On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.     

Sensitivity of oocyte-expressed epithelial Na+ channel to glibenclamide

The effect of glibenclamide on heterologously expressed amiloride-sensitive sodium channels (ENaCs) was investigated in Xenopus oocytes. The ENaC is a heteromer and consists of alpha-, beta- and gamma-subunits and the alpha- and beta-subunits have previously been shown to confer sensitivity to glibenclamide. We coexpressed either colonic rat alpha- (ralpha) or guinea-pig alpha-subunit (gpalpha) with Xenopus betagamma-subunits. The gpalphaxbetagamma was significantly stimulated by glibenclamide (100 microM) (184+/-15%), whereas the ralpha-combination was slightly down-regulated by the sulfonylurea (79+/-4%). The stimulating effect did not interfere with Na(+)-self-inhibition resulting from intracellular accumulation of Na(+)-ions. We exchanged cytosolic termini between both orthologs but the gpalpha-chimera with the termini from rat retained sensitivity to glibenclamide. The effect of glibenclamide on Xenopus ENaC (xENaC) was inhibited by ADP-beta-S but not by ATP-gamma-S, when applied intracellularly. Intracellular loading with Na(+)-ions after inhibition of Na(+)/K(+)-ATPases with ouabain prevented an up-regulation of ENaC activity by glibenclamide. Pretreatment of oocytes expressing xENaC with edelfosine (ET-18-OCH(3)) slightly reduced stimulation of I(ami) (118+/-12%; control: 132+/-9%) while phosphatidylinositol-4,5-biphosphate (PIP(2)) significantly reduced the effect of glibenclamide to 101+/-3%.     

Specific and nonspecific effects of protein kinase C on the epithelial Na (+) channel

The Xenopus oocyte expression system was used to explore the mechanisms of inhibition of the cloned rat epithelial Na(+) channel (rENaC) by PKC (Awayda, M.S., I.I. Ismailov, B.K. Berdiev, C.M. Fuller, and D.J. Benos. 1996. J. Gen. Physiol. 108:49-65) and to determine whether human ENaC exhibits similar regulation. Effects of PKC activation on membrane and/or channel trafficking were determined using impedance analysis as an indirect measure of membrane area. hENaC-expressing oocytes exhibited an appreciable activation by hyperpolarizing voltages. This activation could be fit with a single exponential, described by a time constant (tau) and a magnitude (DeltaI (V)). A similar but smaller magnitude of activation was also observed in oocytes expressing rENaC. This activation likely corresponds to the previously described effect of hyperpolarizing voltage on gating of the native Na(+) channel (Palmer, L.G., and G. Frindt. 1996. J. Gen. Physiol. 107:35-45). Stimulation of PKC with 100 nM PMA decreased DeltaI(V) in hENaC-expressing oocytes to a plateau at 57.1 +/- 4.9% (n = 6) of baseline values at 20 min. Similar effects were observed in rENaC-expressing oocytes. PMA decreased the amiloride-sensitive hENaC slope conductance (g(Na)) to 21.7 +/- 7.2% (n = 6) of baseline values at 30 min. This decrease was similar to that previously reported for rENaC. This decrease of g (Na) was attributed to a decrease of membrane capacitance (C (m)), as well as the specific conductance (g(m)/C(m )). The effects on g(m)/C(m) reached a plateau within 15 min, at approximately 60% of baseline values. This decrease is likely due to the specific ability of PKC to inhibit ENaC. On the other hand, the decrease of C(m) was unrelated to ENaC and is likely an effect of PKC on membrane trafficking, as it was observed in ENaC-expressing as well as control oocytes. At lower PMA concentrations (0.5 nM), smaller changes of C(m) were observed in rENaC- and hENaC-expressing oocytes, and were preceded by larger changes of g(m ) and by changes of g(m)/C(m), indicating specific effects on ENaC. These findings indicate that PKC exhibits multiple and specific effects on ENaC, as well as nonspecific effects on membrane trafficking. Moreover, these findings provide the electrophysiological basis for assessing channel-specific effects of PKC in the Xenopus oocyte expression system.     

Identification of the Ca2+ blocking site of acid-sensing ion channel (ASIC) 1: implications for channel gating

Acid-sensing ion channels ASIC1a and ASIC1b are ligand-gated ion channels that are activated by H+ in the physiological range of pH. The apparent affinity for H+ of ASIC1a and 1b is modulated by extracellular Ca2+ through a competition between Ca2+ and H+. Here we show that, in addition to modulating the apparent H+ affinity, Ca2+ blocks ASIC1a in the open state (IC50 approximately 3.9 mM at pH 5.5), whereas ASIC1b is blocked with reduced affinity (IC50 > 10 mM at pH 4.7). Moreover, we report the identification of the site that mediates this open channel block by Ca2+. ASICs have two transmembrane domains. The second transmembrane domain M2 has been shown to form the ion pore of the related epithelial Na+ channel. Conserved topology and high homology in M2 suggests that M2 forms the ion pore also of ASICs. Combined substitution of an aspartate and a glutamate residue at the beginning of M2 completely abolished block by Ca2+ of ASIC1a, showing that these two amino acids (E425 and D432) are crucial for Ca2+ block. It has previously been suggested that relief of Ca2+ block opens ASIC3 channels. However, substitutions of E425 or D432 individually or in combination did not open channels constitutively and did not abolish gating by H+ and modulation of H+ affinity by Ca2+. These results show that channel block by Ca2+ and H+ gating are not intrinsically linked.     

A novel spliced variant of the epithelial Na+ channel delta-subunit in the human brain

The amiloride-sensitive epithelial Na+ channel regulates Na+ homeostasis in cells. Recently, we described that the delta-subunit is a candidate molecule for a pH sensor in the human brain. Here, an N-terminal spliced variant of the delta-subunit is cloned from human brain, and designated as the delta2-subunit, which is expressed with the original delta-subunit (delta1-subunit) at the same level in the human brain. Functional analyses revealed that the physiological and pharmacological properties (interaction with accessory betagamma-subunits, activation by acidic pH, amiloride sensitivity) of the delta2-subunit were similar to those of the delta1-subunit. In conclusion, the activities of both subunits may be involved in the mechanism underlying pH sensing in the human brain.     

Diversity of Channels Generated by Different Combinations of Epithelial Sodium Channel Subunits

The epithelial sodium channel is a multimeric protein formed by three homologous subunits: α, β, and γ; each subunit contains only two transmembrane domains. The level of expression of each of the subunits is markedly different in various Na⁺ absorbing epithelia raising the possibility that channels with different subunit composition can function in vivo. We have examined the functional properties of channels formed by the association of α with β and of α with γ in the Xenopus oocyte expression system using two-microelectrode voltage clamp and patch-clamp techniques. We found that αβ channels differ from αγ channels in the following functional properties: (a) αβ channels expressed larger Na⁺ than Li⁺ currents (I_Na+/I_Li+ 1.2) whereas αγ channels expressed smaller Na⁺ than Li⁺ currents (I_Na+/I_Li+ 0.55); (b) the Michaelis Menten constants (K _m) of activation of current by increasing concentrations of external Na⁺ and Li⁺ of αβ channels were larger (K _m > 180 mM) than those of αγ channels (K _m of 35 and 50 mM, respectively); (c) single channel conductances of αβ channels (5.1 pS for Na⁺ and 4.2 pS for Li⁺) were smaller than those of αγ channels (6.5 pS for Na⁺ and 10.8 pS for Li⁺); (d) the half-inhibition constant (K _i) of amiloride was 20-fold larger for αβ channels than for αγ channels whereas the K _i of guanidinium was equal for both αβ and αγ. To identify the domains in the channel subunits involved in amiloride binding, we constructed several chimeras that contained the amino terminus of the γ subunit and the carboxy terminus of the β subunit. A stretch of 15 amino acids, immediately before the second transmembrane domain of the β subunit, was identified as the domain conferring lower amiloride affinity to the αβ channels. We provide evidence for the existence of two distinct binding sites for the amiloride molecule: one for the guanidium moiety and another for the pyrazine ring. At least two subunits α with β or γ contribute to these binding sites. Finally, we show that the most likely stoichiometry of αβ and αγ channels is 1α:1β and 1α:1γ, respectively.     

Interaction of pyrethroids with the Na channel in mammalian neuronal cells in culture

The interaction of a series of pyrethroids with the Na⁺ channel of mouse neuroblastoma cells has been followed using both an electrophysiological and a ²²Na⁺ influx approach. By themselves, pyrethroids do not stimulate ²²Na⁺ entry through the Na⁺ channel (or the stimulation they give is too small to be analyzed). However, they stimulate ²²Na⁺ entry when used in conjuction with other toxins specific for the gating system of the channel. These include batrachotoxin, veratridine, dihydrograyanotoxin II or polypeptide toxins like sea anemone and scorpion toxins. This stimulatory effect is fully inhibited by tetrodotoxin with a dissociation constant of 1.6 nM for the tetrodotoxin-receptor complex. Half-maximum saturation of the pyrethroid receptor on the Na⁺ channel is observed in the micromolar range for the most active pyrethroids, Decis and RU 15525. The synergism observed between the effect of pyrethroids on ²²Na⁺ influx on the one hand, and the effects of sea anemone toxin II, Androctonus scorpion toxin II, batrachotoxin, veratridine and dihydrograyanotoxin II on the other, indicates that the binding component for pyrethroids on the Na⁺ channel is distinct from the other toxin receptors. It is also distinct from the tetrodotoxin receptor.Some of the pyrethroids used in this study bind to the Na⁺ channel but are unable to stimulate ²²Na⁺ entry. These inactive compounds behave as antagonists of the active pyrethroids.An electrophysiological approach has shown that pyrethroids by themselves are active on the Na⁺ channel of mammalian neurones, and essentially confirm the conclusions made from ²²Na⁺ flux measurements.Pyrethroids are also active on C9 cells in which Na⁺ channels are ‘silent’, that is, not activatable by electrical stimulation. Pyrethroids chemically activate the silent Na⁺ channel in a manner similar to that with veratridine, batrachotoxin, or polypeptide toxins, which are known to slow down the inactivation process of a functional Na⁺ channel.     

Functional role of the N-terminus of Na,K-ATPase α-subunit as an inactivation gate of palytoxin-induced pump channel

The N-terminus of the Na⁺,K⁺-ATPase α-subunit shows some homology to that of Shaker-B K⁺ channels; the latter has been shown to mediate the N-type channel inactivation in a ball-and-chain mechanism. When the Torpedo Na⁺,K⁺-ATPase is expressed in Xenopus oocytes and the pump is transformed into an ion channel with palytoxin (PTX), the channel exhibits a time-dependent inactivation gating at positive potentials. The inactivation gating is eliminated when the N-terminus is truncated by deleting the first 35 amino acids after the initial methionine. The inactivation gating is restored when a synthetic N-terminal peptide is applied to the truncated pumps at the intracellular surface. Truncated pumps generate no electrogenic current and exhibit an altered stoichiometry for active transport. Thus, the N-terminus of the α-subunit appears to act like an inactivation gate and performs a critical step in the Na⁺,K⁺-ATPase pumping function.     

Permeability properties of ENaC selectivity filter mutants.

The epithelial Na(+) channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na(+) absorption. The amiloride-sensitive ENaC is highly selective for Na(+) and Li(+) ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the alphaS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K(+) ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted alphaS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position alpha589, indicating that alphaS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na(+) and Li(+). These findings demonstrate the critical role of the pore size at the alphaS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the alphaS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the alphaS589 side chain is oriented toward the subunit-subunit interface and that substitution of alphaS589 by larger residues increases the pore diameter by adding extra volume at the subunit-subunit interface.     

Epithelial Na+ channel delta subunit mediates acid-induced ATP release in the human skin

The amiloride-sensitive epithelial Na⁺ channel (ENaC) regulates Na⁺ homeostasis in cells and across epithelia. Although we described that ENaCδ is a candidate molecule for a pH sensor in the human brain, the physiological and pathological roles of ENaCδ in non-neuronal tissues are still unknown. Here we show a novel physiological function of ENaCδ in peripheral tissues in humans. Expression analyses at the level of mRNA clearly revealed that ENaCδ was abundantly expressed in human epidermis and keratinocytes. In addition, ENaCδ protein was detected in there. In cultured keratinocytes, acidic stress (pH 5.0) evoked ATP release, which was significantly reduced in the presence of 100 μM amiloride or 10 μM benzamil. In conclusion, ENaCδ may be involved in the mechanism underlying pH sensing followed by the regulation of cell viability in the human skin.     

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K⁺ _ATP channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na⁺/K⁺-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na⁺/K⁺-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na⁺/K⁺-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K⁺ _ATP channels. 5-HD did not affect the ouabain sensitivity of Na⁺/K⁺-ATPase. Glyburide had both activating and inhibitory effects on Na⁺/K⁺-ATPase at concentrations used to block plasma membrane K⁺ _ATP channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na⁺/K⁺-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K⁺ _ATP channels, when the relation of this channel to Na⁺/K⁺-ATPase is being studied.     

Expression of skeletal muscle Na(V)1.4 Na channel isoform in canine cardiac Purkinje myocytes

BACKGROUND AND AIM: The action potential plateau of Purkinje fibers is particularly sensitive to tetrodotoxin (TTX) and this could be due to a TXX-sensitive Na(+) current. The expression of TTX-sensitive neuronal Na(V)1.1 and Na(V)1.2 isoforms has been reported in canine Purkinje myocytes. Our aim was to investigate by means of biochemical and functional techniques whether the TTX-sensitive skeletal Na(V)1.4 isoform is also expressed in canine cardiac Purkinje myocytes. METHODS AND RESULTS: Using Na(V)1.4 specific primers, a PCR product corresponding to Na(V)1.4 was amplified from canine Purkinje fibers RNA and confirmed by sequencing and megablast of the gene bank. Confocal indirect immunostaining using anti-Na(V)1.4 antibody demonstrates distinct sarcolemmal staining pattern compared to that of the cardiac isoform Na(V)1.5. Expression of Na(V)1.4 in tsA201 cells yielded a TTX-sensitive Na(+) current with an IC(50) of 10nM. CONCLUSIONS: These results demonstrate the expression of the TTX-sensitive Na(V)1.4 channel in canine cardiac Purkinje myocytes. This novel finding suggests a role of Na(V)1.4 channel in Purkinje myocytes and thus has important clinical implications for the mechanisms and management of ventricular arrhythmias originating in the Purkinje network.     

The Na+ cycle of extreme alkalophiles: A secondary Na+/H+ antiporter and Na+/solute symporters

Extremely alkalophilic bacteria that grow optimally at pH 10.5 and above are generally aerobic bacilli that grow at mesophilic temperatures and moderate salt levels. The adaptations to alkalophily in these organisms may be distinguished from responses to combined challenges of high pH together with other stresses such as salinity or anaerobiosis. These alkalophiles all possess a simple and physiologically crucial Na⁺ cycle that accomplishes the key task of pH homeostasis. An electrogenic, secondary Na⁺/H⁺ antiporter is energized by the electrochemical proton gradient formed by the proton-pumping respiratory chain. The antiporter facilitates maintenance of a pH_in that is two or more pH units lower than pH_out at optimal pH values for growth. It also largely converts the initial electrochemical proton gradient formed by respiration into an electrochemical sodium gradient that energizes motility as well as a plethora of Na⁺/solute symporters. These symporters catalyze solute accumulation and, importantly, reentry of Na⁺. The extreme nonmarine alkalophiles exhibit no primary sodium pumping dependent upon either respiration or ATP. ATP synthesis is not part of their Na⁺ cycle. Rather, the specific details of oxidative phosphorylation in these organisms are an interesting analogue of the same process in mitochondria, and may utilize some common features to optimize energy transduction.     

Predominance of poorly reopening single Na+ channels and lack of slow Na+ inactivation in neonatal cardiocytes

Summary Elementary Na⁺ currents through single cardiac Na⁺ channels were recorded at –50 mV in cell-attached patches from neonatal rat cardiocytes kept at holding potentials between –100 and –120 mV.Na⁺ channel activity may occur as burst-like, closely-timed repetitive openings with shut times close to 0.5–0.6 msec, indicating that an individual Na⁺ channel may reopen several times during step depolarization. A systematic quantiative analysis in 19 cell-attached patches showed that reopening may be quite differently pronounced. The majority, namely 16 patches, contained Na⁺ channels with a low tendency to reopen. This was evidenced from the average value for the mean number of openings per sequence, 2.5. Strikingly different results were obtained in a second group of three patches. Here, a mean number of openings per sequence of 3.42, 3.72, and 5.68 was found. Ensemble averages from the latter group of patches revealed macroscopic Na⁺ currents with a biexponential decay phase. Reconstructed Na⁺ currents from patches with poorly reopening Na⁺ channels were devoid of a slow decay component. This strongly suggests that reopening may be causally related to slow Na⁺ inactivation. Poorly pronounced reopening and, consequently, the lack of slow Na⁺ inactivation could be characteristic features of neonatal cardiac Na⁺ channels.     

Characterization of a partially degraded Na+ channel from urinary tract epithelium

Summary The mammalian urinary bladder contains in its apical membrane and cytoplasmic vesicles, a cation-selective channel or activating fragment which seems to partition between the apical membrane and the luminal (or vesicular space). To determine whether it is an activating fragment or whole channel, we first demonstrate that solution known to contain this moiety can be concentrated and when added back to the bladder causes a conductance increase, with a percent recovery of 139±25%. Next, we show that using tip-dip bilayer techniques (at 21°C) and a patch-clamp recorder, the addition of concentrated solution resulted in the appearance of discrete current shots, consistent with the incorporation of a channel (as opposed to an activating fragment) into the bilayer. The residency time of the channel in the bilayer was best described by the sum of two exponentials, suggesting that the appearance of the channel involves an association of the channel with the membrane before insertion. The channel is cation selective and more conductive to K⁺ than Na⁺ (by a factor of 1.6). It has a linearI–V relationship, but a singlechannel conductance that saturates as KCl concentration is raised. This saturation is best described by the Michaelis-Menten equation with aK _m of 160mm KCl and aG _max of 20 pS. The kinetics of the channel are complex, showing at least two open and two closed states.Since the characteristics of this channel are similar to a channel produced by the degradation of amiloride-sensitive Na⁺ channels by the proteolytic enzyme kallikrein (which is released by the cortical collecting duct of the kidney), we suggest that this channel then is not synthesized by the cell but is rather a degraded form of the epithelial Na⁺ channel.     

The Na+/H+ antiporter of the thermohalophilic bacterium Rhodothermus marinus

In the thermohalophilic bacterium Rhodothermus marinus, the NADH:quinone oxidoreductase (complex I) is encoded by two single genes and two operons, one of which contains the genes for five complex I subunits, nqo10-nqo14, a pterin carbinolamine dehydratase, and a putative single subunit Na+/H+ antiporter. Here we report that the latter encodes indeed a functional Na+/H+ antiporter, which is able to confer resistance to Na+, but not to Li+ to an Escherichia coli strain defective in Na+/H+ antiporters. In addition, an extensive amino acid sequence comparison with several single subunit Na+/H+ antiporters from different groups, namely NhaA, NhaB, NhaC, and NhaD, suggests that this might be the first member of a new type of Na+/H+ antiporters, which we propose to call NhaE.     

Species differences in bacterial NhaA Na/H exchangers

Bacteria have adapted their NhaA Na⁺/H⁺ exchangers responsible for salt homeostasis to their different habitats. We present an electrophysiological and kinetic analysis of NhaA from Helicobacter pylori and compare it to the previously investigated exchangers from Escherichia coli and Salmonella typhimurium. Properties of all three transporters are described by a simple model using a single binding site for H⁺ and Na⁺. We show that H.pylori NhaA only has a small acidic shift of its pH-dependent activity profile compared to the other transporters and discuss why a more drastic change in its pH activity profile is not physiologically required.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

Acetylcholine-Induced Na+ influx in the mouse lacrimal gland acinar cells: Demonstration of multiple Na+ transport mechanisms by intracellular Na+ activity measurements

Summary In the isolated, superfused mouse lacrimal gland, intracellular Na⁺ activities (aNa _i ) of the acinar cells were directly measured with double-barreled Na⁺-selective microelectrodes. In the nonstimulated conditionaNa _i was 6.5±0.5 mM and membrane potential (V _m ) was –38.9±0.4 mV. Addition of 1 mM ouabain or superfusion with a K⁺-free solution slightly depolarized the membrane and caused a gradual increase inaNa _i . Stimulation with acetylcholine (ACh, 1 M) caused a membrane hyperpolarization by about 20 mV and an increase inaNa _i by about 9 mM in 5 min. The presence of amiloride (0.1 mM) reduced the ACh-induced increase inaNa _i by approximately 50%, without affectingV _m and input resistance in both nonstimulated and ACh-stimulated conditions. Acid loading the acinar cells by an addition/withdrawal of 20 mM NH₄Cl or by replacement of Tris⁺-buffer saline solution with HCO ₃ ^– /CO₂-buffered solution increasedaNa _i by a few mM. Superfusion with a Cl^–-free NO ₃ ^– solution or 1 mM furosemide or 0.5 mM bumetanide-containing solution had little effect on the restingaNa _i levels, however, it reduced the ACh-induced increase inaNa _i by about 30%. Elimination of metabolite anions (glutamate, fumarate and pyruvate) from the superfusate reduced both the restingaNa _i and the ACh-induced increase inaNa _i .The present results suggest the presence of multiple Na⁺ entry mechanisms activated by ACh, namely, Na⁺/H⁺ exchange, Na-K-Cl cotransport and organic substrate-coupled Na⁺ transport mechanisms.