title: Membrane-permeable calmodulin inhibitors (e.g. W-7/W-13) bind to membranes, changing the electrostatic surface potential: dual effect of W-13 on epidermal growth factor receptor activation.
abstract: Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca2+/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca2+/CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR; W-13 inhibits epidermal growth factor-dependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due to W-13 inhibition of Ca2+/CaM, but the latter results could be due to binding of W-13 to the plasma membrane.
authors.affiliation: Department of Physiology and Biophysics, Health Science Center, State University of New York at Stony Brook, Stony Brook, New York 11794-8661, USA.
authors.forename: Parijat
authors.initials: P
authors.lastname: Sengupta
authors.forename: María José
authors.initials: MJ
authors.lastname: Ruano
authors.forename: Francesc
authors.initials: F
authors.lastname: Tebar
authors.forename: Urszula
authors.initials: U
authors.lastname: Golebiewska
authors.forename: Irina
authors.initials: I
authors.lastname: Zaitseva
authors.forename: Carlos
authors.initials: C
authors.lastname: Enrich
authors.forename: Stuart
authors.initials: S
authors.lastname: McLaughlin
authors.forename: Antonio
authors.initials: A
authors.lastname: Villalobo
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D002462:Cell Membrane / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D008564:Membrane Potentials; D051379:Mice; D008954:Models, Biological; D008956:Models, Chemical; D010743:Phospholipids / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D055672:Static Electricity; D013449:Sulfonamides / Q000494:pharmacology*; D013499:Surface Properties; D013997:Time Factors
chemical_list: D002147:Calmodulin; D004791:Enzyme Inhibitors; D010743:Phospholipids; D013449:Sulfonamides; C017967:W 7; C033772:N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide; D066246:ErbB Receptors