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Some members of the epithelial Na+ channel/degenerin (ENaC/DEG) family of ion channels have been detected in mammalian brain. Therefore, we examined the RNA and protein expression of these channels in another part of the central nervous system, the rabbit retina. We next sought to demonstrate physiological evidence for an amiloride-sensitive current in Müller glia, which, on the basis of a previous study, are thought to express alpha-ENaC (Golestaneh N, de Kozak Y, Klein C, and Mirshahi M. Glia 33: 160-168, 2001). RT-PCR of retinal RNA revealed the presence of alpha-, beta-, gamma-, and delta-ENaC as well as acid-sensing ion channel (ASIC)1, ASIC2, ASIC3, and ASIC4. Immunohistochemical localization with antibodies against alpha-ENaC and beta-ENaC showed labeling in Müller cells and neurons, respectively. The presence of alpha-ENaC, beta-ENaC, and ASIC1 was detected by Western blotting. Cultured Müller cells were whole cell patch clamped. These cells exhibited an inward Na+ current that was blocked by amiloride. These data demonstrate for the first time both the expression of a variety of ENaC and ASIC subunits in the rabbit retina as well as distinct cellular expression patterns of specific subunits in neurons and glia.

Members of the degenerin/epithelial Na(+) channel superfamily of ion channels subserve many functions, ranging from whole body sodium handling to mechanoelectrical transduction. We studied brain Na(+) channel 2 (BNaC-2) in planar lipid bilayers to examine its single channel properties and regulation by Ca(2+). Upon incorporation of vesicles made from membranes of oocytes expressing either wild-type (WT) BNaC-2 or BNaC-2 with a gain-of-function (GF) point mutation (G433F), functional channels with different properties were obtained. WT BNaC-2 resided in a closed state with short openings, whereas GF BNaC-2 was constitutively activated; a decrease in the pH in the trans compartment of the bilayer activated WT BNaC-2 and decreased its permeability for Na(+) over K(+). Moreover, these maneuvers made the WT channel more resistant to amiloride. In contrast, GF BNaC-2 did not respond to a decrease in pH, and its amiloride sensitivity and selectivity for Na(+) over K(+) were unaffected by this pH change. Buffering the bathing solutions with EGTA to reduce the free [Ca(2+)] to <10 nm increased WT single channel open probability 10-fold, but not that of GF BNaC-2. Ca(2+) blocked both WT and GF BNaC-2 in a dose- and voltage-dependent fashion; single channel conductances were unchanged. A drop in pH reduced the ability of Ca(2+) to inhibit these channels. These results show that BNaC-2 is an amiloride-sensitive sodium channel and suggest that pH activation of these channels could be, in part, a consequence of H(+) "interference" with channel regulation by Ca(2+).

Gating differences occur between the alpha-subunits of the bovine and rat clones of an amiloride-sensitive epithelial Na+ channel (ENaC). Deletion of the carboxy terminus of bovine alpha-ENaC (alpha-bENaC) at R567 converted the gating properties to that of rat alpha-ENaC (alpha-rENaC). The equivalent truncation in alpha-rENaC was without effect on the gating of the rat homologue. The addition of actin to ENaC channels composed of either alpha-rENaC or alpha-bENaC alone produced a twofold reduction in conductance and an increase in open probability. Neither alpha-rENaC (R613X) nor alpha-bENaC (R567X) was responsive to actin. Using a chimera consisting of alpha-rENaC1-615 and alpha-bENaC570-650, we examined several different carboxy-terminal truncation mutants plus and minus actin. When incorporated into planar bilayers, the gating pattern of this construct was identical to wild-type (wt) alpha-bENaC. Premature stop mutations proximal to E685X produced channels with gating patterns like alpha-rENaC. Actin had no effect on the E631X truncation, whereas more distal truncations all interacted with actin, as did wt alpha-bENaC. Key findings were confirmed using channels expressed in Xenopus oocytes and studied by cell-attached patch-clamp recording. Our results suggest that the site of actin regulation at the carboxy terminus of the chimera is located between residues 631 and 644.

We tested the hypothesis that an arginine-rich region immediately following the second transmembrane domain may constitute part of the inner mouth of the epithelial Na+ channel (ENaC) pore and, hence, influence conduction and/or selectivity properties of the channel by expressing double point mutants in Xenopus oocytes. Double point mutations of arginines in this post-M2 region of the human alpha-ENaC (alpha-hENaC) led to a decrease and increase in the macroscopic conductance of alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, respectively, but had no effect on the single-channel conductance of either double point mutant. However, the apparent equilibrium dissociation constant for Na+ was decreased for both alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, and the maximum amiloride-sensitive Na+ current was decreased for alphaR586E,R587Ebetagamma-hENaC and increased for alphaR589E,R591Ebetagamma-hENaC. The relative permeabilities of Li+ and K+ vs. Na+ were increased 11.25- to 27.57-fold for alphaR586E,R587Ebetagamma-hENaC compared with wild type. The relative ion permeability of these double mutants and wild-type ENaC was inversely related to the crystal diameter of the permeant ions. Thus the region of positive charge is important for the ion permeation properties of the channel and may form part of the pore itself.

The mechanism by which the cytoskeletal protein actin affects the conductance of amiloride-sensitive epithelial sodium channels (ENaC) was studied in planar lipid bilayers. In the presence of monomeric actin, we found a decrease in the single-channel conductance of alpha-ENaC that did not occur when the internal [Ca2+]free was buffered to <10 nM. An analysis of single-channel kinetics demonstrated that Ca2+ induced the appearance of long-lived closed intervals separating bursts of channel activity, both in the presence and in the absence of actin. In the absence of actin, the duration of these bursts and the time spent by the channel in its open, but not in its short-lived closed state, were inversely proportional to [Ca2+]. This, together with a lengthening of the interburst intervals, translated into a dose-dependent decrease in the single-channel open probability. In contrast, a [Ca2+]-dependent decrease in alpha-ENaC conductance in the presence of actin was accompanied by lengthening of the burst intervals with no significant changes in the open or closed (both short- and long-lived) times. We conclude that Ca2+ acts as a "fast-to-intermediate" blocker when monomeric actin is present, producing a subsequent attenuation of the apparent unitary conductance of the channel.

Gene expression, protein expression, and function of amiloride-sensitive sodium channels were examined in human lymphocytes from normal individuals and individuals with Liddle's disease. Using reverse transcriptase polymerase chain reactions, expression of all three cloned epithelial sodium channel (ENaC) subunits was detected in lymphocytes. Polyclonal antibodies to bovine alpha-ENaC bound to the plasma membrane of normal and Liddle's lymphocytes. A quantitative analysis of fluorescence-tagged ENaC antibodies indicated a 2.5-fold greater surface binding of the antibodies to Liddle's lymphocytes compared with normal lymphocytes. The relative binding intensity increased significantly (25%; p < 0.001) for both normal and Liddle's cells after treatment with 40 microM 8-CPT-cAMP. Amiloride-sensitive whole cell currents were recorded under basal and cAMP-treated conditions for both cell types. Liddle's cells had a 4.5-fold larger inward sodium conductance compared with normal cells. A specific 25% increase in the inward sodium current was observed in normal cells in response to cAMP treatment. Outside-out patches from both cell types under both treatment conditions revealed no obvious differences in the single channel conductance. The P(open) was 4.2 +/- 3.9% for patches from non-Liddle's cells, and 27.7 +/- 5.4% in patches from Liddle's lymphocytes. Biochemical purification of a protein complex, using the same antibodies used for the immunohistochemistry, yielded a functional sodium channel complex that was inhibited by amiloride when reconstituted into lipid vesicles and incorporated into planar lipid bilayers. These four independent methodologies yielded findings consistent with the hypotheses that human lymphocytes express functional, regulatable ENaC and that the mutation responsible for Liddle's disease induces excessive channel expression.

Epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) are co-localized in the apical membrane of many epithelia. These channels are essential for electrolyte and water secretion and/or reabsorption. In cystic fibrosis airway epithelia, a hyperactivated epithelial Na(+) conductance operates in parallel with defective Cl(-) secretion. Several groups have shown that CFTR down-regulates ENaC activity, but the mechanisms and the regulation of CFTR by ENaC are unknown. To test the hypothesis that ENaC and CFTR regulate each other, and to identify the region(s) of ENaC involved in the interaction between CFTR and ENaC, rENaC and its mutants were co-expressed with CFTR in Xenopus oocytes. Whole cell macroscopic sodium currents revealed that wild type (wt) alphabetagamma-rENaC-induced Na(+) current was inhibited by co-expression of CFTR, and further inhibited when CFTR was activated with a cAMP-raising mixture (CKT). Conversely, alphabetagamma-rENaC stimulated CFTR-mediated Cl(-) currents up to approximately 6-fold. Deletion mutations in the intracellular tails of the three rENaC subunits suggested that the carboxyl terminus of the beta subunit was required both for the down-regulation of ENaC by activated CFTR and the up-regulation of CFTR by ENaC. However, both the carboxyl terminus of the beta subunit and the amino terminus of the gamma subunit were essential for the down-regulation of rENaC by unstimulated CFTR. Interestingly, down-regulation of rENaC by activated CFTR was Cl(-)-dependent, while stimulation of CFTR by rENaC was not dependent on either cytoplasmic Na(+) or a depolarized membrane potential. In summary, there appear to be at least two different sites in ENaC involved in the intermolecular interaction between CFTR and ENaC.

The epithelial Na(+) channel (ENaC) is a low-conductance channel that is highly selective for Na(+) and Li(+) over K(+) and impermeable to anions. The molecular basis underlying these conduction properties is not well known. Previous studies with the ENaC subunits demonstrated that the M2 region of alpha-ENaC is critical to channel function. Here we examine the effects of reversing the negative charges of highly conserved amino acids in alpha-subunit human ENaC (alpha-hENaC) M1 and M2 domains. Whole cell and single-channel current measurements indicated that the M2 mutations E568R, E571R, and D575R significantly decreased channel conductance but did not affect Na(+):K(+) permeability. We observed no functional perturbations from the M1 mutation E108R. Whole cell amiloride-sensitive current recorded from oocytes injected with the M2 alpha-hENaC mutants along with wild-type (wt) beta- and gamma-hENaC was low (46-93 nA) compared with the wt channel (1-3 microA). To determine whether this reduced macroscopic current resulted from a decreased number of mutant channels at the plasma membrane, we coexpressed mutant alpha-hENaC subunits with green fluorescent protein-tagged beta- and gamma-subunits. Confocal laser scanning microscopy of oocytes demonstrated that plasma membrane localization of the mutant channels was the same as that of wt. These experiments demonstrate that acidic residues in the second transmembrane domain of alpha-hENaC affect ion permeation and are thus critical components of the conductive pore of ENaC.

The hypothesis that actin interactions account for the signature biophysical properties of cloned epithelial Na(+) channels (ENaC) (conductance, ion selectivity, and long mean open and closed times) was tested using planar lipid bilayer reconstitution and patch clamp techniques. We found the following. 1) In bilayers, actin produced a more than 2-fold decrease in single channel conductance, a 5-fold increase in Na(+) versus K(+) permselectivity, and a substantial increase in mean open and closed times of wild-type alphabetagamma-rENaC but had no effect on a mutant form of rENaC in which the majority of the C terminus of the alpha subunit was deleted (alpha(R613X)betagamma-rENaC). 2) When alpha(R613X)betagamma-rENaC was heterologously expressed in oocytes and single channels examined by patch clamp, 12.5-pS channels of relatively low cation permeability were recorded. These characteristics were identical to those recorded in bilayers for either alpha(R613X)betagamma-rENaC or wild-type alphabetagamma-rENaC in the absence of actin. Moreover, we show that rENaC subunits tightly associate, forming either homo- or heteromeric complexes when prepared by in vitro translation or when expressed in oocytes. Finally, we show that alpha-rENaC is properly assembled but retained in the endoplasmic reticulum compartment. We conclude that actin subserves an important regulatory function for ENaC and that planar bilayers are an appropriate system in which to study the biophysical and regulatory properties of these cloned channels.

An epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. To elucidate the function of the cytoplasmic, NH(2) terminus of rat ENaC (rENaC) subunits, a series of mutant cDNAs was constructed and the cRNAs for all three subunits were expressed in Xenopus oocytes. Amiloride-sensitive Na(+) currents (I(Na)) were measured by the two-electrode voltage clamp technique. Deletion of the cytoplasmic, NH(2) terminus of alpha (Delta2-109), beta (Delta2-49), or gamma-rENaC (Delta2-53) dramatically reduced I(Na). A series of progressive, NH(2)-terminal deletions of alpha-rENaC were constructed to identify motifs that regulate I(Na). Deletion of amino acids 2-46 had no effect on I(Na): however, deletion of amino acids 2-51, 2-55, 2-58, and 2-67 increased I(Na) by approximately 4-fold. By contrast, deletion of amino acids 2-79, 2-89, 2-100, and 2-109 eliminated I(Na). To evaluate the mechanism whereby Delta2-67-alpha-rENaC increased I(Na), single channels were evaluated by patch clamp. The single-channel conductance and open probability of alpha,beta,gamma-rENaC and Delta2-67-alpha,beta,gamma-rENaC were similar. However, the number of active channels in the membrane increased from 6 +/- 1 channels per patch with alpha,beta,gamma-rENaC to 11 +/- 1 channels per patch with Delta2-67-alpha,beta,gamma-rENaC. Laser scanning confocal microscopy confirmed that there were more Delta2-67-alpha,beta, gamma-rENaC channels in the plasma membrane than alpha,beta, gamma-rENaC channels. Deletion of amino acids 2-67 in alpha-rENaC reduced the endocytic retrieval of channels from the plasma membrane and increased the half-life of the channel in the membrane from 1.1 +/- 0.2 to 3.5 +/- 1.1 h. We conclude that the cytoplasmic, NH(2) terminus of alpha-, beta-, and gamma-rENaC is required for channel activity. The cytoplasmic, NH(2) terminus of alpha-rENaC contains two key motifs. One motif regulates the endocytic retrieval of the channel from the plasma membrane. The second motif is required for channel activity.

Application of recombinant DNA technology and electrophysiology to the study of amiloride-sensitive Na+ channels has resulted in an enormous increase in the understanding of the structure-function relationships of these channels. Moreover, this knowledge has permitted the elucidation of the physiological roles of these ion channels in cellular processes as diverse as transepithelial salt and water movement, taste perception, volume regulation, nociception, neuronal function, mechanosensation, and even defaecation. Although members of this ever-growing superfamily of ion channels (the Deg/ENaC superfamily) share little amino acid identity, they are all organized similarly, namely, two short N- and C-termini, two short membrane-spanning segments, and a very large extracellular loop domain. In this brief Topical Review, we discuss the structural features of each domain of this Deg/ENaC superfamily and, using ENaC as a model, show how each domain relates to overall channel function.

Human astrocytoma cells were studied using whole cell patch-clamp recording. An inward, amiloride-sensitive Na+ current was identified in four continuous cell lines originally derived from human glioblastoma cells (CH235, CRT, SKMG-1, and U251-MG) and in three primary cultures of cells obtained from glioblastoma multiforme tumors (up to 4 passages). In addition, cells freshly isolated from a resected medulloblastoma tumor displayed this same characteristic inward current. In contrast, amiloride-sensitive currents were not observed in normal human astrocytes, low-grade astrocytomas, or juvenile pilocytic astrocytomas. The only amiloride-sensitive Na+ channels thus far molecularly identified in brain are the brain Na+ channels (BNaCs). RT-PCR analyses demonstrated the presence of mRNA for either BNaC1 or BNaC2 in these tumors and in normal astrocytes. These results indicate that the functional expression of amiloride-sensitive Na+ currents is a characteristic feature of malignant brain tumor cells and that this pathway may be a potentially useful target for therapeutic intervention.

Protons regulate electrogenic sodium absorption in a variety of epithelia, including the cortical collecting duct, frog skin, and urinary bladder. Recently, three subunits (alpha, beta, gamma) coding for the epithelial sodium channel (ENaC) were cloned. However, it is not known whether pH regulates Na+ channels directly by interacting with one of the three ENaC subunits or indirectly by interacting with a regulatory protein. As a first step to identifying the molecular mechanisms of proton-mediated regulation of apical membrane Na+ permeability in epithelia, we examined the effect of pH on the biophysical properties of ENaC. To this end, we expressed various combinations of alpha-, beta-, and gamma-subunits of ENaC in Xenopus oocytes and studied ENaC currents by the two-electrode voltage-clamp and patch-clamp techniques. In addition, the effect of pH on the alpha-ENaC subunit was examined in planar lipid bilayers. We report that alpha,beta,gamma-ENaC currents were regulated by changes in intracellular pH (pHi) but not by changes in extracellular pH (pHo). Acidification reduced and alkalization increased channel activity by a voltage-independent mechanism. Moreover, a reduction of pHi reduced single-channel open probability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited alpha, beta-ENaC, alpha,gamma-ENaC, and alpha-ENaC currents. We conclude that pHi but not pHo regulates ENaC and that the alpha-ENaC subunit is regulated directly by pHi.

The hypothesis that amiloride-sensitive Na+ channels (ENaC) are involved in cell volume regulation was tested. Anisosmotic ND-20 media (ranging from 70 to 450 mosM) were used to superfuse Xenopus oocytes expressing alpha beta gamma-rat ENaC (alpha beta gamma-rENaC). Whole cell currents were reversibly dependent on external osmolarity. Under conditions of swelling (70 mosM) or shrinkage (450 mosM), current amplitude decreased and increased, respectively. In contrast, there was no change in current amplitude of H2O-injected oocytes to the above osmotic insults. Currents recorded from alpha beta gamma-rENaC-injected oocytes were not sensitive to external Cl- concentration or to the K+ channel inhibitor BaCl2. They were sensitive to amiloride. The concentration of amiloride necessary to inhibit one-half of the maximal rENaC current expressed in oocytes (Ki; apparent dissociation constant) decreased in swollen cells and increased in shrunken oocytes. The osmotic pressure-induced Na+ currents showed properties similar to those of stretch-activated channels, including inhibition by Gd3+ and La3+, and decreased selectivity for Na+. alpha beta gamma-rENaC-expressing oocytes maintained a nearly constant cell volume in hypertonic ND-20. The present study is the first demonstration that alpha beta gamma-rENaC heterologously expressed in Xenopus oocytes may contribute to oocyte volume regulation following shrinkage.

The action of aldosterone to increase apical membrane permeability in responsive epithelia is thought to be due to activation of sodium channels. Aldosterone stimulates methylation of a 95-kDa protein in apical membrane of A6 cells, and we have previously shown that methylation of a 95-kDa protein in the immunopurified Na+ channel complex increases open probability of these channels in planar lipid bilayers. We report here that aldosterone stimulates carboxylmethylation of the beta subunit of xENaC in A6 cells. In vitro translated beta subunit, but not alpha or gamma, serves as a substrate for carboxylmethylation. Carboxylmethylation of ENaC reconstituted in planar lipid bilayers leads to an increase in open probability only when beta subunit is present. When the channel complex is immunoprecipitated from A6 cells and analyzed by Western blot with antibodies to the three subunits of xENaC, all three subunits are recognized as constituents of the complex. The results suggest that Na+ channel activity in A6 cells is regulated, in part, by carboxylmethylation of the beta subunit of xENaC.

Two independent signal transduction pathways regulate lymphocyte amiloride-sensitive sodium channels (ASSCs), one utilizing cAMP as a second messenger and the other utilizing a GTP-binding protein. This implies that two plasma membrane receptors play a role in the regulation of lymphocyte ASSCs. In this study, we tested the hypothesis that alpha1- and alpha2-adrenergic receptors independently regulate lymphocyte ASSCs via the two previously identified second messengers. Direct measurements indicated that norepinephrine increased lymphocyte cAMP and activated ASSCs. The alpha2-specific inhibitor, yohimbine, blocked this activation, thereby linking alpha2-adrenergic receptors to ASSC regulation via cAMP. The alpha1-specific ligand, terazosin, acted as an agonist and activated lymphocyte ASSCs but inhibited ASSC current that had been preactivated by norepinephrine or 8-(4-chlorophenylthio) (CPT)-cAMP. Terazosin had no effect on the lymphocyte whole cell ASSC currents preactivated by treatment with pertussis toxin. This finding indirectly links alpha1-adrenergic receptors to lymphocyte ASSC regulation via GTP-binding proteins. Terazosin had no direct inhibitory or stimulatory effects on alpha,beta,gamma-endothelial sodium channels reconstituted into planar lipid bilayers and expressed in Xenopus oocytes, ruling out a direct interaction between terazosin and the channels. These findings support the hypothesis that both alpha1- and alpha2-adrenergic receptors independently regulate lymphocyte ASSCs via GTP-binding proteins and cAMP, respectively.

Several studies have indicated that olfactory responses are impeded by amiloride. Therefore, it was of interest to see whether, and if so which, olfactory epithelial cellular compartments have amiloride-sensitive structures. Using ultrastructural methods that involved rapid freezing, freeze-substitution and low temperature embedding of olfactory epithelia, this study shows that, in the rat, this tissue is immunoreactive to antibodies against amiloride sensitive Na(+)-channels. However, microvilli of olfactory supporting cells, as opposed to receptor cilia, contained most of the immunoreactive sites. Apices from which the microvilli sprout and receptor cell dendritic knobs had much less if any of the amiloride-antibody binding sites. Using a direct ligand-binding cytochemical method, this study also confirms earlier ones that showed that olfactory receptor cell cilia have Na+, K(+)-ATPase. It is proposed that supporting cell microvilli and the receptor cilia themselves have mechanisms, different but likely complementary, that participate in regulating the salt concentration around the receptor cell cilia. In this way, both structures help to provide the ambient mucous environment for receptor cells to function properly. This regulation of the salt concentration of an ambient fluid environment is a function that the olfactory epithelium shares with cells of transporting epithelia, such as those of kidney.

Using the Xenopus oocyte expression system, we examined the mechanisms by which the beta- and gamma-subunits of an epithelial Na+ channel (ENaC) regulate alpha-subunit channel activity and the mechanisms by which beta-subunit truncations cause ENaC activation. Expression of alpha-ENaC alone produced small amiloride-sensitive currents (-43 +/- 10 nA, n = 7). These currents increased > 30-fold with the coexpression of beta- and gamma-ENaC to -1,476 +/- 254 nA (n = 20). This increase was accompanied by a 3.1- and 2.7-fold increase of membrane fluorescence intensity in the animal and vegetal poles of the oocyte, respectively, with use of an antibody directed against the alpha-subunit of ENaC. Truncation of the last 75 amino acids of the beta-subunit COOH terminus, as found in the original pedigree of individuals with Liddle's syndrome, caused a 4.4-fold (n = 17) increase of the amiloride-sensitive currents compared with wild-type alpha beta gamma-ENaC. This was accompanied by a 35% increase of animal pole membrane fluorescence intensity. Injection of a 30-amino acid peptide with sequence identity to the COOH terminus of the human beta-ENaC significantly reduced the amiloride-sensitive currents by 40-50%. These observations suggest a tonic inhibitory role on the channel's open probability (Po) by the COOH terminus of beta-ENaC. We conclude that the changes of current observed with coexpression of the beta- and gamma-subunits or those observed with beta-subunit truncation are likely the result of changes of channel density in combination with large changes of Po.

1. Conductance of heterotrimeric rat epithelial Na+ channels (alpha, beta, gamma-rENaCs) for Li+ and Na+ in planar lipid bilayers was a non-linear function of ion concentration, with a maximum of 30.4 +/- 2.9 pS and 18.5 +/- 1.9 pS at 1 M Li+ and Na+, respectively. 2. The alpha, beta, gamma-rENaC conductance measured in symmetrical mixtures of Na(+)-Li+ (1 M) exhibited an anomalous mole fraction dependence, with a minimum at 4:1 Li+ to Na+ molar ratio. 3. Permeability ratios PK/PNa and PLi/PNa of the channel calculated from the bionic reversal potentials were dependent on ion concentration: PK/PNa was 0.11 +/- 0.01, and PLi/PNa was 1.6 +/- 0.3 at 50 mM; PK/PNa was 0.04 +/- 0.01 and PLi/PNa was 2.5 +/- 0.4 at 3 M, but differed from the ratios of single-channel conductances in symmetrical Li+, Na+ or K+ solutions. The permeability sequence determined by either method was Li+ > Na+ > K+ >> Rb+ Cs+. 4. Predictions of a model featuring two binding sites and three energy barriers (2S3B), and allowing double occupancy, developed on the basis of single ion current-voltage relationships, are in agreement with the observed conductance maximum in single ion experiments, conductance minimum in the mole fraction experiments, non-linearity of the current-voltage curves in bionic experiments, and the concentration dependence of permeability ratios. 5. Computer simulations using the 2S3B model recreate the ion concentration dependencies of single-channel conductance observed for the immunopurified bovine renal amiloride-sensitive Na+ channel, and short-circuit current in frog skin, thus supporting the hypothesis that ENaCs form a core conduction unit of epithelial Na+ channels.

Limited information is available regarding domains within the epithelial Na+ channel (ENaC) which participate in amiloride binding. We previously utilized the anti-amiloride antibody (BA7.1) as a surrogate amiloride receptor to delineate amino acid residues that contact amiloride, and identified a putative amiloride binding domain WYRFHY (residues 278-283) within the extracellular domain of alpharENaC. Mutations were generated to examine the role of this sequence in amiloride binding. Functional analyses of wild type (wt) and mutant alpharENaCs were performed by cRNA expression in Xenopus oocytes and by reconstitution into planar lipid bilayers. Wild type alpharENaC was inhibited by amiloride with a Ki of 169 nM. Deletion of the entire WYRFHY tract (alpharENaC Delta278-283) resulted in a loss of sensitivity of the channel to submicromolar concentrations of amiloride (Ki = 26.5 microM). Similar results were obtained when either alpharENaC or alpharENaC Delta278-283 were co-expressed with wt beta- and gammarENaC (Ki values of 155 nM and 22.8 microM, respectively). Moreover, alpharENaC H282D was insensitive to submicromolar concentrations of amiloride (Ki = 6.52 microM), whereas alpharENaC H282R was inhibited by amiloride with a Ki of 29 nM. These mutations do not alter ENaC Na+:K+ selectivity nor single-channel conductance. These data suggest that residues within the tract WYRFHY participate in amiloride binding. Our results, in conjunction with recent studies demonstrating that mutations within the membrane-spanning domains of alpharENaC and mutations preceding the second membrane-spanning domains of alpha-, beta-, and gammarENaC alters amiloride's Ki, suggest that selected regions of the extracellular loop of alpharENaC may be in close proximity to residues within the channel pore.

Streaming potentials across cloned epithelial Na+ channels (ENaC) incorporated into planar lipid bilayers were measured. We found that the establishment of an osmotic pressure gradient (Deltapi) across a channel-containing membrane mimicked the activation effects of a hydrostatic pressure differential (DeltaP) on alphabetagamma-rENaC, although with a quantitative difference in the magnitude of the driving forces. Moreover, the imposition of a Deltapi negates channel activation by DeltaP when the Deltapi was directed against DeltaP. A streaming potential of 2.0 +/- 0.7 mV was measured across alphabetagamma-rat ENaC (rENaC)-containing bilayers at 100 mM symmetrical [Na+] in the presence of a 2 Osmol/kg sucrose gradient. Assuming single file movement of ions and water within the conduction pathway, we conclude that between two and three water molecules are translocated together with a single Na+ ion. A minimal effective pore diameter of 3 A that could accommodate two water molecules even in single file is in contrast with the 2-A diameter predicted from the selectivity properties of alphabetagamma-rENaC. The fact that activation of alphabetagamma-rENaC by DeltaP can be reproduced by the imposition of Deltapi suggests that water movement through the channel is also an important determinant of channel activity.

Atomic force microscopy (AFM) was used for high-resolution imaging of the apical distribution of epithelial Na+ channels in A6 renal epithelial cells. A6 cells grown on coverslips were labeled with antibodies generated against an amiloride-sensitive epithelial Na+ channel complex purified from bovine renal medulla that had been conjugated to 8-nm colloidal gold particles before preparation for AFM. AFM revealed that there was a marked increase in the height of the microvilli in cells labeled with the anti-epithelial Na+ channel antibodies compared with unlabeled cells or cells labeled with rabbit nonimmune immunoglobulin G conjugated to colloidal gold particles. We interpret this apparent increase in microvillar height to be due to anti-epithelial Na+ channel antibody binding to the apical microvilli. These data demonstrate that epithelial Na+ channels are restricted to the apical microvilli in Na+-transporting renal epithelial cells. Furthermore, they demonstrate the applicability of using AFM for high-resolution imaging of the cell surface distribution of epithelial ion channels.

We have generated two site-directed mutants, K504E and K515E, in the alpha subunit of an amiloride-sensitive bovine epithelial Na+ channel, alpha bENaC. The region in which these mutations lie is in the large extracellular loop immediately before the second membrane-spanning domain (M2) of the protein. We have found that when membrane vesicles prepared from Xenopus oocytes expressing either K504E or K515E alpha bENaC are incorporated into planar lipid bilayers, the gating pattern, cation selectivity, and amiloride sensitivity of the resultant channel are all altered as compared to the wild-type protein. The mutated channels exhibit either a reduction or a complete lack of its characteristic burst-type behavior, significantly reduced Na+:K+ selectivity, and an approximately 10-fold decrease in the apparent inhibitory equilibrium dissociation constant (Ki) for amiloride. Single-channel conductance for Na+ was not affected by either mutation. On the other hand, both K504E and K515E alpha bENaC mutants were significantly more permeable to K+, as compared to wild type. These observations identify a lysine-rich region between amino acid residues 495 and 516 of alpha bENaC as being important to the regulation of fundamental channel properties.

Cystic fibrosis (CF) airway epithelia exhibit enhanced Na+ reabsorption in parallel with diminished Cl- secretion. We tested the hypothesis that actin plays a role in the regulation of a cloned epithelial Na+ channel (ENaC) by the cystic fibrosis transmembrane conductance regulator (CFTR). We found that immunopurified bovine tracheal CFTR coreconstituted into a planar lipid bilayer with alpha,beta,gamma-rat ENaC (rENaC) decreased single-channel open probability (Po) of rENaC in the presence of actin by over 60%, a significantly greater effect than was observed in the absence of actin (approximately 20%). In the presence of actin, protein kinase A plus ATP activated both CFTR and rENaC, but CFTR was activated in a sustained manner, whereas the activation of rENaC was transitory. ATP alone could also activate ENaC transiently in the presence ofactin but had no effect on CFTR. Stabilizing short actin filaments at a fixed length with gelsolin (at a ratio to actin of 2:1) produced a sustained activation of alpha,beta,gamma-rENaC in both the presence or absence of CFTR. Gelsolin alone (i.e., in the absence of actin) had no effect on the conductance or Po of either CFTR or rENaC. We have also found that short actin filaments produced their modulatory action on alpha-rENaC independent of the presence of the beta- or gamma-rENaC subunits. In contrast, CFTR did not affect any properties of the channel formed by alpha-rENaC alone, i.e., in the absence of beta- or gamma-rENaC. These results indicate that CFTR can directly downregulate single Na+ channel activity, which may account for the observed differences between Na+ transport in normal and CF-affected airway epithelia. Moreover, the presence of actin confers an enhanced modulatory ability of CFTR on Na+ channels.

Protein kinase A (PKA)- and G protein-mediated regulation of immunopurified adult rabbit alveolar epithelial type II (ATII) cell proteins that exhibit amiloride-sensitive Na+ channel activity was studied in planar lipid bilayers and freshly isolated ATII cells. Addition of the catalytic subunit of PKA + ATP increased single channel open probability from 0.42 +/- 0.05 to 0.82 +/- 0.07 in a voltage-independent manner, without affecting unitary conductance. This increase in open probability of the channels was mainly due to a decrease in the time spent by the channel in its closed state. The apparent inhibition constant for amiloride increased from 8.0 +/- 1.8 microM under control conditions to 15 +/- 3 microM after PKA-induced phosphorylation; that for ethylisopropylamiloride increased from 1.0 +/- 0.4 to 2.0 +/- 0.5 microM. Neither pertussis toxin (PTX) nor guanosine 5'-O-(3-thiotriphosphate) affected ATII Na+ channel activity in bilayers. Moreover, PTX failed to affect amiloride-inhibitable 22Na+ uptake in freshly isolated ATII cells. In vitro, ADP ribosylation induced by PTX revealed the presence of a specifically ribosylated band at 40-45 kDa in the total solubilized ATII cell protein fraction, but not in the immunopurified fraction. Moreover, the immunopurified channel was downregulated in response to guanosine 5'-O-(3-thiotriphosphate)-mediated activation of the exogenous G alpha(i-2), but not G(oA), G alpha(i-1), or G alpha(i-3), protein added to the channel. This effect occurred only in the presence of actin. These results suggest that amiloride-sensitive Na+ channels in adult alveolar epithelia regulated by PKA-mediated phosphorylation also retain the ability to be regulated by G alpha([i-2), but not G alpha([i-1) or G alpha(i-3), protein.

A family of novel epithelial Na+ channels (ENaCs) have recently been cloned from several different tissues. Three homologous subunits (alpha, beta, gamma-ENaCs) from the core conductive unit of Na(+)-selective, amiloride-sensitive channels that are found in epithelia. We here report the results of a study assessing the regulation of alpha,beta,gamma-rENaC by Ca2+ in planar lipid bilayers. Buffering of the bilayer bathing solutions to [Ca2+] < 1 nM increased single-channel open probability by fivefold. Further investigation of this phenomenon revealed that Ca2+ ions produced a voltage-dependent block, affecting open probability but not the unitary conductance of ENaC. Imposing a hydrostatic pressure gradient across bilayers containing alpha,beta,gamma-rENaC markedly reduced the sensitivity of these channels to inhibition by [Ca2+]. Conversely, in the nominal absence of Ca2+, the channels lost their sensitivity to mechanical stimulation. These results suggest that the previously observed mechanical activation of ENaCs reflects a release of the channels from block by Ca2+.

We have recently cloned the alpha subunit of a bovine amiloride-sensitive Na+ channel (alphabENaC). This subunit shares extensive homology with both rat and human alphaENaC subunits but shows marked divergence at the C terminus beginning at amino acid 584 of the 697-residue sequence. When incorporated into planar lipid bilayers, alphabENaC almost exclusively exhibits a main transition to 39 picosiemens (pS) with very rare 13 pS step transitions to one of two subconductance states (26 and 13 pS). In contrast, the alpha subunit of the rat renal homolog of ENaC (alpharENaC) has a main transition step to 13 pS that is almost constituitively open, with a second stepwise transition of 26 to 39 pS. A deletion mutant of alphabENaC, encompassing the entire C-terminal region (R567X), converts the kinetic behavior of alphabENaC to that of alpharENaC, i. e. a transition to 13 pS followed by a second 26 pS transition to 39 pS. Chemical cross-linking of R567X restores the wild-type alphabENaC gating pattern, whereas treatment with the reducing agent dithiothreitol produced only 13 pS transitions. In contrast, an equivalent C-terminal truncation of alpharENaC (R613X) had no effect on the gating pattern of alpharENaC. These results are consistent with the hypothesis that interactions between the C termini of alphabENaC account for the different kinetic behavior of this member of the ENaC family of Na+ channels.

Cytoskeletal elements play an important role in the regulation of ion transport in epithelia. We have studied the effects of actin filaments of different length on the alpha, beta, gamma-rENaC (rat epithelial Na+ channel) in planar lipid bilayers. We found the following. 1) Short actin filaments caused a 2-fold decrease in unitary conductance and a 2-fold increase in open probability (Po) of alpha,beta,gamma-rENaC. 2) alpha,beta,gamma-rENaC could be transiently activated by protein kinase A (PKA) plus ATP in the presence, but not in the absence, of actin. 3) ATP in the presence of actin was also able to induce a transitory activation of alpha, beta,gamma-rENaC, although with a shortened time course and with a lower magnitude of change in Po. 4) DNase I, an agent known to prohibit elongation of actin filaments, prevented activation of alpha,beta,gamma-rENaC by ATP or PKA plus ATP. 5) Cytochalasin D, added after rundown of alpha,beta,gamma-rENaC activity following ATP or PKA plus ATP treatment, produced a second transient activation of alpha,beta,gamma-rENaC. 6) Gelsolin, a protein that stabilizes polymerization of actin filaments at certain lengths, evoked a sustained activation of alpha,beta,gamma-rENaC at actin/gelsolin ratios of <32:1, with a maximal effect at an actin/gelsolin ratio of 2:1. These results suggest that short actin filaments activate alpha, beta,gamma-rENaC. PKA-mediated phosphorylation augments activation of this channel by decreasing the rate of elongation of actin filaments. These results are consistent with the hypothesis that cloned alpha,beta,gamma-rENaCs form a core conduction unit of epithelial Na+ channels and that interaction of these channels with other associated proteins, such as short actin filaments, confers regulation to channel activity.

The recent immunopurification and cloning of various lung Na+ channel proteins has provided the necessary tools to study Na+ transport at a fundamental level across a number of epithelial tissues. Various macroscopic measurements of Na+ transport have shown that Na+ ions enter the cytoplasm of alveolar cells mainly through amiloride-inhibitable Na+ channels. Molecular biology studies have shown the existence of three Na+ channel subunit mRNAs (alpha-, beta-, and gamma-rENaC) in mature fetal (FDLE) and adult alveolar type II (ATII) cells. Patch-clamp studies have demonstrated the existence of various types of amiloride-inhibitable Na+ channels, located in the apical membranes of FDLE and ATII cells. beta-Agonists and agents that enhance intracellular adenosine 3',5'-cyclic monophosphate levels increase the open probability of these channels, leading to increased Na+ transport across the alveolar epithelium in vivo. Immunopurification of a putative channel protein from adult ATII cells showed that it contains an amiloride-binding subunit with a molecular mass of 150 kDa. When this protein was reconstituted in planar lipid bilayers, it exhibited single channels with a conductance of 25 pS, which were moderately selective for Na+ over K+. The open probability of these channels was increased by the addition of protein kinase A (PKA) and ATP, and was decreased to the same extent by addition of [N-ethyl-N-isopropyl]-2'-4'-amiloride (EIPA) and amiloride (1 microM each) in the apical side of the bilayer, in agreement with the results of patch-clamp studies in ATII cells. Exposure of rats to sublethal hyperoxia increased alpha-rENaC mRNA and the functional expression of Na+ channels in alveolar epithelial cells and limited alveolar edema. These findings indicate that alveolar epithelial channels contain at least one family of amiloride-sensitive Na+ channel proteins, which displays a number of unique properties, including sensitivity to EIPA.

We examined the regulation of a cloned epithelial Na+ channel (alpha beta gamma-rENaC) by protein kinase A (PKA) and protein kinase C (PKC). Experiments were performed in Xenopus oocytes and in planar lipid bilayers. At a holding potential of -100 mV, amiloride-sensitive current averaged -1,279 +/- 111 nA (n = 7) in alpha beta gamma-rENaC-expressing oocytes. Currents in water-injected oocytes were essentially unresponsive to 10 microM amiloride. A 1-h stimulation of PKC with 100 nM of PMA inhibited whole-cell currents in Xenopus oocytes to 17.1 +/- 1.8, and 22.1 +/- 2.6% of control (n = 7), at holding potentials of -100 and +40 mV, respectively. Direct injection of purified PKC resulted in similar inhibition to that observed with PMA. Additionally, the inactive phorbol ester, phorbol-12-myristate-13-acetate, 4-O-methyl, was without effect on alpha beta gamma-rENaC currents. Pretreatment with the microtubule inhibitor colchicine (100 microM) did not modify the inhibitory effect of PMA; however, pretreatment with 20 microM cytochalasin B decreased the inhibitory action of PMA to < 20% of that previously observed. In vitro-synthesized alpha beta gamma-rENaC formed an amiloride-sensitive Na(+)-selective channel when incorporated into planar lipid bilayers. Addition of PKC, diacyl-glycerol, and Mg-ATP to the side opposite that which amiloride blocked, decreased the channel's open probability (Po) from 0.44 +/- 0.06 to 0.13 +/- 0.03 (n = 9). To study the effects of PKA on alpha beta gamma-rENaC expressed in Xenopus oocytes, cAMP levels were elevated with 10 microM forskolin and 1 mM isobutyl-methyl-xanthine. This cAMP-elevating cocktail did not cause any stimulation of alpha beta gamma-rENaC currents in either the inward or outward directions. This lack of activation was also observed in oocytes preinhibited with PMA and in oocytes pretreated with cytochalasin B and PMA. Neither alpha-rENaC nor alpha beta gamma-rENaC incorporated into planar lipid bilayers could be activated with PKA and Mg-ATP added to either side of the membrane, as Po remained at 0.63 +/- 0.06 (n = 7) and 0.45 +/- 0.05 (n = 9), respectively. We conclude that: alpha beta gamma-rENaC is inhibited by PKC, and that alpha beta gamma-rENaC is not activated by PKA.

Amiloride-sensitive Na+ channels play a vital role in many important physiological processes such as delineation of the final urine composition, sensory transduction, and whole-body Na+ homeostasis. These channels display a wide range of biophysical properties, and are regulated by cAMP-mediated second messenger systems. The first of these channels has recently been cloned. This cloned amiloride-sensitive Na+ channel is termined ENaC (Epithelial Na+ Channel) and, in heterologous cellular expression systems, displays a single channel conductance of 4 to 7 pS, a high PNa/PK (> 10), a high amiloride sensitivity (Ki(amil) = 150 nM), and relatively long open and closed times. ENaC may form the core conduction element of many of these functionally diverse forms of Na+ channel. The kinetic and regulatory differences between these channels may be due, in large measure, to unique polypeptides that associate with the core element, forming a functional channel unit.

The mechanoelectrical transduction channels on hair cells have been suggested to be operated by tip links that are stretched when the hair bundle is deflected in the direction of the tallest row of stereocilia. Localising these channels is therefore an important test of this hypothesis. The transduction channels are known to be amiloride-sensitive and immunogold labelling with antibodies raised against the amiloride-sensitive epithelial Na+ channel from kidney (alpha NaCh), has suggested that sites with similar characteristics are located in the region where the tips of the shorter stereocilia appear to come into contact with the sides of the adjacent taller stereocilia rather than being associated directly with the tip links. Now, further immunocytochemical experiments have been performed to determine if amiloride and dihydrostreptomycin, both of which can block transduction, can affect this labelling. Immunofluorescent labelling of the stereocilia is obtained when surface preparations of the organ of Corti are fixed and incubated with alpha NaCh followed by an appropriate secondary antibody. This labelling is abolished by trypsinization prior to fixation but retained if the tissue is pretreated with amiloride and then trypsinized in its presence. Because amiloride is known to protect amiloride-binding sites from degradation by trypsin, these results suggest that alpha NaCh is revealing amiloride-binding sites on the stereocilia. Similarly, immunofluorescent labelling of the stereocilia is abolished if cochlear tissue is pretreated with dihydrostreptomycin (DHS) and fixed in its presence prior to incubation with alpha NaCh. Quantitative analysis of colloidal gold labelling using transmission electron microscopy shows that DHS treatment produces a significant reduction in the number of gold particles on stereocilia, especially in the region of contact between them. These results suggest that anti-Na+ recognises a site with characteristics similar to the mechanoelectrical transduction channels.

Cystic fibrosis airway epithelia exhibit enhanced Na+ reabsorption in parallel with diminished Cl- secretion. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) directly affects epithelial Na+ channel activity by co-incorporating into planar lipid bilayers immunopurified bovine tracheal CFTR and either heterologously expressed rat epithelial Na+ channel ( alpha,b eta,gamma-rENaC) or an immunopurified bovine renal Na+ channel protein complex. The single channel open probability (Po) of rENaC was decreased by 24% in the presence of CFTR. Protein kinase A (PKA) plus ATP activated CFTR, but did not have any effect on rENaC. CFTR also decreased the extent of elevation of the renal Na+ channel Po following PKA-mediated phosphorylation. Moreover, the presence of CFTR prohibited the inward rectification of the gating of this renal Na+ channel normally induced by PKA-mediated phosphorylation, thus down-regulating inward Na+ current. This interaction between CFTR and Na+ channels occurs independently of whether or not wild-type CFTR is conducting anions. However, the nonconductive CFTR mutant, G551D CFTR, cannot substitute for the wild-type molecule. Our results indicate that CFTR can directly down-regulate single Na+ channel activity, thus accounting, at least in part, for the observed differences in Na+ transport between normal and cystic fibrosis-affected airway epithelia.

The action of aldosterone to increase apical membrane permeability in responsive epithelia is thought to be due to activation of sodium channels. This channel is regulated, in part, by G-proteins, but it is not known if this mechanism is regulated by aldosterone. We report that aldosterone stimulates the expression of the 41-kDa alphai3 subunit of the heterotrimeric GTP-binding proteins in A-6 cells. Both mRNA and the total amount of this protein are increased by aldosterone. The G-protein is palmitoylated in response to the steroid, and the newly synthesized subunit is found to co-localize with the sodium channel. Aldosterone stimulation of sodium transport is significantly inhibited by inhibition of palmitoylation. These results suggest that aldosterone regulates sodium channel activity in epithelia through stimulation of the expression and post-translational targeting of a channel regulatory G-protein subunit.

A cloned rat epithelial Na+ channel (rENaC) was studied in planar lipid bilayers. Two forms of the channel were examined: channels produced by the alpha subunit alone and those formed by alpha, beta, and gamma subunits. The protein was derived from two sources: either from in vitro translation reaction followed by Sephadex column purification or from heterologous expression in Xenopus oocytes and isolation of plasma membranes. We found that either alpha-rENaC alone or alpha- in combination with beta- and gamma-rENaC, produced highly Na(+)-selective (PNa/PK = 10), amiloride-sensitive (Kamili = 170 nM), and mechanosensitive cation channels in planar bilayers. alpha-rENaC displayed a complicated gating mechanism: there was a nearly constitutively open 13-picosiemens (pS) state and a second 40-pS level that was achieved from the 13-pS level by a 26-pS transition. alpha-, beta-, gamma-rENaC showed primarily the 13-pS level. alpha-rENaC and alpha,beta,gamma-rENaC channels studied by patch clamp displayed the same gating pattern, albeit with > 2-fold lowered conductance levels, i.e. 6 and 18 pS, respectively. Upon treatment of either channel with the sulfhydryl reducing agent dithiothreitol, both channels fluctuated among three independent 13-pS sublevels. Bathing each channel with a high salt solution (1.5 M NaCl) produced stochastic openings of 19 and 38 pS in magnitude between all three conductance levels. Different combinations of alpha-, beta-, and gamma-rENaC in the reconstitution mixture did not produce channels of intermediate conductance levels. These findings suggest that functional ENaC is composed of three identical conducting elements and that their gating is concerted.

Liddle's disease is an autosomal dominant genetic disorder characterized by severe low renin hypertension ("pseudoaldosteronism") that has been genetically linked to a locus on chromosome 16 encoding the beta-subunit of an amiloride-sensitive Na+ channel (ASSC) (15). Peripheral blood lymphocytes (PBL) express ASSC that are functionally indistinguishable from those expressed by Na(+)-reabsorbing renal epithelial cells (3, 5). The amiloride-sensitive Na+ conductance in PBL from affected and unaffected individuals from the original Liddle's pedigree was examined using whole cell patch clamp. Typically, the basal Na+ currents in cells from affected individuals were maximally activated. Basal Na+ currents in cells from unaffected individuals were minimal and could be maximally activated by superfusion with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Affected cells could not be further stimulated with CPT-cAMP. Superfusion with a supermaximal concentration of amiloride (2 microM) inhibited both the cAMP-activated Na+ conductance in unaffected cells and the constitutively activated inward conductance in affected cells. Cytosolic addition of a peptide identical to the terminal 10 amino acids of the truncated beta-subunit normalized the cAMP-mediated but not the pertussis toxin-induced regulation of the mutant ASSC. The findings show that lymphocyte ASSC are constitutively activated in affected individuals, that a mutation of the beta-subunit alters ASSC responsiveness to specific regulatory effectors, and that the cellular mechanism responsible for the pathophysiology of Liddle's disease is abnormal regulation of Na+ channel activity. These findings have important diagnostic and therapeutic implications and provide a cellular phenotype for the diagnosis of pseudoaldosteronism.

Stretch-mediated regulation of amiloride-sensitive Na+ channels was examined in Epstein-Barr virus-transformed human B lymphocytes. Cation conductances were measured using whole cell patch-clamp techniques. Stretch activation, induced by increasing the hydrostatic pressure of the bath solution, immediately and reversibly increased both inward and outward ionic conductances once a threshold of 2-5 mmH2O was reached. Ionic substitutions confirmed that stretch enhanced membrane conductivity for both Na+ and K+. Amiloride (2 microM) completely prevented the response to elevated hydrostatic pressure; however, when amiloride was applied after stretch-induced activation, the sensitivity to amiloride was dramatically decreased (inhibitor concentration that reduces whole cell current by 50% of approximately 20 microM). Evidence that the currents induced by stretch were mediated by Na+ channels was provided by the lack of response to stretch in lymphocytes from patients with Liddle's syndrome, which is caused by expression of a truncated mutant of the beta-subunit of the amiloride-sensitive Na+ channel. Pretreatment with colchicine (0.5 mM, 30 min) prevented stretch-induced activation, which shows evidence of the involvement of the cytoskeleton. These data indicate that stretch regulates the conductance of amiloride-sensitive Na+ channels in immortalized human B lymphocytes and also alters its cationic selectivity and its sensitivity to amiloride.

The hypothesis that amiloride-sensitive Na+ channel complexes immunopurified from bovine renal papillary collecting tubules contain, as their core conduction component, an ENaC subunit, was tested by functional and immunological criteria. Disulfide bond reduction with dithiothreitol (DTT) of renal Na+ channels incorporated into planar lipid bilayers caused a reduction of single channel conductance from 40 pS to 13 pS, and uncoupled PKA regulation of this channel. The cation permeability sequence, as assessed from bi-ionic reversal potential measurements, and apparent amiloride equilibrium dissociation constant (K(amil)i) of the Na+ channels were unaltered by DTT treatment. Like ENaC, the DTT treated renal channel became mechanosensitive, and displayed a substantial decrease in K(amil)i following stretch (0.44 +/- 0.12 microM versus 6.9 +/- 1.0 microM). Moreover, stretch activation induced a loss in the channel's ability to discriminate between monovalent cations, and even allowed Ca2+ to permeate. Polyclonal antibodies generated against a fusion protein of alpha bENaC recognized a 70 kDa polypeptide component of the renal Na+ channel complex. These data suggest that ENaC is present in the immunopurified renal Na+ channel protein complex, and that PKA sensitivity is conferred by other associated proteins.

Purified bovine renal papillary Na+ channels, when reconstituted into planar lipid bilayers, reside in three conductance states: a 40-pS main state, and two subconductive states (12-13 pS and 24-26 pS). The activity of these channels is regulated by phosphorylation and by G-proteins. Protein kinase A (PKA)-induced phosphorylation increased channel activity by increasing the open state time constants from 160 +/- 30 (main conductance), and 15 +/- 5 ms (both lower conductances), respectively, to 365 +/- 30 ms for all of them. PKA phosphorylation also altered the closed time of the channel from 250 +/- 30 ms to 200 +/- 35 ms, thus shifting the channel into a lower-conductance, long open time mode. PKA phosphorylation increased the PNa:PK of the channel from 7:1 to 20:1, and shifted the amiloride inhibition curve to the right (apparent K(i)amil from 0.7 to 20 microM). Pertussis toxin-induced ADP-ribosylation of either phosphorylated of either phosphorylated or nonphosphorylated channels decreased the PNa:PK to 2:1 and 4:1, respectively, and altered K(i)amil to 8 and 2 microM for phosphorylated and nonphosphorylated channels, respectively. GTP-gamma-S treatment of either phosphorylated or nonphosphorylated channels resulted in an increase of PNa:PK to 30:1 and 10:1, respectively, and produced a leftward shift in the amiloride dose-response curve, altering K(i)amil to 0.5 and 0.1 microM, respectively. These results suggest that amiloride-sensitive renal Na+ channel biophysical characteristics are not static, but depend upon the biochemical state of the channel protein and/or its associated G-protein.

We have previously demonstrated that apical Na+ channels in A6 renal epithelial cells are associated with spectrin-based membrane cytoskeleton proteins and that the lateral mobility of these channels, as determined by fluorescence photobleach recovery (FPR) analysis, is severely restricted by this association (Smith et al., 1991. Proc. Natl. Acad. Sci. USA 88:6971-6975). Recent data indicate that the actin component of the cytoskeleton may play a role in modulating Na+ channel activity (Cantiello et al., 1991. Am. J. Physiol. 261:C882-C888); however, it is unknown if the Na+ channel's linkage to the spectrin-based membrane cytoskeleton is also involved in regulating channel activity. In this study, we have used FPR to examine if the linkage of the Na+ channels to the membrane cytoskeleton is a site for modulation of Na+ channel activity in filter grown A6 cells by vasopressin and aldosterone. We hypothesized that if the linkage of the Na+ channels to the membrane cytoskeleton is a site for regulation of Na+ channel activity by vasopressin and aldosterone, then hormone-mediated changes in either the membrane cytoskeleton or the affinity of the Na+ channel for the membrane cytoskeleton, should be reflected in changes in the lateral mobility and/or mobile fraction of Na+ channels on the cell surface. FPR revealed that although the rates of lateral mobility were not affected, there was a twofold increase in mobility fraction (f) of apical Na+ channels in aldosterone-treated (16 hr) monolayers (f = 32.31 +/- 5.42%) when compared to control (unstimulated) (f = 14.2 +/- 0.77%) and vasopressin-treated (20 min) (f = 12.7 +/- 2.4%) monolayers. The twofold increase in mobile fraction of Na+ channels corresponds to the average increase in Na+ transport in response to aldosterone in A6 cells. The aldosterone-induced increase in Na+ transport and mobile fraction can be inhibited by the methylation inhibitor, 3-deazaadenosine, consistent with the hypothesis that a methylation event is involved in aldosterone induced upregulation of Na+ transport. We propose that the membrane cytoskeleton is involved in the aldosterone-mediated activation of epithelial Na+ channels.

Purified bovine renal epithelial Na+ channels when reconstituted into planar lipid bilayers displayed a specific orientation when the membrane was clamped to -40 mV (cis-side) during incorporation. The trans-facing portion of the channel was extracellular (i.e., amiloride-sensitive), whereas the cis-facing side was intracellular (i.e., protein kinase A-sensitive). Single channels had a main state unitary conductance of 40 pS and displayed two subconductive states each of 12-13 pS, or one of 12-13 pS and the second of 24-26 pS. Elevation of the [Na+] gradient from the trans-side increased single-channel open probability (Po) only when the cis-side was bathed with a solution containing low [Na+] (< 30 mM) and 10-100 microM [Ca2+]. Under these conditions, Po saturated with increasing [Na+]trans. Buffering of the cis compartment [Ca2+] to nearly zero (< 1 nM) with 10 mM EGTA increased the initial level of channel activity (Po = 0.12 +/- 0.02 vs 0.02 +/- 0.01 in control), but markedly reduced the influence of both cis- and trans-[Na+] on Po. Elevating [Ca2+]cis at constant [Na+] resulted in inhibition of channel activity with an apparent [KiCa2+] of 10-100 microM. Protein kinase C-induced phosphorylation shifted the dependence of channel Po on [Ca2+]cis to 1-3 microM at stationary [Na+]. The direct modulation of single-channel Po by Na+ and Ca2+ demonstrates that the gating of amiloride-sensitive Na2+ channels is indeed dependent upon the specific ionic environment surrounding the channels.

Patch-clamp experiments have demonstrated an amiloride-sensitive Na+ conductance in human B lymphoid cells. We measured whole cell currents in rat lymphocytes and observed a similar Na(+)-specific inward conductance. The presence of 400 microM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate in the bath significantly increased the inward current, and this adenosine 3',5'-cyclic monophosphate activation was abolished by 2 microM amiloride. We immunopurified a protein complex from rat lymphocyte membranes using an anti-bovine kidney Na+ channel antibody. The complex consisted of five distinct polypeptides with apparent M(r) values of 110,000, 92,000, 59,000, 48,000, and 42,000. This putative channel complex was incorporated into planar lipid bilayers, where we observed single Na+ channel activity that was blocked by amiloride in a concentration-dependent manner. The addition of protein kinase A and ATP to the "intracellular" solution elicited a twofold increase in channel activity. Reverse transcription-polymerase chain reaction analysis was used to determine if the rat lymphocytes express the message for the recently cloned Na+ channel of the rat colon (rENaC). Primers for the alpha-subunit of rENaC identified no message in the lymphocyte RNA, while primers for the beta-subunit of the clone produced low levels of the expected product. Thus it appears that a rENaC-like beta-subunit may be an essential component of the lymphocyte Na+ channel that was isolated. At the same time, this channel is different from those recently cloned in that it does not include an alpha-subunit homologous to that of rENaC.

A bovine homologue of the rat and human epithelial Na+ channel subunits, alpha-rENaC and alpha-hENaC, was cloned. The cDNA clone, termed alpha-bENaC, was isolated from a bovine renal papillary collecting duct cDNA expression library. The bovine cDNA is 3,584 base pairs (bp) long, has an open reading frame of 2,094 bp encoding a 697-amino acid protein, and is 75-85% homologous to its rat and human counterparts. In vitro translation of the transcribed cRNA yields an 80-kDa polypeptide and one at 92 kDa in the presence of pancreatic microsomes. The clone exhibits consensus sequences for N-linked glycosylation and for phosphorylation by protein kinase C, but not for protein kinase A. After expression in Xenopus laevis oocytes, a small amiloride-sensitive Na+ conductance that exhibited inward rectification and a reversal potential greater than +30 mV, consistent with the predicted equilibrium potential for Na+, was identified. The expressed alpha-bENaC-associated Na+ current was not responsive to elevations in adenosine 3',5'-cyclic monophosphate but could be stimulated by phorbol 12-myristate 13-acetate, an activator of protein kinase C. alpha-bENaC also formed amiloride-sensitive chimeric channels when coexpressed with the rat beta- and gamma-ENaC subunits in Xenopus oocytes. alpha-bENaC therefore represents a novel isoform of a growing family of epithelial Na+ channels.

We investigated the cellular and molecular events associated with the increase in sodium transport across the alveolar epithelium of rats exposed to hyperoxia (85% O2 for 7 days followed by 100% O2 for 4 days). Alveolar type II (ATII) cell RNA was isolated and probed with a cDNA for one of the rat colonic epithelial sodium channel subunits (alpha rENaC). The alpha rENaC mRNA (3.7-kb transcript) increased 3-fold in ATII cell RNA isolated from rats exposed to 85% O2 for 7 days and 6-fold after 4 days of subsequent exposure to 100% O2. In situ hybridization revealed increased expression of alpha rENaC mRNA transcripts in both airway and alveolar epithelial cells of hyperoxic rats. When immunostained with a polyclonal antibody to kidney sodium channel protein, ATII cells from hyperoxic rats exhibited a significant increase in the amount of immunogenic protein present in both the plasma membrane and the cytoplasm. When patched in the whole-cell mode, ATII cells from hyperoxic rats exhibited amiloride and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride (EIPA)-sensitive currents that were 100% higher compared with those obtained from air-breathing rats. Single-channel sodium currents (mean conductance of 25 pS) were seen in ATII cells patched in both the inside-out and cell-attached modes. The number and open probability of these channels increased significantly during exposure to hyperoxia. Exposure to sublethal hyperoxia up-regulated both alpha rENaC mRNA and the functional expression of sodium channels in ATII cells.

Some non-sensory epithelia of the inner ear were examined for the localization of immunoreactivity to polyclonal antibodies raised against amiloride-sensitive Na+ channels from the bovine kidney. The pre-embedding immunogold technique was used for this purpose. Labelings were found on the membrane of the endolymphatic surface of strial marginal cells, epithelial cells of spiral prominence and Reissner's membrane, and ampullar dark cells. In contrast, no labeling was found on the luminal membrane of mesothelial cells of Reissner's membrane, the cells lining the supra-strial perilymphatic space, transitional cells and ampullar ceiling cells. Since the antibodies used may also label non-selective cation channels and non-functional sodium channel precursors as suggested by others, it was not possible to determine the labelings are solely due to amiloride-sensitive Na+ channels. However, the observed result was consistent with the previous studies that amiloride blocks ion transport in strial marginal cells and the semicircular canal. It is therefore likely that the observed labeling includes amiloride-sensitive Na+ channels. These labeled ion channels in a variety of epithelial cells lining the endolymphatic space could be important in the inner ear fluid regulation.

We have previously cloned a bovine renal epithelial channel homologue (alpha-bENaC) belonging to the epithelial Na+ channel (ENaC) family. With the use of a rabbit nuclease-treated in vitro translation system, mRNA coding for alpha-bENaC was translated and the polypeptide products were reconstituted into liposomes. On incorporation into planar lipid bilayers, in vitro-translated alpha-bENaC protein 1) displayed voltage-independent Na+ channel activity with a single-channel conductance of 40 pS, 2) was mechanosensitive in that the single-channel open probability was maximally activated with a hydrostatic pressure gradient of 0.26 mmHg across the bilayer, 3) was blocked by low concentrations of amiloride [apparent inhibitory constant of amiloride (K(i)amil approximately 150 nM], and 4) was cation selective with a Li+:Na+:K+ permselectivity of 2:1:0.14 under nonstretched conditions. These pharmacological and selectivity characteristics were altered to a lower amiloride affinity (K(i)amil > 25 microM) and a lack of monovalent cation selectivity in the presence of a hydrostatic pressure gradient. This observation of stretch activation (SA) of alpha-bENaC was confirmed in dual electrode recordings of heterologously expressed alpha-bENaC whole cell currents in Xenopus oocytes swelled by the injection of 15 nl of a 100 mM KCl solution. We conclude that alpha-bENaC, and by analogy other ENaCs, represent a novel family of cloned SA channels.

Low-amiloride-affinity (L-type) Na+ channels have been functionally and immunologically localized to alveolar type II (ATII) cells. Purified rabbit ATII epithelial cells were isolated by elastase digestion and solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate. The solubilized proteins were purified by ion-exchange chromatography, followed by immunoaffinity purification over a column to which rabbit polyclonal antibodies raised against purified bovine renal Na+ channel protein were bound. The proteins eluted from the immunoaffinity column were assayed for specific binding of [3H]Br-benzamil and reconstituted into planar lipid bilayers. Sequential purification steps gave a final enrichment in specific [3H]Br-benzamil binding of > 2,000 compared with the homogenate. Single-channel currents of 25 pS were recorded from the immunopurified rabbit ATII cell protein. Addition of the catalytic subunit of protein kinase A (PKA) plus ATP to the presumed cytoplasmic side of the bilayer resulted in a significant increase in the single-channel open probability (Po), from 0.40 +/- 0.14 to 0.8 +/- 0.12, without altering single-channel conductance. The addition of amiloride or ethylisopropyl amiloride (EIPA) to the side opposite that in which PKA acts reduced Po with no change in single-channel conductance. Rabbit ATII Na+ channels in bilayers had an inhibitory constant for amiloride of 8 microM and 1 microM for EIPA. These data confirm the presence of L-type Na+ channels in adult mammalian ATII cells.

Patch clamp methods were used to characterize sodium channels on the apical membrane of Ambystoma distal nephron. The apical membranes were exposed by everting and perfusing initial collecting tubules in vitro. In cell-attached patches, we observed channels whose mean inward unitary current averaged 0.39 +/- 0.05 pA (9 patches). The conductance of these channels was 4.3 +/- 0.2 pS. The unitary current approached zero at a pipette voltage of -92 mV. When clamped at the membrane potential the channel expressed a relatively high open probability (0.46). These characteristics, together with observation that doses of 0.5 to 2 microM amiloride reversibly inhibited the channel activity, are consistent with the presence of the high amiloride affinity, high sodium selectivity channel reported for rat cortical collecting tubule and cultured epithelial cell lines. We used antisodium channel antibodies to identify biochemically the epithelial sodium channels in the distal nephron of Ambystoma. Polyclonal antisodium channel antibodies generated against purified bovine renal, high amiloride affinity epithelial sodium channel specifically recognized 110, 57, and 55 kDa polypeptides in Ambystoma and localized the channels to the apical membrane of the distal nephron. A polyclonal antibody generated against a synthetic peptide corresponding to the C-terminus of Apx, a protein associated with the high amiloride affinity epithelial sodium channel expressed in A6 cells, specifically recognized a 170 kDa polypeptide. These data corroborate that the apically restricted sodium channels in Ambystoma are similar to the high amiloride affinity, sodium selective channels expressed in both A6 cells and the mammalian kidney.

In mammals, transduction of sodium stimuli occurs via amiloride-sensitive sodium channels. In rat, gustatory physiological sensitivity to sodium stimuli develops gradually during the early postnatal period. In addition, if pregnant rats are subjected to dietary sodium restriction during gestation, their offspring fail to develop normal gustatory physiological responses to sodium and sensitivity to amiloride. In the present study, we used polyclonal antibodies raised against amiloride-sensitive sodium channels to ascertain whether gustatory function is correlated with the immunological presence of the transduction apparatus for sodium stimuli in the taste buds of neonatal rats and adult offspring of sodium-restricted dams. The results indicate that antiamiloride-sensitive sodium channel antisera bind cells within taste buds of neonatal and adult rats, regardless of maternal dietary condition. Therefore, despite the functional absence of taste system amiloride-sensitive sodium channels, the antigenic determinants of these channels are expressed. These data suggest that the onset of normal gustatory sodium sensitivity in neonatal normal rats results from the progressive activation of existing, quiescent channels. Furthermore, they rule out the possibility that the failure to synthesize channel protein underlies the lack of gustatory sodium and amiloride sensitivity in the offspring of sodium-restricted rats.