To study and define the early time-dependent response (≤6 h) of blocker-sensitive epithelial Na+channels (ENaCs) to stimulation of Na+ transport by aldosterone, we used a new modified method of blocker-induced noise analysis to determine the changes of single-channel current ( i Na) channel open probability ( P o), and channel density ( N T) under transient conditions of transport as measured by macroscopic short-circuit currents ( I sc). In three groups of experiments in which spontaneous baseline rates of transport averaged 1.06, 5.40, and 15.14 μA/cm2, stimulation of transport occurred due to increase of blocker-sensitive channels. N T varied linearly over a 70-fold range of transport (0.5–35 μA/cm2). Relatively small and slow time-dependent but aldosterone-independent decreases of P o occurred during control (10–20% over 2 h) and aldosterone experimental periods (10–30% over 6 h). When the P o of control and aldosterone-treated tissues was examined over the 70-fold extended range of Na+ transport, P o was observed to vary inversely with I sc, falling from ∼0.5 to ∼0.15 at the highest rates of Na+ transport or ∼25% per 3-fold increase of transport. Because decreases of P o from any source cannot explain stimulation of transport by aldosterone, it is concluded that the early time-dependent stimulation of Na+ transport in A6 epithelia is due exclusively to increase of apical membrane N T.

Blocker-induced noise analysis and laser scanning confocal microscopy were used to test the idea that cAMP-mediated vesicle exocytosis/endocytosis may be a mechanism for regulation of functional epithelial Na+ channels (ENaCs) at apical membranes of A6 epithelia. After forskolin stimulation of Na+ transport and labeling apical membranes with the fluorescent dye N-(3-triethylammoniumpropyl)4-(6-4 diethylaminophenyl) hexatrienyl pyridinium dibromide (FM 4-64), ENaC densities (N(T)) decreased exponentially (time constant approximately 20 min) from mean values of 320 to 98 channels/cell within 55 min during washout of forskolin. Two populations of apical membrane-labeled vesicles appeared in the cytosol within 55 min, reaching mean values near 18 vesicles/cell, compared with five vesicles per cell in control, unstimulated tissues. The majority of cAMP-dependent endocytosed vesicles remained within a few micrometers of the apical membranes for the duration of the experiments. A minority of vesicles migrated to >5 microm below the apical membrane. Because steady states require identical rates of endocytosis and exocytosis, and because forskolin increased endocytic rates by fivefold or more, cAMP/protein kinase A acts kinetically not only to increase rates of cycling of vesicles at the apical membranes, but also principally to increase exocytic rates. These observations are consistent with and support, but do not prove, that vesicle trafficking is a mechanism for cAMP-mediated regulation of apical membrane channel densities in A6 epithelia.

The serum and glucocorticoid induced kinase 1 (SGK1) participates in the regulation of sodium reabsorption in the distal segment of the renal tubule, where it may modify the function of the epithelial sodium channel (ENaC). The molecular mechanism underlying SGK1 regulation of ENaC in renal epithelial cells remains controversial. We have addressed this issue in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible system. Expression of a constitutively active mutant of SGK1 (SGK1T S425D) induced a sixfold increase in amiloride-sensitive short-circuit current (I sc). Using noise analysis we demonstrate that SGK1 effect on I sc is due to a fourfold increase in the number of functional ENaCs in the membrane and a 43% increase in channel open probability. Impedance analysis indicated that SGK1T S425D increased the absolute value of cell equivalent capacitance by an average of 13.7%. SGK1T S425D also produced a 1.6–1.9-fold increase in total and plasma membrane subunit abundance, without changing the half-life of channels in the membrane. We conclude that in contrast to aldosterone, where stimulation of transport can be explained simply by an increase in channel synthesis, SGK1 effects are more complex and involve at least three actions: (1) increase of ENaC open probability; (2) increase of subunit abundance within apical membranes and intracellular compartments; and (3) activation of one or more pools of preexistent channels within the apical membranes and/or intracellular compartments.

Weak channel blocker-induced noise analysis was used to determine the way in which the steroids aldosterone and corticosterone stimulated apical membrane Na+ entry into the cells of tissue-cultured A6 epithelia. Among groups of tissues grown on a variety of substrates, in a variety of growth media, and with cells at passages 73–112, the steroids stimulated both amiloride-sensitive and amiloride-insensitive Na+ transport as measured by short-circuit currents in chambers perfused with either growth medium or a Ringer solution. From baseline rates of blocker-sensitive short-circuit current between 2 and 7 μA/cm2, transport was stimulated about threefold in all groups of experiments. Single channel currents averaged near 0.3 pA (growth medium) and 0.5 pA (Ringer) and were decreased 6–20% from controls by steroid due to the expected decreases of fractional transcellular resistance. Irrespective of baseline transport rates, the steroids in all groups of tissues stimulated transport by increase of the density of blocker-sensitive epithelial Na+ channels (ENaCs). Channel open probability was the same in control and stimulated tissues, averaging ∼0.3 in all groups of tissues. Accordingly, steroid-mediated increases of open channel density responsible for stimulation of Na+ transport are due to increases of the apical membrane pool of functional channels and not their open probability.

A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged alpha- or beta-subunits of the epithelial Na(+) channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy revealed a predominantly subapical distribution of EGFP-labeled subunits, suggesting a readily accessible pool of subunits available to participate in Na(+) transport. The basal level of Na(+) transport in the clonal lines was enhanced two- to fourfold relative to the parent line. Natriferic responses to insulin or aldosterone were similar in magnitude to the parent line, while forskolin-stimulated Na(+) transport was 64% greater than control in both the alpha- and beta-transfected lines. In response to forskolin, EGFP-labeled channel subunits traffic to the apical membrane. These data suggest that channel regulators, not the channel per se, form the rate-limiting step in response to insulin or aldosterone stimulation, while the number of channel subunits is important for basal as well as cAMP-stimulated Na(+) transport.