A Gating Model for the Archeal Voltage-Dependent K Channel KvAP in DPhPC and POPE:POPG Decane Lipid Bilayers

Voltage-dependent K⁺ (Kv) channels form the basis of the excitability of nerves and muscles. KvAP is a well-characterized archeal Kv channel that has been widely used to investigate many aspects of Kv channel biochemistry, biophysics, and structure. In this study, a minimal kinetic gating model for KvAP function in two different phospholipid decane bilayers is developed. In most aspects, KvAP gating is similar to the well-studied eukaryotic Shaker Kv channel: conformational changes occur within four voltage sensors, followed by pore opening. Unlike the Shaker Kv channel, KvAP possesses an inactivated state that is accessible from the pre-open state of the channel. Changing the lipid composition of the membrane influences multiple gating transitions in the model, but, most dramatically, the rate of recovery from inactivation. Inhibition by the voltage sensor toxin VSTx1 is most easily explained if VSTx1 binds only to the depolarized conformation of the voltage sensor. By delaying the voltage sensor's return to the hyperpolarized conformation, VSTx1 favors the inactivated state of KvAP.     

Cyclic AMP-dependent protein kinase activation of cystic fibrosis transmembrane conductance regulator chloride channels in planar lipid bilayers.

Membrane vesicles, prepared from mouse NIH-3T3 fibroblasts and Chinese hamster ovary cells expressing high levels of cystic fibrosis transmembrane conductance regulator (CFTR), were fused with Mueller-Rudin planar lipid bilayers. Upon addition of the catalytic subunit of cAMP-dependent protein kinase and ATP, low conductance Cl(-)-selective ion channels were observed in 10 of 16 experiments. The channels had a linear current-voltage relationship and a unitary conductance of approximately 6.5 pS. The channels were more permeable to Cl- than to I- and showed no appreciable time-dependent voltage activation. In contrast, addition of cAMP-dependent protein kinase and ATP to lipid bilayers fused with vesicles prepared from mock transfected (n = 14) cells failed to activate Cl- channels. These data support the conclusion that CFTR is a Cl- channel. They indicate that it can be reconstituted in a planar lipid bilayer and that the biophysical and regulatory properties are very similar to those observed in the native cell membrane. These data also argue against the requirement for loosely associated factors for regulation or function of the channel.     

Membrane phospholipid composition affects function of potassium channels from rabbit colon epithelium

We tested the effects of membrane phospholipids on the functionof high-conductance,Ca²⁺-activatedK⁺ channels from the basolateralcell membrane of rabbit distal colon epithelium by reconstituting thesechannels into planar bilayers consisting of different 1:1 mixtures ofphosphatidylethanolamine (PE), phosphatidylcholine (PC),phosphatidylserine (PS), and phosphatidylinositol (PI). At low ambientK⁺ concentrations single-channelconductance is higher in PE/PS and PE/PI bilayers than in PE/PCbilayers. At high K⁺concentrations this difference in channel conductance is abolished. Introducing the negatively charged SDS into PE/PC bilayersincreases channel conductance, whereas the positively chargeddodecyltrimethylammonium has the opposite effect. All these findingsare consistent with modulation of channel current by the charge of thelipid membrane surrounding the channel. But theK⁺ that permeates the channelsenses only a small fraction of the full membrane surface potential ofthe charged phospholipid bilayers, equivalent to separation of theconduction pathway from the charged phospholipid head groups by 20 Å. This distance appears to insulate the channel entrancefrom the bilayer surface potential, suggesting large dimensions of thechannel-forming protein. In addition, in PE/PC and PE/PI bilayers, butnot in PE/PS bilayers, the open-state probability of the channeldecreases with time ("channel rundown"), indicating thatphospholipid properties other than surface charge are required tomaintain channel fluctuations.

     

Antibacterial peptide pleurocidin forms ion channels in planar lipid bilayers

Pleurocidin, a 25-residue alpha helical cationic peptide, isolated from skin mucous secretions of the winter flounder, displays a strong anti-microbial activity and appears to play a role in innate host defence. This peptide would be responsible for pore formation in the membrane of bacteria leading to lysis and therefore death. In this study, we investigated the behaviour of pleurocidin in different planar lipid bilayers to determine its mechanism of membrane permeabilisation. Macroscopic conductance experiments showed that pleurocidin did not display a pore-forming activity in neutral phosphatidylcholine/phosphatidylethanolamine (PC/PE) lipid bilayers. However, in 7:3:1 PC/PE/phosphatidylserine (PS) lipid bilayers, pleurocidin showed reproducible I/V curves at different peptide concentrations. This activity is confirmed by single-channel experiments since well-defined ion channels were obtained if the lipid mixture was containing an anionic lipid (PS). The ion channel characteristics such as-no voltage dependence, only one unitary conductance, linear relation ship current-voltage-, are not in favour of the membrane permeabilisation according to the barrel model but rather by the toroidal pore formation.     

The effect of the polar moiety of lipids on bilayer conductance induced by uncouplers of oxidative phosphorylation

Summary Bilayer membranes were formed from decane, cholesterol, and three lipids isolated fromStaphylococcus aureus: positively charged lysyl phosphatidylglycerol (LysPG), negatively charged phosphatidylglycerol (PG), and neutral diglucosyldiglyceride (DiGluDiGly). The uncouplers of oxidative phosphorylation, 2,4-dinitrophenol (DNP) and 3-t-butyl,5-chloro,2-chloro,4-nitrosalicylanilide (S 13), increased the electrical conductance of all three differently charged bilayers. S 13 was found to be the most effective reagent of the known uncouplers in increasing conductance of the bilayers. The conductance induced by uncouplers was investigated as a function of pH and uncoupler concentration. The pH of maximum conductance for each uncoupling agent was dependent on both the uncoupler and the lipid; it was lower for each uncoupler in LysPG and higher in PG compared to DiGluDiGly bilayers. At a pH below the optimum for LysPG, the conductance of the positively charged membrane was 500 times and of the neutral one 10 times higher than that of the negatively charged bilayer at equal uncoupler concentration and pH. Above the pH optimum for DiGluDiGly, the conductance was approximately equal for the positive and neutral membranes, but was lower in PG bilayers. Conductance depended linearly on uncoupler concentration. The bilayer conductance induced by S 13 was entirely due to increased proton permeability in all three lipids. The findings are consistent with the role of uncouplers as carriers for protons across the hydrocarbon interior of lipid membranes. The differences in conductance of differently charged lipid bilayers at equal uncoupler concentration, as well as the change of pH optimum of conductance with lipid charge, can be explained in terms of an electrostatic energy contribution of the fixed lipid charges to the distribution of the uncoupler anion between the aqueous and the membrane phases.     

Cardiac calcium channels in planar lipid bilayers. L-type channels and calcium-permeable channels open at negative membrane potentials

Planar lipid bilayer recordings were used to study Ca channels from bovine cardiac sarcolemmal membranes. Ca channel activity was recorded in the absence of nucleotides or soluble enzymes, over a range of membrane potentials and ionic conditions that cannot be achieved in intact cells. The dihydropyridine-sensitive L-type Ca channel, studied in the presence of Bay K 8644, was identified by a detailed comparison of its properties in artificial membranes and in intact cells. L-type Ca channels in bilayers showed voltage dependence of channel activation and inactivation, open and closed times, and single-channel conductances in Ba2+ and Ca2+ very similar to those found in cell-attached patch recordings. Open channels were blocked by micromolar concentrations of external Cd2+. In this cell-free system, channel activity tended to decrease during the course of an experiment, reminiscent of Ca2+ channel "rundown" in whole-cell and excised-patch recordings. A purely voltage-dependent component of inactivation was observed in the absence of Ca2+ stores or changes in intracellular Ca2+. Millimolar internal Ca2+ reduced unitary Ba2+ influx but did not greatly increase the rate or extent of inactivation or the rate of channel rundown. In symmetrical Ba2+ solutions, unitary conductance saturated as the Ba2+ concentration was increased up to 500 mM. The bilayer recordings also revealed activity of a novel Ca2+-permeable channel, termed "B-type" because it may contribute a steady background current at negative membrane potentials, which is distinct from L-type or T-type Ca channels previously reported. Unlike L-type channels, B-type channels have a small unitary Ba2+ conductance (7 pS), but do not discriminate between Ba2+ and Ca2+, show no obvious sensitivity to Bay K 8644, and do not run down. Unlike either L- or T-type channels, B-type channels did not require a depolarization for activation and displayed mean open times of greater than 100 ms.     

Permeability and electrical properties of planar lipid membranes from thylakoid lipids.

Electrical measurements were carried out on planar lipid membranes from thylakoid lipids. The specific capacitance of membranes formed from decane-containing monogalactosyldiacylglycerol (MGDG), which accounts for 57% of the total lipid content of thylakoids, showed that it adopted a bilayer structure. Solvent-free bilayers of MGDG were not formed, with very rare exceptions, indicating that decane is required to stabilize the planar conformation. However, this cone-shaped lipid produces bilayer structures in combination with other cylindrical thylakoid lipids even in the absence of organic solvent. We compared the properties of solvent-free and decane-containing bilayers from MGDG, soybean lecithin, and the quaternary mixture of lipids similar to that found in vivo. The conductance of decane-MGDG was 26 times higher than that of decane-lecithin. The flux through the decane-lecithin bilayer was found to be slightly dependent on pH, whereas the decane-MGDG membrane was not. The specific conductance of bilayers formed from the quaternary mixture of lipids was 5 to 10 times larger than lecithin (with alkane or not). Further experiments with bilayers made in the presence of a KCl gradient showed that decane-MGDG, decane-MGDG/DGDG/SQDG/PG, and solvent-free MGDG/DGDG/SQDG/PG were cation-selective. The permeability coefficient for potassium ranged from 4.9 to 8.3 x 10(-11) cm s-1. The permeability coefficient for protons in galactolipids, however, was determined to be about six orders of magnitude higher than the value for potassium ions. The HCl permeation mechanism through the lipid membranes was determined from diffusion potentials measured in HCl gradients. Our results suggest that HCl was not transported as neutral molecules. The data is discussed with regard to the function of galactolipids in the ion transport through thylakoid membranes.     

Antibacterial peptide pleurocidin forms ion channels in planar lipid bilayers

Pleurocidin, a 25-residue α helical cationic peptide, isolated from skin mucous secretions of the winter flounder, displays a strong anti-microbial activity and appears to play a role in innate host defence. This peptide would be responsible for pore formation in the membrane of bacteria leading to lysis and therefore death. In this study, we investigated the behaviour of pleurocidin in different planar lipid bilayers to determine its mechanism of membrane permeabilisation. Macroscopic conductance experiments showed that pleurocidin did not display a pore-forming activity in neutral phosphatidylcholine/phosphatidylethanolamine (PC/PE) lipid bilayers. However, in 7:3:1 PC/PE/phosphatidylserine (PS) lipid bilayers, pleurocidin showed reproducible I/V curves at different peptide concentrations. This activity is confirmed by single-channel experiments since well-defined ion channels were obtained if the lipid mixture was containing an anionic lipid (PS). The ion channel characteristics such as—no voltage dependence, only one unitary conductance, linear relation ship current-voltage—, are not in favour of the membrane permeabilisation according to the barrel model but rather by the toroidal pore formation.     

Probability of alamethicin conductance states varies with nonlamellar tendency of bilayer phospholipids.

With few exceptions, membrane lipids are usually regarded as a kind of filler or passive solvent for membrane proteins. Yet, cells exquisitely control membrane composition. Many phospholipids found in plasma membrane bilayers favor packing into inverted hexagonal bulk phases. It was suggested that the strain of forcing such lipids into a bilayer may affect membrane protein function, such as the operation of transmembrane channels. To investigate this, we have inserted the peptide alamethicin into bilayer membranes composed of lipids of empirically determined inverted hexagonal phase "spontaneous radii" Ro, which will have expectably different degrees of strain when forced into bilayer form. We observe a correlation between measured Ro and the relative probabilities of different conductance states. States of higher conductance are more probable in dioleoylphosphatidylethanolamine, the lipid of highest curvature, 1/Ro, than in dioleoylphosphatidylcholine, the lipid of lowest curvature.     

bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo–electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2–20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2–0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid–protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.     

The effect of local anesthetics on the electric properties of black flat bilayers

The effect of some local anesthetics on conductance of bilayer lipid membranes made of egg yolk lecithin has been studied. Low concentrations of anesthetics were shown to gradually increase the integral conductance of bilayers. This effect is connected with the protonophore activity of the tested anesthetics and it caused the stabilization of the bilayers. High concentrations of anesthetics caused destabilization of membranes and formed zones of nonselective increased conductance.     

Phosphatidylethanolamine-phosphatidylglycerol bilayer as a model of the inner bacterial membrane

Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) are the main lipid components of the inner bacterial membrane. A computer model for such a membrane was built of palmitoyloleoyl PE (POPE) and palmitoyloleoyl PG (POPG) in the proportion 3:1, and sodium ions (Na+) to neutralize the net negative charge on each POPG (POPE-POPG bilayer). The bilayer was simulated for 25 ns. A final 10-ns trajectory fragment was used for analyses. In the bilayer interfacial region, POPEs and POPGs interact readily with one another via intermolecular hydrogen (H) bonds and water bridges. POPE is the main H-bond donor in either PEPE or PEPG H-bonds; PGPG H-bonds are rarely formed. Almost all POPEs are H-bonded and/or water bridged to either POPE or POPG but PE-PG links are favored. In effect, the atom packing in the near-the-interface regions of the bilayer core is tight. Na+ does not bind readily to lipids, and interlipid links via Na+ are not numerous. Although POPG and POPE comprise one bilayer, their bilayer properties differ. The average surface area per POPG is larger and the average vertical location of the POPG phosphate group is lower than those of POPE. Also, the alkyl chains of POPG are more ordered and less densely packed than the POPE chains. The main conclusion of this study is that in the PE-PG bilayer PE interacts more strongly with PG than with PE. This is a likely molecular-level event behind a regulating mechanism developed by the bacteria to control its membrane permeability and stability consisting in changes of the relative PG/PE concentration in the membrane.     

Recombinant CLIC1 (NCC27) assembles in lipid bilayers via a pH-dependent two-state process to form chloride ion channels with identical characteristics to those observed in Chinese hamster ovary cells expressing CLIC1

CLIC1 (NCC27) is an unusual, largely intracellular, ion channel that exists in both soluble and membrane-associated forms. The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration. Soluble E. coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells. The process of membrane integration of CLIC1 is pH-dependent. Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer. These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics. This has four times the conductance of the SCSK and fast kinetics that characterize the native channel. This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins.     

Effect of phosphatidylserine on unitary conductance and Ba2+ block of the BK Ca2+-activated K+ channel: re-examination of the surface charge hypothesis

Incorporation of BK Ca2+-activated K+ channels into planar bilayers composed of negatively charged phospholipids such as phosphatidylserine (PS) or phosphatidylinositol (PI) results in a large enhancement of unitary conductance (gch) in comparison to BK channels in bilayers formed from the neutral zwitterionic lipid, phospatidylethanolamine (PE). Enhancement of gch by PS or PI is inversely dependent on KCl concentration, decreasing from 70% at 10 mM KCl to 8% at 1,000 mM KCl. This effect was explained previously by a surface charge hypothesis (Moczydlowski, E., O. Alvarez, C. Vergara, and R. Latorre. 1985. J. Membr. Biol. 83:273-282), which attributed the conductance enhancement to an increase in local K+ concentration near the entryways of the channel. To test this hypothesis, we measured the kinetics of block by external and internal Ba2+, a divalent cation that is expected to respond strongly to changes in surface electrostatics. We observed little or no effect of PS on discrete blocking kinetics by external and internal Ba2+ at 100 mM KCl and only a small enhancement of discrete and fast block by external Ba2+ in PS-containing membranes at 20 mM KCl. Model calculations of effective surface potential sensed by the K+ conduction and Ba2+-blocking reactions using the Gouy-Chapman-Stern theory of lipid surface charge do not lend support to a simple electrostatic mechanism that predicts valence-dependent increase of local cation concentration. The results imply that the conduction pore of the BK channel is electrostatically insulated from the lipid surface, presumably by a lateral distance of separation (>20 A) from the lipid head groups. The lack of effect of PS on apparent association and dissociation rates of Ba2+ suggest that lipid modulation of K+ conductance is preferentially coupled through conformational changes of the selectivity filter region that determine the high K+ flux rate of this channel relative to other cations. We discuss possible mechanisms for the effect of anionic lipids in the context of specific molecular interactions of phospholipids documented for the KcsA bacterial potassium channel and general membrane physical properties proposed to regulate membrane protein conformation via energetics of bilayer stress.     

Interaction between filamentous actin and lipid bilayer causes the increase of syringomycin E channel-forming activity

The effect of filamentous (F) actin on the channel-forming activity of syringomycin E (SRE) in negatively charged and uncharged bilayer lipid membranes (BLM) was studied. F-actin did not affect the membrane conductance in the absence of SRE. No changes in SRE-induced membrane conductance were observed when the above agents were added to the same side of BLM. However, the opposite side addition of F-actin and SRE provokes a multiple increase in membrane conductance. The similar voltage dependence of membrane conductance, equal values of single channel conductance and the effective gating charge of the channels upon F-actin action suggests that the actin-dependent increase in BLM conductance may result from an increase in the number of opened SRE-channels. BLM conductance kinetics depends on the sequence of SRE and F-actin addition, suggesting that actin-dependent rise of conductance may be induced by BLM structural changes that follow F-actin adsorption. F-actin exerted similar effect on membrane conductance of both negatively charged and uncharged bilayers, as well as on conductance of BLM with high ionic strength bathing solution, suggesting the major role for hydrophobic interactions in F-actin adsorption on lipid bilayer.     

Bilayer reconstitution of voltage-dependent ion channels using a microfabricated silicon chip

Painted bilayers containing reconstituted ion channels serve as a well defined model system for electrophysiological investigations of channel structure and function. Horizontally oriented bilayers with easy solution access to both sides were obtained by painting a phospholipid:decane mixture across a cylindrical pore etched into a 200-microm thick silicon wafer. Silanization of the SiO(2) layer produced a hydrophobic surface that promoted the adhesion of the lipid mixture. Standard lithographic techniques and anisotropic deep-reactive ion etching were used to create pores with diameters from 50 to 200 microm. The cylindrical structure of the pore in the partition and the surface treatment resulted in stable bilayers. These were used to reconstitute Maxi K channels in the 100- and 200-microm diameter pores. The electrophysiological characteristics of bilayers suspended in microchips were comparable with that of other bilayer preparations. The horizontal orientation and good voltage clamping properties make the microchip bilayer method an excellent system to study the electrical properties of reconstituted membrane proteins simultaneously with optical probes.     

A stimulus-activated conductance in isolated taste epithelial membranes.

Membrane vesicles isolated from the cutaneous taste epithelium of the catfish were incorporated into phospholipid bilayers on the tips of patch pipettes. Voltage-dependent conductances were observed in approximately 50% of the bilayers and single-channel currents having conductances from 8 to greater than 250 pS were recorded. In 40% of the bilayers displaying no voltage-dependent conductances, micromolar concentrations of L-arginine, a potent stimulus for one class of catfish amino acid taste receptors, activated a nonselective cation conductance. The L-arginine-gated conductance was concentration-dependent, showing half-maximal activation in response to approximately 15 microM L-arginine. L-Arginine-activated channels had unitary conductances of 40-50 pS and reversed between -6 and +18 mV with pseudointracellular solution in the pipette and Ringer in the bath. L-Alanine, a potent stimulus for the other major class of catfish amino acid taste receptors, did not alter bilayer conductance. D-Arginine, which is a relatively ineffective taste stimulus for catfish but a good cross-adapter of the L-arginine-induced neural response, had no effect on bilayer conductance at concentrations below 200 microM. However, increasing concentrations of D-arginine from 1 to 100 microM progressively suppressed the L-arginine-activated conductance, suggesting that D-arginine competed for the L-arginine receptor, but did not activate the associated cation channel. This interpretation is consonant with recent biochemical binding studies in this system. These results suggest that L-arginine taste receptor proteins in the catfish are part of or closely coupled to cation-selective channels which are opened by L-arginine binding.     

How Does a Voltage Sensor Interact with a Lipid Bilayer? Simulations of a Potassium Channel Domain

The nature of voltage sensing by voltage-activated ion channels is a key problem in membrane protein structural biology. The way in which the voltage-sensor (VS) domain interacts with its membrane environment remains unclear. In particular, the known structures of Kv channels do not readily explain how a positively charged S4 helix is able to stably span a lipid bilayer. Extended (2 x 50 ns) molecular dynamics simulations of the high-resolution structure of the isolated VS domain from the archaebacterial potassium channel KvAP, embedded in zwitterionic and in anionic lipid bilayers, have been used to explore VS/lipid interactions at atomic resolution. The simulations reveal penetration of water into the center of the VS and bilayer. Furthermore, there is significant local deformation of the lipid bilayer by interactions between lipid phosphate groups and arginine side chains of S4. As a consequence of this, the electrostatic field is "focused" across the center of the bilayer.     

Calcium channel activity in a purified dihydropyridine-receptor preparation of skeletal muscle

A purified dihydropyridine-receptor complex (DHPR) of skeletal muscle consisting of a major polypeptide of Mr 150K under reducing conditions induces divalent cation selective channels when incorporated into planar lipid bilayers. Channels were inserted into preformed planar bilayers by two techniques: (i) direct dilution of detergent-solubilized DHPR into the aqueous chambers adjacent to the bilayer membrane or (ii) reconstitution of DHPR into phospholipid vesicles followed by fusion of the preformed vesicles to the planar bilayer membrane. Unlike native membrane preparations of t-tubules, which only have one major Ca channel type of slope conductance of 12 pS in symmetrical 100 mM Ba, the purified DHPR complex induced at least two channel types with conductances of 12-14 and 22 pS. Some recordings suggest that these two channels are statistically coupled in time, i.e., that they may correspond to substrates of the same DHPR channel. Activity was found to occur spontaneously in the absence of the Ca channel agonist Bay k 8644. The 12-14-pS channel from DHPR exhibits voltage-dependent kinetics, is highly selective for barium ions, and was inhibited by micromolar nitrendipine. The 12-14-pS DHPR channel appears to be identical with functional Ca channels previously described in native t-tubules.     

Mechanosensitivity of an epithelial Na+ channel in planar lipid bilayers: release from Ca2+ block.

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+.