ELECTROPHYSIOLOGICAL CHARACTERIZATION OF THE RAT EPITHELIAL NA- EFFECTS OF NA+ AND CA2+( CHANNEL (RENAC) EXPRESSED IN MDCK CELLS )

The epithelial Na+ channel (ENaC), composed of three subunits (alpha, beta, and gamma), is expressed in several epithelia and plays a critic al role in salt and water balance and in the regulation of blood press ure. Little is known, however, about the electrophysiological properti es of this cloned channel when expressed in epithelial cells. Using wh ole-cell and single channel current recording techniques, we have now characterized the rat alpha beta gamma ENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole- cell patch-clamp configuration, the alpha beta gamma rENaC-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). Thi s conductance was completely and reversibly inhibited by 10 mu M amilo ride, with a Ki of 20 nM at a membrane potential of -103 mV; the amilo ride inhibition was slightly voltage dependent. Amiloride-sensitive wh ole-cell current of MDCK cells expressing alpha beta or alpha gamma su bunits alone was -115.2 +/- 1.4 pA and -52.1 +/- 24.5 pA at -143 mV, r espectively, similar to the whole-cell Na+ current of untransfected ce lls. Relaxation analysis of the amiloride-sensitive current after volt age steps suggested that the channels were activated by membrane hyper polarization. Ion selectivity sequence of the Na+ conductance was Li> Na+ >> K+ = N-methyl-D-glucamine+ (NMDG(+)). Using excised outside-o ut patches, amiloride-sensitive single channel conductance, likely res ponsible for the macroscopic Na+ channel current, was found to be simi lar to 5 and 8 pS when Na+ and Li+ were used as a charge carrier, resp ectively. K+ conductance through the channel was undetectable. The cha nnel activity, defined as a product of the number of active channel (n ) and open probability (P-o), was increased by membrane hyperpolarizat ion. Both whole-cell Na+ current and conductance were saturated with i ncreased 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+ conduc tance (with Li+ as a charge carrier) was inhibited by the addition of ionomycin (1 mu M) and Ca2+ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 mu M Ca2+ caused a biphasic inhib ition, 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 mu M was accompanied by a decrease in channel activity. Increasin g cytosolic Ca2+ to 10 mu M exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca2+ concentrations from (1 nM to 10 mu M. 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+.