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Article

Effects of Na⁺ and Ca²⁺

Toru Ishikawa^*,,, Yoshinori Marunaka^*,,, and Daniela Rotin^*,,

From the ^* Hospital for Sick Children, Division of Respiratory Research, Toronto, Ontario M5G 1X8, Canada; and the Department of Biochemistry, and Department of Paediatrics, University of Toronto, Toronto, Ontario, Canada

The epithelial Na⁺ channel (ENaC), composed of three subunits (, β, and ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat βENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the βrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at –143 mV (–1,466.2 ± 297.5 pA) than did untransfected cells (–47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 µM amiloride, with a Ki of 20 nM at a membrane potential of –103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing β or subunits alone was –115.2 ± 41.4 pA and –52.1 ± 24.5 pA at –143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-D-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be 5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P_o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP_o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 µM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 µM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 µM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 µM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 µM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.

Key Words: epithelial Na⁺ channel • patch-clamp • Madin-Darby canine kidney cells • Na⁺ • Ca²⁺

Abbreviations: ENaC, epithelial Na⁺ channel; I-V, current–voltage; MDCK, Madin-Darby canine kidney; NMDG, N-methyl- D-glucamine

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