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J. Biol. Chem., Vol. 277, Issue 7, 4900-4905, February 15, 2002

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Activation of Large Conductance Sodium Channels upon Expression of Amiloride-sensitive Sodium Channel in Sf9 Insect Cells^*

U. Subrahmanyeswara Rao, Randy E. Steimle, and Premalatha Balachandran

From the Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198

The amiloride-sensitive epithelial sodium channels (ENaC) mediate Na⁺ reabsorption in epithelial tissues including distal nephron, colon, lung, and secretory glands and plays a critical role in pathophysiology of hypertension and cystic fibrosis. The ENaC is a multimeric protein composed of -ENaC, -ENaC, and -ENaC subunits. To study the biochemical properties of the channel, the subunit cDNAs of rat colon ENaC (rENaC) were subcloned into baculoviruses, and the corresponding proteins were expressed in Sf9 insect cells. The functional characteristics of the expressed rENaC were studied in planar lipid bilayers. The results show that expression of -rENaC and -rENaC in Sf9 insect cells results in the generation of cation-selective large conductance channels. Although the large conductance channels observed in the -rENaC-containing membranes were unaffected by amiloride, the large conductance channels found in -rENaC complex-containing membranes exhibited voltage-dependent flickering in the presence of micromolar amiloride. Possible implications of these observations are discussed.

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