The epithelial Na + channel (ENaC), composed of three subunits (α, β, and γ), 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 αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-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 αβ or αγ 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 Ca 2+ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca 2+ 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 Ca 2+ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca 2+ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca 2+ 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 Ca 2+ .

It is currently believed that a nonselective cation (NSC) channel, which responds to arginine vasotocin (an antidiuretic hormone) and stretch, regulates Na + absorption in the distal nephron. However, the mechanisms of regulation of this channel remain incompletely characterized. To study the mechanisms of regulation of this channel, we used renal epithelial cells (A6) cultured on permeable supports. The apical membrane of confluent monolayers of A6 cells expressed a 29-pS channel, which was activated by stretch or by 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase. This channel had an identical selectivity for Na + , K + , Li + , and Cs + , but little selectivity for Ca 2+ (P Ca /P Na < 0.005) or Cl − (P Cl /P Na < 0.01), identifying it as an NSC channel. Stretch had no additional effects on the open probability ( P o ) of the IBMX-activated channel. This channel had one open (“ O ”) and two closed (short “ C S ” and long “ C L ”) states under basal, stretch-, or IBMX-stimulated conditions. Both stretch and IBMX increased the P o of the channel without any detectable changes in the mean open or closed times. These observations led us to the conclusion that a kinetic model “ C L ↔ C S ↔ O ” was the most suitable among three possible linear models. According to this model, IBMX or stretch would decrease the leaving rate of the channel for C L from C S , resulting in an increase in P o . Cytochalasin D pretreatment abolished the response to stretch or IBMX without altering the basal activity. H89 (an inhibitor of cAMP-dependent protein kinase) completely abolished the response to both stretch and IBMX, but, unlike cytochalasin D, also diminished the basal activity. We conclude that: ( a ) the functional properties of the cAMP-activated NSC channel are similar to those of the stretch-activated one, ( b ) the actin cytoskeleton plays a crucial role in the activation of the NSC channel induced by stretch and cAMP, and ( c ) the basal activity of the NSC channel is maintained by PKA-dependent phosphorylation but is not dependent on actin microfilaments.