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J. Biol. Chem., Vol. 282, Issue 12, 8984-8993, March 23, 2007

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Ca²⁺-activated IK1 Channels Associate with Lipid Rafts upon Cell Swelling and Mediate Volume Recovery^*

Elisabeth T. Barfod, Ann L. Moore, Michael W. Roe¹, and Steven D. Lidofsky²

From the Departments of Pharmacology and Medicine, University of Vermont, Burlington, Vermont 05405

Restoration of cell volume in the continued presence of osmotic stimuli is essential, particularly in hepatocytes, which swell upon nutrient uptake. Responses to swelling involve the Ca²⁺-dependent activation of K⁺ channels, which promote fluid efflux to drive volume recovery; however, the channels involved in hepatocellular volume regulation have not been identified. We found that hypotonic exposure of HTC hepatoma cells evoked the opening of 50 pS K⁺-permeable channels, consistent with intermediate conductance (IK) channels. We isolated from rat liver and HTC cells a cDNA with sequence identity to the coding region of IK1. Swelling-activated currents were inhibited by transfection with a dominant interfering IK1 mutant. The IK channel blockers clotrimazole and TRAM-34 inhibited whole cell swelling-activated K⁺ currents and volume recovery. To determine whether IK1 underwent volume-sensitive localization, we expressed a green fluorescent protein fusion of IK1 in HTC cells. The localization of IK1 was suggestive of distribution in lipid rafts. Consistent with this, there was a time-dependent increase in colocalization between IK1 and the lipid raft ganglioside GM1 on the plasma membrane, which subsequently decreased with volume recovery. Pharmacological disruption of lipid rafts altered the plasma membrane distribution of IK1 and inhibited volume recovery after hypotonic exposure. Collectively, these findings support the hypothesis that IK1 regulates compensatory responses to hepatocellular swelling and suggest that regulation of cell volume involves coordination of signaling from lipid rafts with IK1 function.

Received for publication, August 14, 2006 , and in revised form, January 2, 2007.

^* This work was supported in part by National Institutes of Health (NIH) Grant DK56644 (to S. D. L.). The automated DNA sequencing was performed in the Vermont Cancer Center DNA Analysis Facility and was supported in part by Grant P30CA22435 from the NCI, NIH. Use of the DeltaVision restoration microscope and Volocity 3 software was provided through the Neuroscience Imaging Core supported by NIH Grant P20 RR16435 from the Center of Biomedical Research Excellence program of the National Center for Research Resources. Laser scanning confocal microscopy was performed at the University of Vermont Microscopy Imaging Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Current address: AMB M172, University of Chicago, 5841 South Maryland Ave., Chicago, IL 60637.

² To whom correspondence should be addressed: Dept. of Medicine, University of Vermont, Given C-327, Burlington, VT 05405-0001. Tel.: 802-656-8696; Fax: 802-656-8031; E-mail: steven.lidofsky{at}uvm.edu.

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