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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Institut National de la Santé et de la Recherche Médicale Unité 467 and 2Proteomic Core Facilities, Institut Fédératif de Recherche 94, Faculté de Médecine, Université Paris-Descartes, Paris; 3Department of Physics, University of Warsaw, Warsaw, Poland; and 4Institut National de la Santé et de la Recherche Médicale U561, Hôpital Saint Vincent de Paul, Université Paris-Descartes, Paris, France
Submitted 2 May 2005 ; accepted in final form 19 July 2005
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.
heat shock; geldanamycin; cellular stress; channel trafficking; transepithelial chloride transport
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