different steps in CFTR traffic. In kinetic studies wiskostatin appeared to accelerate the initial rate of CFTR endocytosis as well as inhibit its recycling back to the cell surface over longer time periods. Our studies implicate a role for N-WASP-mediated actin polymerization in regulating CFTR surface expression and channel activity.

Src homology domain 2 adaptors affect adherence of Salmonella enterica serovar Typhimurium to non-phagocytic cells

“…Accordingly, Nck and Shc could affect receptor trafficking through their interactions with the actin cytoskeleton. For example, N-WASP has recently been shown to be critical for surface localization of CFTR by affecting both endocytosis and endocytic recycling of CFTR (Ganeshan et al, 2007). Because Nck is a potent upstream regulator of N-WASP (Tomasevic et al, 2007) it may also be involved in these processes.…”

“…Is there evidence that Nck and Shc are involved in receptor trafficking? The actin cytoskeleton actively participates in the recycling of certain G-protein-coupled receptors, the transferrin receptor, glucose transporter 4 and CFTR (Ganeshan et al, 2007;Jiang et al, 2002;Stanasila et al, 2006;Yan et al, 2005). Accordingly, Nck and Shc could affect receptor trafficking through their interactions with the actin cytoskeleton.…”

Modulation of TMEM16A-Channel Activity as Ca2+ Activated Cl− Conductance via the Interaction With Actin Cytoskeleton in Murine Portal Vein

“…The membrane expression of cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channel is decreased by actin disruption (13). Actin disruption attenuates CFTR Cl − current and activates BK Ca current and ClC-2 current (10,13,14).…”

VIP as a Corrector of CFTR Trafficking and Membrane Stability

“…The tyrosine based motif interacts with the clathrin adapter complex AP-2 to enter into clathrin coated pits. CFTR also interacts with actin-binding proteins like myosin VI and to N-SWAP to recycle back to the plasma membrane via recycling endosomes (Okiyoneda and Lukacs, 2007;Swiatecka-Urban et al, 2004;Ganeshan et al, 2006). Conflicting results exist regarding the role of cAMP or other second messengers in CFTR recycling probably due to disparity in epithelial cells studied or the use of over-expressing systems which might saturate the normal pathway for CFTR trafficking.…”