title: Association between Hsp90 and the ClC-2 chloride channel upregulates channel function.
abstract: The voltage-dependent ClC-2 chloride channel has been implicated in a variety of physiological functions, including fluid transport across specific epithelia. ClC-2 is activated by hyperpolarization, weakly acidic external pH, intracellular Cl-, and cell swelling. To add more insight into the mechanisms involved in ClC-2 regulation, we searched for associated proteins that may influence ClC-2 activity. With the use of immunoprecipitation of ClC-2 from human embryonic kidney-293 cells stably expressing the channel, followed by electrophoretic separation of coimmunoprecipitated proteins and mass spectrometry identification, Hsp70 and Hsp90 were unmasked as possible ClC-2 interacting partners. Association of Hsp90 with ClC-2 was confirmed in mouse brain. Inhibition of Hsp90 by two specific inhibitors, geldanamycin or radicicol, did not affect total amounts of ClC-2 but did reduce plasma membrane channel abundance. Functional experiments using the whole cell configuration of the patch-clamp technique showed that inhibition of Hsp90 reduced ClC-2 current amplitude and impaired the intracellular Cl- concentration [Cl-]-dependent rightward shift of the fractional conductance. Geldanamycin and radicicol increased both the slow and fast activation time constants in a chloride-dependent manner. Heat shock treatment had the opposite effect. These results indicate that association of Hsp90 with ClC-2 results in greater channel activity due to increased cell surface channel expression, facilitation of channel opening, and enhanced channel sensitivity to intracellular [Cl-]. This association may have important pathophysiological consequences, enabling increased ClC-2 activity in response to cellular stresses such as elevated temperature, ischemia, or oxidative reagents.
authors.affiliation: INSERM, Unité 467, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75015 Paris, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Hinzpeter
authors.forename: Joanna
authors.initials: J
authors.lastname: Lipecka
authors.forename: Franck
authors.initials: F
authors.lastname: Brouillard
authors.forename: Maryvonne
authors.initials: M
authors.lastname: Baudoin-Legros
authors.forename: Michal
authors.initials: M
authors.lastname: Dadlez
authors.forename: Aleksander
authors.initials: A
authors.lastname: Edelman
authors.forename: Janine
authors.initials: J
authors.lastname: Fritsch
mesh_terms: D000818:Animals; D016227:Benzoquinones; D000090007:CLC-2 Chloride Channels; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000378:metabolism* / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D018869:Heat-Shock Response / Q000502:physiology; D006801:Humans; D047468:Immunoprecipitation; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007668:Kidney / Q000166:cytology; D047029:Lactams, Macrocyclic; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018408:Patch-Clamp Techniques; D021381:Protein Transport / Q000502:physiology; D011809:Quinones / Q000494:pharmacology; D051381:Rats; D015854:Up-Regulation
chemical_list: D016227:Benzoquinones; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D002712:Chlorides; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D007783:Lactones; D018942:Macrolides; D011809:Quinones; C035359:monorden; C001277:geldanamycin