Extracellular histidine residues crucial for Na+ self-inhibition of epithelial Na+ channels

Epithelial Na(+) channels (ENaC) participate in the regulation of extracellular fluid volume homeostasis and blood pressure. Channel activity is regulated by both extracellular and intracellular Na(+). The down-regulation of ENaC activity by external Na(+) is referred to as Na(+) self-inhibition. We investigated the structural determinants of Na(+) self-inhibition by expressing wild-type or mutant ENaCs in Xenopus oocytes and analyzing changes in whole-cell Na(+) currents following a rapid increase of bath Na(+) concentration. Our results indicated that wild-type mouse alphabetagammaENaC has intrinsic Na(+) self-inhibition similar to that reported for human, rat, and Xenopus ENaCs. Mutations at His(239) (gammaH239R, gammaH239D, and gammaH239C) in the extracellular loop of the gammaENaC subunit prevented Na(+) self-inhibition whereas mutations of the corresponding His(282) in alphaENaC (alphaH282D, alphaH282R, alphaH282W, and alphaH282C) significantly enhanced Na(+) self-inhibition. These results suggest that these two histidine residues within the extracellular loops are crucial structural determinants for Na(+) self-inhibition.     

Extracellular protons regulate human ENaC by modulating Na+ self-inhibition

The epithelial Na(+) channel, ENaC, is exposed to a wide range of proton concentrations in the kidney, lung, and sweat duct. We, therefore, tested whether pH alters ENaC activity. In Xenopus oocytes expressing human alpha-, beta-, and gammaENaC, amiloride-sensitive current was altered by protons in the physiologically relevant range (pH 8.5-6.0). Compared with pH 7.4, acidic pH increased ENaC current, whereas alkaline pH decreased current (pH(50) = 7.2). Acidic pH also increased ENaC current in H441 epithelia and in human primary airway epithelia. In contrast to human ENaC, pH did not alter rat ENaC current, indicating that there are species differences in ENaC regulation by protons. This resulted predominantly from species differences in gammaENaC. Maneuvers that lock ENaC in a high open-probability state ("DEG" mutation, proteolytic cleavage) abolished the effect of pH on human ENaC, indicating that protons alter ENaC current by modulating channel gating. Previous work showed that ENaC gating is regulated in part by extracellular Na(+) ("Na(+) self-inhibition"). Based on several observations, we conclude that protons regulate ENaC by altering Na(+) self-inhibition. First, protons reduced Na(+) self-inhibition in a dose-dependent manner. Second, ENaC regulation by pH was abolished by removing Na(+) from the extracellular bathing solution. Third, mutations that alter Na(+) self-inhibition produced corresponding changes in ENaC regulation by pH. Together, the data support a model in which protons modulate ENaC gating by relieving Na(+) self-inhibition. We speculate that this may be an important mechanism to facilitate epithelial Na(+) transport under conditions of acidosis.     

External nickel inhibits epithelial sodium channel by binding to histidine residues within the extracellular domains of alpha and gamma subunits and reducing channel open probability

Epithelial sodium channels (ENaC) are regulated by various intracellular and extracellular factors including divalent cations. We studied the inhibitory effect and mechanism of external Ni(2+) on cloned mouse alpha-beta-gamma ENaC expressed in Xenopus oocytes. Ni(2+) reduced amiloride-sensitive Na(+) currents of the wild type mouse ENaC in a dose-dependent manner. The Ni(2+) block was fast and partially reversible at low concentrations and irreversible at high concentrations. ENaC inhibition by Ni(2+) was accompanied by moderate inward rectification at concentrations higher than 0.1 mm. ENaC currents were also blocked by the histidine-reactive reagent diethyl pyrocarbonate. Pretreatment of the oocytes with the reagent reduced Ni(2+) inhibition of the remaining current. Mutations at alphaHis(282) and gammaHis(239) located within the extracellular loops significantly decreased Ni(2+) inhibition of ENaC currents. The mutation alphaH282D or double mutations alphaH282R/gammaH239R eliminated Ni(2+) block. All mutations at gammaHis(239) eliminated Ni(2+)-induced inward current rectification. Ni(2+) block was significantly enhanced by introduction of a histidine at alphaArg(280). Lowering extracellular pH to 5.5 and 4.4 decreased or eliminated Ni(2+) block. Although alphaH282C-beta-gamma channels were partially inhibited by the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), alpha-beta-gamma H239C channels were insensitive to MTSET. From patch clamp studies, Ni(2+) did not affect unitary current but decreased open probability when perfused into the recording pipette. Our results suggest that external Ni(2+) reduces ENaC open probability by binding to a site consisting of alphaHis(282) and gammaHis(239) and that these histidine residues may participate in ENaC gating.     

Functional role of extracellular loop cysteine residues of the epithelial Na+ channel in Na+ self-inhibition

The epithelial Na(+) channel (ENaC) is typically formed by three homologous subunits (alpha, beta, and gamma) that possess a characteristic large extracellular loop (ECL) containing 16 conserved cysteine (Cys) residues. We investigated the functional role of these Cys residues in Na(+) self-inhibition, an allosteric inhibition of ENaC activity by extracellular Na(+). All 16 Cys residues within alpha and gamma ECLs and selected beta ECL Cys residues were individually mutated to alanine or serine residues. The Na(+) self-inhibition response of wild type and mutant channels expressed in Xenopus oocytes was determined by whole cell voltage clamp. Individual mutation of eight alpha (Cys-1, -4, -5, -6, -7, -10, -13, or -16), one beta (Cys-7), and nine gamma (Cys-3, -4, -6, -7, -10, -11, -12, -13, or -16) residues significantly reduced the magnitude of Na(+) self-inhibition. Na(+) self-inhibition was eliminated by simultaneous mutations of either the last three alpha ECL Cys residues (Cys-14, -15, and -16) or Cys-7 within both alpha and gamma ECLs. By analyzing the Na(+) self-inhibition responses and the effects of a methanethiosulfonate reagent on channel currents in single and double Cys mutants, we identified five Cys pairs within the alphaECL (alphaCys-1/alphaCys-6, alphaCys-4/alphaCys-5, alphaCys-7/alphaCys-16, alphaCys-10/alphaCys-13, and alphaCys-11/alphaCys-12) and one pair within the gammaECL (gammaCys-7/gammaCys-16) that likely form intrasubunit disulfide bonds. We conclude that approximately half of the ECL Cys residues in the alpha and gamma ENaC subunits are required to establish the tertiary structure that ensures a proper Na(+) self-inhibition response, likely by formation of multiple intrasubunit disulfide bonds.     

Block of tetrodotoxin-resistant Na+ channel pore by multivalent cations: gating modification and Na+ flow dependence

Tetrodotoxin-resistant (TTX-R) Na(+) channels are much less susceptible to external TTX but more susceptible to external Cd(2+) block than tetrodotoxin-sensitive (TTX-S) Na(+) channels. Both TTX and Cd(2+) seem to block the channel near the "DEKA" ring, which is probably part of a multi-ion single-file region adjacent to the external pore mouth and is involved in the selectivity filter of the channel. In this study we demonstrate that other multivalent transitional metal ions such as La(3+), Zn(2+), Ni(2+), Co(2+), and Mn(2+) also block the TTX-R channels in dorsal root ganglion neurons. Just like Cd(2+), the blocking effect has little intrinsic voltage dependence, but is profoundly influenced by Na(+) flow. The apparent dissociation constants of the blocking ions are always significantly smaller in inward Na(+) currents than those in outward Na(+) current, signaling exit of the blocker along with the Na(+) flow and a high internal energy barrier for "permeation" of these multivalent blocking ions through the pore. Most interestingly, the activation and especially the inactivation kinetics are slowed by the blocking ions. Moreover, the gating changes induced by the same concentration of a blocking ion are evidently different in different directions of Na(+) current flow, but can always be correlated with the extent of pore block. Further quantitative analyses indicate that the apparent slowing of channel activation is chiefly ascribable to Na(+) flow-dependent unblocking of the bound La(3+) from the open Na(+) channel, whereas channel inactivation cannot happen with any discernible speed in the La(3+)-blocked channel. Thus, the selectivity filter of Na(+) channel is probably contiguous to a single-file multi-ion region at the external pore mouth, a region itself being nonselective in terms of significant binding of different multivalent cations. This region is "open" to the external solution even if the channel is "closed" ("deactivated"), but undergoes imperative conformational changes during the gating (especially the inactivation) process of the channel.     

Identification of epithelial Na+ channel (ENaC) intersubunit Cl- inhibitory residues suggests a trimeric alpha gamma beta channel architecture

The extracellular domain of the epithelial Na(+) channel (ENaC) is exposed to a wide range of anion concentrations in the kidney. We have previously demonstrated that extracellular Cl(-) inhibits ENaC activity. To identify sites involved in Cl(-) inhibition, we mutated residues in the extracellular domain of α-, β-, and γENaC that are homologous to the Cl(-) binding site in acid-sensing ion channel 1a and tested the effect of Cl(-) on the activity of ENaC expressed in Xenopus oocytes. We identified two Cl(-) inhibitory sites in ENaC. One is formed by residues in the thumb domain of αENaC and the palm domain of βENaC. Mutation of residues at this interface decreased Cl(-) inhibition and decreased Na(+) self-inhibition. The second site is formed by residues at the interface of the thumb domain of βENaC and the palm domain of γENaC. Mutation of these residues also decreased Cl(-) inhibition yet had no effect on Na(+) self-inhibition. In contrast, mutations in the thumb domain of γENaC and palm of αENaC had little or no effect on Cl(-) inhibition or Na(+) self-inhibition. The data demonstrate that Cl(-) inhibits ENaC activity by two distinct Na(+)-dependent and Na(+)-independent mechanisms that correspond to the two functional Cl(-) inhibitory sites. Furthermore, based on the effects of mutagenesis on Cl(-) inhibition, the additive nature of mutations, and on differences in the mechanisms of Cl(-) inhibition, the data support a model in which ENaC subunits assemble in an αγβ orientation (listed clockwise when viewed from the top).     

Evidence for a modulatory effect of external potassium ions on ionic current mediated by the cardiac Na+-Ca2+ exchanger

The aim of this study was to investigate whether or not the activity of the cardiac Na(+)-Ca(2+) exchanger might be directly sensitive to external K(+) concentration ([K(+)](e)). Measurements of whole-cell exchanger current (I(NaCa)) were made at 37 degrees C from guinea-pig isolated ventricular myocytes, using whole-cell patch clamp recording with major interfering conductances blocked. Changing [K(+)](e) from 0 to 5mM significantly reduced both outward and inward exchange currents in a time-dependent manner. Various [K(+)](e) between 1 and 15 mM were tested and the inhibitory effect was observed to be concentration-dependent. At steady-state, 5mM [K(+)](e) decreased the density of Ni(2+)-sensitive current by 52.8+/-4.3% (mean+/-S.E.M., n=6) and of 0Na0Ca-sensitive current by 39.0+/-4.4% (n=5). The possibility that the inhibitory effect of external K(+) on I(NaCa) might wholly or in part be secondary to activation of the sarcolemmal Na(+)-K(+) pump was investigated by testing the effect of K(+) addition in the presence of a high concentration of strophanthidin (500 microM). Ni(2+)-sensitive I(NaCa) was still observed to be sensitive to external K(+) (I(NaCa) decreased by 39.4+/-9.4%, n=4), suggesting that the inhibitory effect could occur independently of activation of the Na(+)-K(+) pump. The effect of external K(+) on I(NaCa) was verified using a baby hamster kidney (BHK) cell line stably expressing the cardiac Na(+)-Ca(2+) exchanger isoform, NCX1. Similar to native I(NaCa), NCX1 current was also suppressed by [K(+)](e). However, [K(+)](e) did not alter current amplitude in untransfected BHK cells. The effect of [K(+)](e) on I(NaCa) could not be attributed to simply adding any monovalent cation back to the external solution, since it was not reproduced by application of equimolar Li(+), Cs(+) and TEA(+). Rb(+), however, could mimic the effect of K(+). Collectively, these data suggest that external K(+) at physiologically and pathologically relevant concentrations might be able to modulate directly the activity of the cardiac Na(+)-Ca(2+) exchanger.     

Cl- interference with the epithelial Na+ channel ENaC

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A and ATP-regulated Cl- channel that also controls the activity of other membrane transport proteins, such as the epithelial Na+ channel ENaC. Previous studies demonstrated that cytosolic domains of ENaC are critical for down-regulation of ENaC by CFTR, whereas others suggested a role of cytosolic Cl- ions. We therefore examined in detail the anion dependence of ENaC and the role of its cytosolic domains for the inhibition by CFTR and the Cl- channel CLC-0. Coexpression of rat ENaC with human CFTR or the human Cl- channel CLC-0 caused inhibition of amiloride-sensitive Na+ currents after cAMP-dependent stimulation and in the presence of a 100 mM bath Cl- concentration. After activation of CFTR by 3-isobutyl-1-methylxanthine and forskolin or expression of CLC-0, the intracellular Cl- concentration was increased in Xenopus oocytes in the presence of a high bath Cl- concentration, which inhibited ENaC without changing surface expression of alpha beta gammaENaC. In contrast, a 5 mM bath Cl- concentration reduced the cytosolic Cl- concentration and enhanced ENaC activity. ENaC was also inhibited by injection of Cl- into oocytes and in inside/out macropatches by exposure to high cytosolic Cl- concentrations. The effect of Cl- was mimicked by Br-, Br-, NO3(-), and I-. Inhibition by Cl- was reduced in trimeric channels with a truncated COOH terminus of betaENaC and gammaENaC, and it was no longer detected in dimeric alpha deltaCbeta ENaC channels. Deletion of the NH2 terminus of alpha-, beta-, or gammaENaC, mutations in the NH2-terminal phosphatidylinositol bisphosphate-binding domain of betaENaC and gammaEnaC, and activation of phospholipase C, all reduced ENaC activity but allowed for Cl(-)-dependent inhibition of the remaining ENaC current. The results confirm a role of the carboxyl terminus of betaENaC for Cl(-)-dependent inhibition of the Na+ channel, which, however, may only be part of a complex regulation of ENaC by CFTR.     

Epithelial Na+ channels are activated by laminar shear stress

The degenerin/epithelial Na+ channel (ENaC) superfamily is a group of structurally related ion channels that are involved in diverse biological processes, including responses to mechanical stimuli. In renal cortical collecting ducts, changes in rates of perfusion affect Na+ reabsorption through an amiloride-sensitive pathway, suggesting that ENaC may be a mechanosensitive channel. In this study, we examined whether ENaC expressed in oocytes is regulated by laminar shear stress (LSS). A 1.8-mm (internal diameter) perfusion pipette was placed within 0.5-1.0 mm of the oocyte to provide laminar flow across the oocyte surface. LSS induced a dose-dependent and reversible increase in benzamil-sensitive whole cell Na+ currents in oocytes expressing alphabetagamma ENaC. The half-time for activation by LSS was approximately 5 s. Repetitive stimulation by LSS of oocytes expressing ENaC was associated with a reduction in the response to LSS. Oocytes expressing alphabetaS518Kgamma, a pore region mutant with a high open probability, were insensitive to LSS. We demonstrated previously that channels with a Cys residue introduced at position alphaSer-580 had a low open probability, but, following modification by [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET), channels exhibited a high open probability. Oocytes expressing alphaS580Cbetagamma ENaC respond to LSS similar to wild type; however, covalent modification by MTSET largely eliminated the response to LSS. Our results suggest that shear stress activates ENaC by modifying the gating properties of the channel.     

Properties of Na+ currents conducted by a skeletal muscle L-type Ca2+ channel pore mutant (SkEIIIK)

Four glutamate residues residing at corresponding positions within the four conserved membrane-spanning repeats of L-type Ca(2+) channels are important structural determinants for the passage of Ca(2+) across the selectivity filter. Mutation of the critical glutamate in Repeat III in the a 1S subunit of the skeletal L-type channel (Ca(v)1.1) to lysine virtually eliminates passage of Ca(2+) during step depolarizations. In this study, we examined the ability of this mutant Ca(v)1.1 channel (SkEIIIK) to conduct inward Na(+) current. When 150 mM Na(+) was present as the sole monovalent cation in the bath solution, dysgenic (Ca(v)1.1 null) myotubes expressing SkEIIIK displayed slowly-activating, non-inactivating, nifedipine-sensitive inward currents with a reversal potential (45.6 ± 2.5 mV) near that expected for Na(+). Ca(2+) block of SkEIIIK-mediated Na(+) current was revealed by the substantial enhancement of Na(+) current amplitude after reduction of Ca(2+) in the external recording solution from 10 mM to near physiological 1 mM. Inward SkEIIIK-mediated currents were potentiated by either ±Bay K 8644 (10 mM) or 200-ms depolarizing prepulses to +90 mV. In contrast, outward monovalent currents were reduced by ±Bay K 8644 and were unaffected by strong depolarization, indicating a preferential potentiation of inward Na(+) currents through the mutant Ca(v)1.1 channel. Taken together, our results show that SkEIIIK functions as a non-inactivating, junctionally-targeted Na(+) channel when Na(+) is the sole monvalent cation present and urge caution when interpreting the impact of mutations designed to ablate Ca(2+) permeability mediated by Ca(v) channels on physiological processes that extend beyond channel gating and permeability.     

Differential interactions of Na+ channel toxins with T-type Ca2+ channels

Two types of voltage-dependent Ca(2+) channels have been identified in heart: high (I(CaL)) and low (I(CaT)) voltage-activated Ca(2+) channels. In guinea pig ventricular myocytes, low voltage-activated inward current consists of I(CaT) and a tetrodotoxin (TTX)-sensitive I(Ca) component (I(Ca(TTX))). In this study, we reexamined the nature of low-threshold I(Ca) in dog atrium, as well as whether it is affected by Na(+) channel toxins. Ca(2+) currents were recorded using the whole-cell patch clamp technique. In the absence of external Na(+), a transient inward current activated near -50 mV, peaked at -30 mV, and reversed around +40 mV (HP = -90 mV). It was unaffected by 30 microM TTX or micromolar concentrations of external Na(+), but was inhibited by 50 microM Ni(2+) (by approximately 90%) or 5 microM mibefradil (by approximately 50%), consistent with the reported properties of I(CaT). Addition of 30 microM TTX in the presence of Ni(2+) increased the current approximately fourfold (41% of control), and shifted the dose-response curve of Ni(2+) block to the right (IC(50) from 7.6 to 30 microM). Saxitoxin (STX) at 1 microM abolished the current left in 50 microM Ni(2+). In the absence of Ni(2+), STX potently blocked I(CaT) (EC(50) = 185 nM) and modestly reduced I(CaL) (EC(50) = 1.6 microM). While TTX produced no direct effect on I(CaT) elicited by expression of hCa(V)3.1 and hCa(V)3.2 in HEK-293 cells, it significantly attenuated the block of this current by Ni(2+) (IC(50) increased to 550 microM Ni(2+) for Ca(V)3.1 and 15 microM Ni(2+) for Ca(V)3.2); in contrast, 30 microM TTX directly inhibited hCa(V)3.3-induced I(CaT) and the addition of 750 microM Ni(2+) to the TTX-containing medium led to greater block of the current that was not significantly different than that produced by Ni(2+) alone. 1 microM STX directly inhibited Ca(V)3.1-, Ca(V)3.2-, and Ca(V)3.3-mediated I(CaT) but did not enhance the ability of Ni(2+) to block these currents. These findings provide important new implications for our understanding of structure-function relationships of I(CaT) in heart, and further extend the hypothesis of a parallel evolution of Na(+) and Ca(2+) channels from an ancestor with common structural motifs.     

Cpt-cAMP activates human epithelial sodium channels via relieving self-inhibition

External Na(+) self-inhibition is an intrinsic feature of epithelial sodium channels (ENaC). Cpt-cAMP regulates heterologous guinea pig but not rat αβγ ENaC in a ligand-gated manner. We hypothesized that cpt-cAMP may eliminate the self-inhibition of human ENaC thereby open channels. Regulation of self-inhibition by this compound in oocytes was analyzed using the two-electrode voltage clamp and Ussing chamber setups. External cpt-cAMP stimulated human but not rat and murine αβγ ENaC in a dose- and external Na(+) concentration-dependent fashion. Intriguingly, cpt-cAMP activated human δβγ more potently than αβγ channels, suggesting that structural diversity in ectoloop between human α, δ, and those ENaC of other species determines the stimulating effects of cpt-cAMP. Cpt-cAMP increased the ratio of stationary and maximal currents. Mutants having abolished self-inhibition (β(ΔV348) and γ(H233R)) almost completely eliminated cpt-cAMP mediated activation of ENaC. On the other hand, mutants both enhancing self-inhibition and elevating cpt-cAMP sensitivity increased the stimulating effects of the compound. This compound, however, could not activate already fully opened channels, e.g., degenerin mutation (αβ(S520C)γ) and the proteolytically cleaved ENaC by plasmin. Cpt-cAMP activated native ENaC to the same extent as that for heterologous ENaC in human lung epithelial cells. Our data demonstrate that cpt-cAMP, a broadly used PKA activator, stimulates human αβγ and δβγ ENaC channels by relieving self-inhibition.     

A new subunit of the epithelial Na+ channel identifies regions involved in Na+ self-inhibition

The epithelial Na+ channel (ENaC) is the apical entry pathway for Na+ in many Na+-reabsorbing epithelia. ENaC is a heterotetrameric protein composed of homologous alpha, beta, and gamma subunits. Mutations in ENaC cause severe hypertension or salt wasting in humans; and consequently, ENaC activity is tightly controlled. According to the concept of Na+ self-inhibition, the extracellular Na+ ion itself can reduce ENaC activity. The molecular basis for Na+ self-inhibition is unknown. Here, we describe cloning of a new ENaC subunit from Xenopus laevis (epsilonxENaC). epsilonxENaC can replace alphaxENaC and formed functional, highly selective, amiloride-sensitive Na+ channels when coexpressed with betaxENaC and gammaxENaC. Channels containing epsilonxENaC showed strong inhibition by extracellular Na+. This Na+ self-inhibition was significantly slower than for alphaxENaC-containing channels. Using site-directed mutagenesis, we show that the proximal part of the large extracellular domain controls the speed of self-inhibition. This suggests that this region is involved in conformational changes during Na+ self-inhibition.     

Na self inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases

The regulation of the open probability of the epithelial Na(+) channel (ENaC) by the extracellular concentration of Na(+), a phenomenon called "Na(+) self inhibition," has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na(+) self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na(+) concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q(10)act = 1.5) activation rate and a strongly temperature-dependant (Q(10)inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na(+) self inhibition depended only on the extracellular Na(+) concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na(+) as well as a reduction of Na(+) outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na(+) self inhibition is an intrinsic property of sodium channels resulting from the expression of the alpha, beta, and gamma subunits of human ENaC in Xenopus oocyte. The extracellular Na(+)-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.     

External Cu2+ inhibits human epithelial Na+ channels by binding at a subunit interface of extracellular domains

Epithelial Na(+) channels (ENaCs) play an essential role in the regulation of body fluid homeostasis. Certain transition metals activate or inhibit the activity of ENaCs. In this study, we examined the effect of extracellular Cu(2+) on human ENaC expressed in Xenopus oocytes and investigated the structural basis for its effects. External Cu(2+) inhibited human αβγ ENaC with an estimated IC(50) of 0.3 μM. The slow time course and a lack of change in the current-voltage relationship were consistent with an allosteric (non pore-plugging) inhibition of human ENaC by Cu(2+). Experiments with mixed human and mouse ENaC subunits suggested that both the α and β subunits were primarily responsible for the inhibitory effect of Cu(2+) on human ENaC. Lowering bath solution pH diminished the inhibition by Cu(2+). Mutations of two α, two β, and two γ His residues within extracellular domains significantly reduced the inhibition of human ENaC by Cu(2+). We identified a pair of residues as potential Cu(2+)-binding sites at the subunit interface between thumb subdomain of αhENaC and palm subdomain of βhENaC, suggesting a counterclockwise arrangement of α, β, and γ ENaC subunits in a trimeric channel complex when viewed from above. We conclude that extracellular Cu(2+) is a potent inhibitor of human ENaC and binds to multiple sites within the extracellular domains including a subunit interface.     

Side chain orientation of residues lining the selectivity filter of epithelial Na+ channels

Epithelial Na(+) channels (ENaCs) selectively conduct Na(+) and Li(+) but exclude K(+). A three-residue tract ((G/S)XS) present within all three subunits has been identified as a key structure forming a putative selectivity filter. We investigated the side chain orientation of residues within this tract by analyzing accessibility of the introduced sulfhydryl groups to thiophilic Cd(2+). Xenopus oocytes were used to express wild-type or mutant mouse alphabetagammaENaCs. The blocking effect of external Cd(2+) was examined by comparing amiloride-sensitive Na(+) currents measured by two-electrode voltage clamp in the absence and presence of Cd(2+) in the bath solution. The currents in mutant channels containing a single Cys substitution at the first or third position within the (G/S)XS tract (alphaG587C, alphaS589C, betaG529C, betaS531C, gammaS546C, and gammaS548C) were blocked by Cd(2+) with varying inhibitory constants (0.06-13 mm), whereas the currents in control channels were largely insensitive to Cd(2+) at concentrations up to 10 mm. The Cd(2+) blocking effects were fast, with time constants in the range of seconds, and were only partially reversible. The blocked currents were restored by 10 mm dithiothreitol. Mutant channels containing alanine or serine substitutions at these sites within the alpha subunit were only poorly and reversibly blocked by 10 mm Cd(2+). These results indicate that the introduced sulfhydryl groups face the conduction pore and suggest that serine hydroxyl groups within the selectivity filter in wild-type ENaCs face the conduction pore and may contribute to cation selectivity by participating in coordination of permeating cations.     

Tissue kallikrein activation of the epithelial Na channel

Epithelial Na Channels (ENaC) are responsible for the apical entry of Na(+) in a number of different epithelia including the renal connecting tubule and cortical collecting duct. Proteolytic cleavage of γ-ENaC by serine proteases, including trypsin, furin, elastase, and prostasin, has been shown to increase channel activity. Here, we investigate the ability of another serine protease, tissue kallikrein, to regulate ENaC. We show that excretion of tissue kallikrein, which is secreted into the lumen of the connecting tubule, is stimulated following 5 days of a high-K(+) or low-Na(+) diet in rats. Urinary proteins reconstituted in a low-Na buffer activated amiloride-sensitive currents (I(Na)) in ENaC-expressing oocytes, suggesting an endogenous urinary protease can activate ENaC. We next tested whether tissue kallikrein can directly cleave and activate ENaC. When rat ENaC-expressing oocytes were exposed to purified tissue kallikrein from rat urine (RTK), ENaC currents increased threefold in both the presence and absence of a soybean trypsin inhibitor (SBTI). RTK and trypsin both decreased the apparent molecular mass of cleaved cell-surface γ-ENaC, while immunodepleted RTK produced no shift in apparent molecular mass, demonstrating the specificity of the tissue kallikrein. A decreased effect of RTK on Xenopus ENaC, which has variations in the putative prostasin cleavage sites in γ-ENaC, suggests these sites are important in RTK activation of ENaC. Mutating the prostasin site in mouse γ-ENaC (γRKRK186QQQQ) abolished ENaC activation and cleavage by RTK while wild-type mouse ENaC was activated and cleaved similar to that of the rat. We conclude that tissue kallikrein can be a physiologically relevant regulator of ENaC activity.     

Na+-Ca2+ exchange in mouse oocytes: modifications in the regulation of intracellular free Ca2+ during oocyte maturation

Increases in intracellular free Ca(2+)+ concentration (Ca(2+)+ oscillations) occur during meiotic maturation and fertilization of mammalian oocytes but little is known about the mechanisms of Ca(2+) homeostasis in these cells. Cells extrude Ca(2+) from the cytosol using two main transport processes, the Ca(2+)-ATPase and the Na(+)-Ca(2+) exchanger. The aim of this study was to determine whether Na(+)-Ca(2+) exchange activity is present in immature and mature mouse oocytes. Na(+)-Ca(2+) exchange can be revealed by altering the Na(+) concentration gradient across the plasma membrane and recording intracellular free Ca(2+) concentrations using Ca(2+)-sensitive fluorescent dyes. Depletion of extracellular Na(+) caused an immediate increase in Ca(2+) concentration in immature oocytes and a delayed increase in mature oocytes. The Na(+) ionophore, monensin, caused an increase in intracellular Ca(2+) in immature oocytes similar to that induced by Na(+)-depleted medium. In mature oocytes, monensin had no effect on intracellular Ca(2+) but the time taken for Ca(2+) to reach a peak value on removal of extracellular Na(+) was significantly decreased. Finally, addition of Ca(2+) to immature oocytes incubated in Ca(2+)-free medium caused an increase in the concentration of intracellular Ca(2+) that was dependent upon the presence of extracellular Na(+). This effect was not seen in mature oocytes. The data show that Na(+)-Ca(2+) exchange occurs in immature and mature mouse oocytes and that Ca(2+) homeostasis in immature oocytes is more sensitive to manipulations that activate Na(+)-Ca(2+) exchange.     

Extracellular pH modulates kinetics of the Na(+),K(+)-ATPase

To investigate effects of pH on the Na(+),K(+)-ATPase, we used the Xenopus oocytes to measure transient charge movements in the absence of extracellular K(+), and steady-state currents mediated by the pump as well as ATPase activity. The activity of purified Na(+), K(+)-ATPase strongly depends on pH, which has been attributed to protonation of intracellular sites. The steady-state current reflects pump activity, the transient charge movement voltage-dependent interaction of external Na(+) ions with the pump molecule and/or conformational changes during Na(+)/Na(+) exchange. The steady-state current exhibits a characteristic voltage dependence with maximum at about 0 mV at low external K(+) (< or =2 mM) and with 50 Na(+). This dependency is not significantly affected by changes in external pH in the range from pH 9 to pH 6. Only below pH 6, the voltage dependence of pump current becomes less steep, and may be attributed to a pH-dependent inhibition of the forward pump cycle by external Na(+). External stimulation of the pump by K(+) in the absence of Na(+) can be described by a voltage-dependent K(m) value with an apparent valency z(K). At higher external pH the z(K) value is reduced. The transient current signal in the absence of external K(+) can be described by the sum of three exponentials with voltage-dependent time constants of about 50 ms, 700 micros and less than 100 micros during pulses to 0 mV. The charge distribution was calculated by integration of the transient current signals. The slowest component and the associated charge distributions do not significantly depend on external pH changes. The intermediate component of the transients is represented by a voltage-dependent rate constant which shows a minimum at about -120 mV and increases with decreasing pH. Nevertheless, the contribution to the charge movement is not altered by pH changes due to a simultaneous increase of the amplitude of this component. We conclude that reduction of external pH counteracts external K(+) and Na(+) binding.