Mouhamed S. Awayda

Dilated Intercellular Spaces and Shunt Permeability in Nonerosive Acid-Damaged Esophageal Epithelium

OBJECTIVES:It has recently been established that patients with nonerosive reflux disease have on biopsy within esophageal epithelium a lesion known as dilated intercellular spaces (DIS).METHODS:To further explore the nature and implications of this lesion, in vitro models of nonerosive acid and acid-pepsin damage were created in Ussing chamber-mounted rabbit esophageal epithelium. Using these models circuit analysis and permeability studies were carried out, the latter using dextran of varying size and human epidermal growth factor (EGF).RESULTS:Luminal HCl, pH 1.1, or HCl, pH 2.0 + pepsin, 1 mg/ml, for 30 min significantly reduced transepithelial electrical resistance (RT) but produced no gross erosions or histologic evidence of cell necrosis. Transmission electron microscopy, however, documented the presence of DIS. Circuit analysis on healthy esophageal epithelium showed that shunt resistance (RS) was much lower than apical membrane, basolateral membrane and transcellular resistances (Ra, Rb, and Rcell, respectively) and approached that of RT. Further, circuit analysis on acid and acid-pepsin damaged tissues showed that the declines in RT resulted from declines in RS. Moreover, the declines in RT (and so RS) were associated with a linear increase in permeability to 4 kD dextrans as well as an increase in permeability to 6 kD EGF and dextrans as large as 20 kD.CONCLUSIONS:In nonerosive acid-damaged esophageal epithelium DIS develop in association with and as a marker of reduced transepithelial resistance and increased shunt permeability. This change in shunt permeability upon acid or acid-pepsin exposure is substantial, permitting dextran molecules as large as 20 kD (33 Å) to diffuse across the epithelium. Also, this shunt leak enables luminal EGF at 6 kD to diffuse across the acid-damaged epithelium and by so doing enables it to access its receptors on epithelial basal cells. We hypothesize that the shunt leak of EGF may in part account for the development of a reparative phenomenon on esophageal biopsy in patients with nonerosive reflux disease known as basal cell hyperplasia.

TRIPLE-BARREL ORGANIZATION OF ENAC, A CLONED EPITHELIAL NA+ CHANNEL

A cloned rat epithelial Na+ channel (rENaC) was studied in planar lipid bilayers. Two forms of the channel were examined: channels produced by the α subunit alone and those formed by α, β, and γ 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 α-rENaC alone or α- in combination with β- and γ-rENaC, produced highly Na+-selective (PNa/PK = 10), amiloride-sensitive (Kiamil = 170 nM), and mechanosensitive cation channels in planar bilayers. α-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. α-, β-, γ-rENaC showed primarily the 13-pS level. α-rENaC and α,β,γ-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 α-, β-, and γ-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.

ENaC-membrane interactions: regulation of channel activity by membrane order.

Recently, it was reported that the epithelial Na+ channel (ENaC) is regulated by temperature (Askwith, C.C., C.J. Benson, M.J. Welsh, and P.M. Snyder. 2001. Proc. Natl. Acad. Sci. USA. 98:6459–6463). As these changes of temperature affect membrane lipid order and lipid–protein interactions, we tested the hypothesis that ENaC activity can be modulated by membrane lipid interactions. Two approaches were used to modulate membrane anisotropy, a lipid order–dependent parameter. The nonpharmacological approach used temperature changes, while the pharmacological one used chlorpromazine (CPZ), an agent known to decrease membrane order, and Gd+3. Experiments used Xenopus oocytes expressing human ENaC. Methods of impedance analysis were used to determine whether the effects of changing lipid order indirectly altered ENaC conductance via changes of membrane area. These data were further corroborated with quantitative morphology on micrographs from oocytes membranes studied via electron microscopy. We report biphasic effects of cooling (stimulation followed by inhibition) on hENaC conductance. These effects were relatively slow (minutes) and were delayed from the actual bath temperature changes. Peak stimulation occurred at a calculated Tmax of 15.2. At temperatures below Tmax, ENaC conductance was inhibited with cooling. The effects of temperature on g Na were distinct from those observed on ion channels endogenous to Xenopus oocytes, where the membrane conductance decreased monoexponentially with temperature (t = 6.2°C). Similar effects were also observed in oocytes with reduced intra- and extracellular [Na+], thereby ruling out effects of self or feedback inhibition. Addition of CPZ or the mechanosensitive channel blocker, Gd+3, caused inhibition of ENaC. The effects of Gd+3 were also attributed to its ability to partition into the outer membrane leaflet and to decrease anisotropy. None of the effects of temperature, CPZ, or Gd+3 were accompanied by changes of membrane area, indicating the likely absence of effects on channel trafficking. However, CPZ and Gd+3 altered membrane capacitance in an opposite manner to temperature, consistent with effects on the membrane-dielectric properties. The reversible effects of both Gd+3 and CPZ could also be blocked by cooling and trapping these agents in the rigidified membrane, providing further evidence for their mechanism of action. Our findings demonstrate a novel regulatory mechanism of ENaC.

Indirect Activation of the Epithelial Na+ Channel by Trypsin

We tested the hypothesis that the serine protease trypsin can indirectly activate the epithelial Na+ channel (ENaC). Experiments were carried out in Xenopus oocytes and examined the effects on the channel formed by all three human ENaC subunits and that formed by Xenopus epsilon and human β and γ subunits (ϵβγENaC). Low levels of trypsin (1–10 ng/ml) were without effects on the oocyte endogenous conductances and were specifically used to test the effects on ENaC. Addition of 1 ng/ml trypsin for 60 min stimulated the amiloride-sensitive human ENaC conductance (gNa) by ∼6-fold. This effect on the gNa was [Na+]-independent, thereby ruling out an interaction with channel feedback inhibition by Na+. The indirect nature of this activation was confirmed in cell-attached patch clamp experiments with trypsin added to the outside of the pipette. Trypsin was comparatively ineffective at activating ϵβγENaC, a channel that exhibited a high spontaneous open probability. These observations, in combination with surface binding experiments, indicated that trypsin indirectly activated membrane-resident channels. Activation by trypsin was also dependent on catalytic activity of this protease but was not accompanied by channel subunit proteolysis. Channel activation was dependent on downstream activation of G-proteins and was blocked by G-protein inhibition by injection of guanyl-5′-yl thiophosphate and by pre-stimulation of phospholipase C. These data indicate a receptor-mediated activation of ENaC by trypsin. This trypsin-activated receptor is distinct from that of protease-activated receptor-2, because the response to trypsin was unaffected by protease-activated receptor-2 overexpression or knockdown.

Associated Proteins and Renal Epithelial Na+ Channel Function

Abstract. 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 (Kamili) of the Na+ channels were unaltered by DTT treatment. Like ENaC, the DTT treated renal channel became mechanosensitive, and displayed a substantial decrease in Kamili following stretch (0.44 ± 0.12 μm versus 6.9 ± 1.0 μm). Moreover, stretch activation induced a loss in the channels ability to discriminate between monovalent cations, and even allowed Ca2+ to permeate. Polyclonal antibodies generated against a fusion protein of αbENaC 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.

Regulation of the epithelial Na+ channel by intracellular Na+

The hypothesis that the intracellular Na+ concentration ([Na+]i) is a regulator of the epithelial Na+ channel (ENaC) was tested with the Xenopus oocyte expression system by utilizing a dual-electrode voltage clamp. [Na+]iaveraged 48.1 ± 2.2 meq ( n = 27) and was estimated from the amiloride-sensitive reversal potential. [Na+]iwas increased by direct injection of 27.6 nl of 0.25 or 0.5 M Na2SO4. Within minutes of injection, [Na+]istabilized and remained elevated at 97.8 ± 6.5 meq ( n = 9) and 64.9 ± 4.4 ( n = 5) meq 30 min after the initial injection of 0.5 and 0.25 M Na2SO4, respectively. This increase of [Na+]icaused a biphasic inhibition of ENaC currents. In oocytes injected with 0.5 M Na2SO4( n = 9), a rapid decrease of inward amiloride-sensitive slope conductance ( g Na) to 0.681 ± 0.030 of control within the first 3 min and a secondary, slower decrease to 0.304 ± 0.043 of control at 30 min were observed. Similar but smaller inhibitions were also observed with the injection of 0.25 M Na2SO4. Injection of isotonic K2SO4(70 mM) or isotonic K2SO4made hypertonic with sucrose (70 mM K2SO4-1.2 M sucrose) was without effect. Injection of a 0.5 M concentration of either K2SO4, N-methyl-d-glucamine (NMDG) sulfate, or 0.75 M NMDG gluconate resulted in a much smaller initial inhibition (<14%) and little or no secondary decrease. Thus increases of [Na+]ihave multiple specific inhibitory effects on ENaC that can be temporally separated into a rapid phase that was complete within 2-3 min and a delayed slow phase that was observed between 5 and 30 min.

Regulation of a cloned epithelial Na+ channel by its β- and γ-subunits

Using the Xenopus oocyte expression system, we examined the mechanisms by which the β- and γ-subunits of an epithelial Na+channel (ENaC) regulate α-subunit channel activity and the mechanisms by which β-subunit truncations cause ENaC activation. Expression of α-ENaC alone produced small amiloride-sensitive currents (-43 ± 10 nA, n = 7). These currents increased >30-fold with the coexpression of β- and γ-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 α-subunit of ENaC. Truncation of the last 75 amino acids of the β-subunit COOH terminus, as found in the original pedigree of individuals with Liddles syndrome, caused a 4.4-fold ( n = 17) increase of the amiloride-sensitive currents compared with wild-type αβγ-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 β-ENaC significantly reduced the amiloride-sensitive currents by 40-50%. These observations suggest a tonic inhibitory role on the channels open probability ( P o) by the COOH terminus of β-ENaC. We conclude that the changes of current observed with coexpression of the β- and γ-subunits or those observed with β-subunit truncation are likely the result of changes of channel density in combination with large changes of P o.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 Liddles 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 channels 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.

The A-kinase anchoring protein 15 regulates feedback inhibition of the epithelial Na+ channel

Protein kinase A anchoring proteins or AKAPs regulate the activity of many ion channels. Protein kinase A (PKA) is a well‐recognized target of AKAPs, with other kinases now emerging as additional targets. We examined the roles of epithelial‐expressed AKAPs in regulating the epithelial Na+ channel (ENaC). Experiments used heterologous expression with AKAP15, AKAP‐KL, and AKAP79 in Xenopus oo‐cytes. Experiments were carried out under high and low Na+ conditions, as Na+ loading is known to affect the baseline activity of ENaC in a PKC‐dependent mechanism. ENaC activity was unaffected by AKAP79 and AKAP‐KL expression. However, oocytes coexpressing AKAP15 exhibited an 80% and 91% reduction in the amiloride‐sensitive, whole‐cell conductance in high and low Na+ conditions, respectively. The reduced channel activity was unaffected by PKA activation or inhibition, indicating a PKA‐independent mechanism. Expression with a membrane‐targeting domain, mutant form of AKAP15 (AKAP15m) prevented the decrease of ENaC activity, but only under low Na+ conditions. In high sodium conditions, coexpression with AKAP15m led to an increase of ENaC activity to levels similar to those observed under low Na+. These results indicate that membrane‐associated AKAP15 reduces ENaC activity whereas the cytoplasmically associated one may participate in the channels feedback inhibition by intracellular Na+, a process known to involve PKC. This hypothesis was further confirmed in coexpression experiments, which demonstrated functional and physical interaction between AKAP15 and PKCα. We propose that AKAP15 regulates ENaC via a novel PKA‐indepen‐dent pathway.—Bengrine, A., Li, J., Awayda, M. S. The A‐kinase anchoring protein 15 regulates feedback inhibition of the epithelial Na+ channel. FASEB J. 21, 1189–1201 (2007)

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of αENaC using the serine protease subtilisin. Trimeric channels formed with αFM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an ∼10-fold decrease in amiloride-sensitive whole cell conductance as compared with αWT (2.2 versus 21.2 microsiemens (μS)). Both αWT and αFM were activated to similar levels by subtilisin cleavage. Channels formed with αFD, a construct that deleted the segment between the two furin sites (Δ175–204), exhibited an intermediate conductance of 13.2 μS. More importantly, αFD retained the ability to be activated by subtilisin to 108.8 ± 20.9 μS, a level not significantly different from that of subtilisin activated αWT (125.6 ± 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of α was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with αLD, a construct that extended the deleted sequence of αFD by 17 amino acids (Δ175–221). Channels with αLD were uncleaved, exhibited low baseline activity (4.1 μS), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex.

Acute Cholesterol-induced Anti-natriuretic Effects ROLE OF EPITHELIAL Na+ CHANNEL ACTIVITY, PROTEIN LEVELS, AND PROCESSING

The epithelial Na+ channel (ENaC) is modulated by membrane lipid composition. However, the effect of an in vivo change of membrane composition is unknown. We examined the effect of a 70-day enhanced cholesterol diet (ECD) on ENaC and renal Na+ handling. Rats were fed a standard chow or one supplemented with 1% cholesterol and 0.5% cholic acid (ECD). ECD animals exhibited marked anti-diuresis and anti-natriuresis (40 and 47%), which peaked at 1–3 weeks. Secondary compensation returned urine output and urinary Na+ excretion to control levels by week 10. During these initial changes, there were no accompanying effects on systolic blood pressure, serum creatinine, or urinary creatinine excretion, indicating that the these effects of ECD preceded those which modify renal filtration and blood pressure. The effects of ECD on ENaC were evaluated by measuring the relative protein content of α, β, and γ subunits. α and γ blots were further examined for subunit cleavage (a process that activates ENaC). No significant changes were observed in α and β levels throughout the study. However, levels of cleaved γ were elevated, suggesting that ENaC was activated. The changes of γ persisted at week 10 and were accompanied by additional subunit fragments, indicating potential changes of γ-cleaving proteases. Enhanced protease activity, and specifically that which could act on the second identified cleavage site in γ, was verified in a newly developed urinary protease assay. These results predict enhanced ENaC activity, an effect that was confirmed in patch clamp experiments of principal cells of split open collecting ducts, where ENaC open probability was increased by 40% in the ECD group. These data demonstrate a complex series of events and a new regulatory paradigm that is initiated by ECD prior to the onset of elevated blood pressure. These events lead to changes of renal Na+ handling, which occur in part by effects on extracellular γ-ENaC cleavage.