Pulmonary Surfactant Proteins Insert Cation-Permeable Channels in Planar Bilayers

Molecular Genetics and Metabolism 70, 295–300 (2000) doi:10.1006/mgme.2000.3022, available online at http://www.idealibrary.com on

Pulmonary Surfactant Proteins Insert Cation-Permeable Channels in Planar Bilayers David G. Oelberg* ,1 and Fang Xu† *Center for Pediatric Research and Department of Pediatrics, Children’s Hospital of The King’s Daughters and Eastern Virginia Medical School, 855 West Brambleton Avenue, Norfolk, Virginia 23510; and †Department of Internal Medicine–Cardiovascular Division, University of Virginia Health Sciences Center, Box 158, Charlottesville, Virginia 22903 Received June 22, 2000

Air breathing creates air-liquid interfaces within the lungs. Ensuing surface tension promotes collapse unless pulmonary surfactant is available to reduce surface tension and stabilize interfaces. Recently, other effects have been linked to pulmonary surfactants that are not sufficiently explained by reduction of surface tension. Decrease of smooth muscle tone (1), modulation of immune response (2), inhibition of bacterial growth (3), and promotion of salt-water clearance by nonventilated lungs (4) are attributable surfactant effects that are not readily explained by surface tension reduction. Despite the recognition that inclusion of surfactant proteins (SPs) in the phospholipid mixture is necessary for many of these salutary effects, mechanism(s) of action beyond reduction of surface tension remain unclear. We have observed that intact pulmonary surfactant containing hydrophobic proteins (PS⫹SP), surfactant protein B (SP-B), and surfactant protein C (SP-C)— unlike synthetic surfactant that lacks all protein components (PS-SP)—suppresses neutrophil activation via membrane depolarization (5). We hypothesize that hydrophobic proteins from PS⫹SP insert in membranes to induce channel activities that modify cellular responses. We tested for in vitro channel insertion by PS⫹SP employing planar lipid bilayers. PS⫹SP was presented to bilayers employing either intact surfactant preparations or extracted surfactant proteins. The observation that PS⫹SP inserts monovalent, cation-selective channels in planar lipid bilayers suggests that some of the observed biological effects of pulmonary surfac-

Pulmonary surfactant, a mixture of lipids and proteins, promotes lung ventilation by reduction of surface tension at air–fluid interfaces. Exogenous surfactants containing hydrophobic proteins induce biological effects in lungs that are not fully explained by reduction of surface tension and are not induced by surfactants lacking proteins. We hypothesized that hydrophobic proteins from surfactant insert in membranes to induce channel activities that contribute to the observed biological effects of surfactant. To test for channel insertion by surfactant, planar lipid bilayers were monitored electrophysiologically in the presence of either intact pulmonary surfactant or extracted surfactant proteins reconstituted with phospholipids or directly added to bilayer lipids prior to membrane casting. In this in vitro model, both intact surfactant and extracted surfactant proteins initiated gated channel activities with slope conductances averaging 40 pS. Observed reversal potentials confirmed monovalent cation conductance, and conductance of smaller monovalent cations was selective. Voltage dependence of channel openings and rectification of channel current were not observed. These results confirm that hydrophobic surfactant proteins induce channel-mediated transport in artificial membranes. We speculate that pulmonary surfactants, in addition to reducing surface tension at air–fluid interfaces, initiate physiological and therapeutic effects in lung by cation channel insertion at exposed epithelial membranes. © 2000 Academic Press

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To whom correspondence should be addressed at Center for Pediatric Research, Eastern Virginia Medical School, 855 West Brambleton Avenue, Norfolk, VA 23510-1001. Fax: (757) 6686476. E-mail: [email protected]. 295

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tants— both physiologic and therapeutic—are explained by cation channel insertion in membrane. METHODS Intact surfactant preparations. To test for channel insertion by intact surfactant preparations in initial studies, two commercially available surfactants— beractant (PS⫹SP) and colfosceril palmitate, hexadecanol, tyloxapol (PS-SP)—were tested. Beractant (Survanta; Abbott Laboratories, Chicago, IL) is a lipid-fortified extract of minced bovine lung containing two of four SPs—SP-B and SP-C (PS⫹SP). Colfosceril palmitate, hexadecanol, tyloxapol (Exosurf; Glaxo Wellcome Inc., Research Triangle Park, NC) is a synthetic lipid preparation functionally resembling natural pulmonary surfactant but lacking SPs (PS-SP). Surfactant protein extraction and reconstitution. To test for the dependency of channel insertion upon SP presence while controlling the lipid environment, channel activity was examined in the presence of phospholipids mimicking the lipid composition of natural pulmonary surfactant reconstituted (PS⫹SP) or not reconstituted (PS-SP) with extracted SPs. SPs and lipid were extracted from beractant following dilution with methanol (2:0.8 (v/v) methanol:H 2O) and solubilization with chloroform (1:2:0.8 CHCl 3:MeOH:H 2O) (6). Phase separation was effected by diluting the mixture with CHCl 3 and H 2O to achieve a final ratio of 2:2:1.8. Modified Folch extraction was completed by separation and filtration of the organic phase followed by evaporation of CHCl 3 under vacuum (43°C). To separate SPs from lipid, organic extract was applied to a Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden) column (2.5 ⫻ 100 cm) equilibrated with 2:1 (v/v) CHCl 3:MeOH and eluted (0.3 ml/min flow) while monitoring effluent for protein at 280 nm (7). Fractions were collected (4.5 ml) and protein was measured with 0.1% SDS (8). One-dimensional SDSPAGE was performed under reducing conditions with 2-mercaptoethanol employing Tris-Tricine 16.5% gels stained with Coomassie blue (9). Apparent molecular weights were estimated by comparing mobilities with polypeptide molecular weight standards (Bio-Rad Laboratories, Hercules, CA). Creation of artificial membranes. Planar lipid bilayers were created by painting phospholipids dissolved in decane across an aperture (250 ␮m diameter) separating cis and trans compartments of

buffered salt solutions. A film of phospholipids (phosphatidylethanolamine:phosphatidylserine (50: 50)) (Avanti Polar Lipids, Alabaster, AL) dissolved in decane (20 mg/ml) was painted across the aperture in a Delrin partition while immersed in buffered electrolyte solutions. Each bilayer was cast in the presence of 1 mM CaCl 2, 5 mM Tris-Hepes (pH 7.4), and 50 –500:50 –500 mM (cis:trans) salt solution comprised of sodium, potassium, or choline chloride salts. Spontaneous thinning of the film was monitored both visually by dissecting microscope and electrically by observation of increasing membrane capacitance. Electrical measurements were made at ambient temperatures by way of agar bridges connecting Ag/AgCl electrodes to cupped solutions of electrolyte on either side of the bilayer. Centrally located areas of thinned film (50 –100 ␮m diameter) constituted the bilayer with capacitances of ⬃200 pF and resistances ⬎100 ⍀/cm 2. Testing of intact surfactant and extracted surfactant proteins for channel insertion. Intact surfactants, beractant (PS⫹SP) or colfosceril palmitate, hexadecanol, tyloxapol (PS-SP), were tested for channel insertion by addition to the cis chamber at a dilution of 0.03– 0.3%. Extracted SPs were tested for channel insertion by either of two methods. By the first method, SPs reconstituted with phospholipids (PS⫹SP) (protein:lipid ratio ⫽ 5:1000) dipalmitoylphosphatidylcholine, phosphatidylglycerol, and phosphatidic acid (7:2:1) were added to the cis chamber at a dilution of 0.03– 0.3%. In separate experiments, dipalmitoylphosphatidylcholine, phosphatidylglycerol, and phosphatidic acid (7:2:1) lacking SPs (PS-SP) were added to the cis chamber at 0.03– 0.3% dilution. By the second method, prior to casting bilayers, extracted SPs were mixed directly with bilayer phospholipids phosphatidylethanolamine: phosphatidylserine (50:50) in CHCl 3 providing a 1:1000 (w/w) protein:lipid mixture. Solvent was removed under N 2, the dried protein:lipid residue was dissolved in decane (20 mg/ml), and the bilayer (containing SPs by direct addition) was cast. Following addition of one of the PS⫹SP or PS-SP preparations to the cis chamber or following direct addition of SPs to the bilayer, the trans chamber was held at virtual ground by an Axopatch 1C patch clamp amplifier with integrating headstage configured in the “bilayer” mode. The amplifier measured current while applying holding potential across the bilayer to clamp voltage. Data were viewed on a digital oscilloscope and stored on a Neurocorder

SURFACTANT INSERTS CHANNELS IN PLANAR BILAYERS

VCR-based recording system for subsequent analysis. Membrane capacitance was manually nulled. Under these conditions and at holding potentials greater than the reversal potential (E rev), incorporation of a cation-selective channel was represented by positive current fluctuations. Upon identification of gated current activity across the bilayer, single channel slope conductance ( g s) was calculated from the equation g s ⫽ dI(V)/dV, where dI(V) is the measured single channel current (difference between total current when channel is open and when it is closed) at an applied potential (V). Average open probability (P o) was estimated by determining the sum of the individual channel openings using the 50% threshold detection method and dividing by the total time analyzed. Voltage dependency was demonstrated by voltage-dependent changes in P o in the presence of transbilayer salt gradients (i.e., asymmetrical salt solutions). Channel rectification was indicated by loss of linearity over the I–V slope. Cation/anion selectivity of a channel was determined from the E rev—applied holding potential at which current activity was absent— calculated by the Goldman-Hodgkin-Katz equation. RESULTS Channel activity induced by intact PS⫹SP. Within 5–20 min of the addition of intact PS⫹SP (beractant) to the stirred cis chamber, current conductance was typically induced in the painted bilayers (Fig. 1). Under both symmetrical (50:50 or 500: 500 mM) and asymmetrical (500:50 mM) NaCl and KCl conditions, conductance at a given holding potential exhibited gating between two defined levels of current with sharp transitions between open and closed states characteristic of single channel activity. Slope conductances averaged 39 ⫾ 6 pS (mean ⫾ SE) across all preparations exhibiting channel insertion (Fig. 2A), and observed E rev for preparations employing asymmetric cis:trans salt compositions confirmed that current was carried by both monovalent cations, Na ⫹ and K ⫹ (Fig. 2A). Other characteristics defining single channel activity—versus simple diffusion or carrier-mediated transport— included randomly occurring dwell times within open and closed states (Fig. 2B). Examining P o as a function of the applied holding potential, dependence of P o upon voltage was not observed. Under 500 mM NaCl:500 mM KCl bathing conditions, small predilection for K ⫹ was observed based on the change in E rev (P K/P Na ⫽ 1.3). Dependence of chan-

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FIG. 1. Single channel currents from intact PS⫹SP incorporated in planar lipid bilayer. Current records are from a bilayer symmetrically cast between 50 and 50 mM (cis:trans) NaCl-buffered solutions. Intact PS⫹SP (beractant) (0.03%) was added to the cis chamber. Single channel currents were recorded at indicated holding potentials. Current openings were indicated by upward deflections at positive potentials and downward at negative potentials.

nels upon Ca 2⫹ presence was suggested by suppression of channel conductance following Ca 2⫹ chelation with EGTA. However, bilayer survival was also limited by Ca 2⫹ chelation, and bilayer instability likely accounted for loss of current conductance. Divalent cation conductance was not observed in the absence of monovalent cations and the presence of symmetrical 50 mM CaCl 2 or MgCl 2 bathing conditions. Channel blockade did not occur in the presence of amiloride (1 mM), Ba 2⫹ (6 mM), or Gd 3⫹ (1 mM). Channel rectification was not observed (Fig. 2A). Channel activity was promoted by increased concentrations of beractant (0.3–1%), application of higher holding potentials (⫾50 –70 mV), and mechanical stirring of the cis compartment—presumably by promoting fusion of channel-forming components with the planar bilayer. In contrast to observed channel insertion by intact PS⫹SP containing SP-B and SP-C, PS-SP (colfosceril palmitate, hexadecanol, tyloxapol) did not initiate channel activity at any time. Channel activity induced by extracted SPs. To test the hypothesis that SPs are necessary, and possibly sufficient, for formation of oligomeric complexes contributing to channel activity in the present model, SP-B and SP-C were extracted from

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beractant and tested for channel insertion. Analysis of eluted fraction 26 by SDS-PAGE under reducing conditions revealed two bands at 3 and 6 kDa, respectively (Fig. 3). These bands corresponded with the reported mobilities of SP-C and SP-B following SDS-PAGE (10). Upon confirming their identity by SDS-PAGE, extracted SPs from fraction 26 were presented to planar lipid bilayers by either of two methods. By the first method, extracted SPs were reconstituted with

FIG. 3. Analysis of SPs extracted from intact PS⫹SP. SDSpolyacrylamide gel electrophoresis was performed of standard proteins (M r) and eluted surfactant extract (Fraction 26). Protein bands at 3 and 6 kDa correspond to SP-C and SP-B, respectively.

lipids— dipalmitoylphosphatidylcholine, phosphatidylglycerol and phosphatidic acid—resembling those of intact pulmonary surfactant. By the second method, extracted SPs were added directly to bilayer phospholipids—phosphatidylethanolamine and phosphatidylserine—prior to bilayer casting. By both methods, SPs inserted selective cation channels of uniform conductances (Fig. 4A) similar to those inserted by intact PS⫹SP. Slope conductances following direct addition and reconstitution were 37 and 39 pS, respectively (Fig. 4B). Observed E rev during 500:50 mM NaCl conditions confirmed cation selectivity nearly identical (E rev ⫽ ⫺58 mV) to that of intact PS⫹SP (results not shown). Na ⫹ was selectively conducted over the larger cation, choline ⫹ (P choline/P Na ⫽ 0.5), and voltage dependence was not observed. Amiloride had no effect on activity, and channel rectification was not apparent (Fig. 4B). In the absence of extracted SPs, reconstituting lipids alone (PS-SP)— dipalmitoylphosphatidylcholine, phosphatidylglycerol, and phosphatidic acid— when presented to lipid bilayers did not induce channel activity. DISCUSSION

FIG. 2. Current–voltage relationships and open probability of channels. (A) Current–voltage relationships of single channels inserted by intact PS⫹SP in asymmetric bilayers during three experiments. Bilayers were cast between 500 and 50 mM (cis: trans) buffered NaCl (closed circles) and KCl (open circles). E rev values equaling ⫺62 and ⫺64 mV, respectively, confirm that channels were selective for monovalent cations Na ⫹ and K ⫹. Slope conductances averaged 38 pS. (B) Channel open time distributions at ⫹70 mV were discriminated at 50% of the open channel current. The smooth curve is a single exponential leastsquares fit of data (time constant ⫽ 8 ms).

These studies provide the first electrophysiological evidence that pulmonary surfactant has the potential to insert monovalent cation channels in native membrane. The insertion of channels by intact PS⫹SP and by extracted SPs added directly to bilayer lipids prior to casting or as reconstituted PS⫹SP and the failure of intact PS-SP and of reconstituting lipids lacking SPs to induce channel activity collectively demonstrate that SPs are critical to channel formation by surfactant preparations. Analysis of SPs extracted from intact PS⫹SP by

SURFACTANT INSERTS CHANNELS IN PLANAR BILAYERS

FIG. 4. Single channel currents from extracted SPs incorporated in planar lipid bilayer. (A) An aliquot of fraction 26 mixed directly with phosphatidylethanolamine:phosphatidylserine (50: 50) prior to casting bilayer between 50 and 50 mM (cis:trans) NaCl inserted single channel currents at indicated holding potentials. (B) Current–voltage relationships of single channels inserted by extracted SPs in symmetric bilayers (50:50 mM NaCl). SPs inserted in bilayers by prior reconstitution with lipids (open circles) or by direct addition (closed circles). Slope conductances equaled 39 and 37 pS, respectively.

us and others indicates that SPs in this surfactant preparation consist of SP-B and SP-C (7,10). Both SP-B and SP-C are hydrophobic lipopolypeptides containing extended ␣-helical regions within their secondary structures. Amphipathic, ␣-helical structures within SP-B and SP-C resemble other monomeric proteins that are recognized to insert in membrane bilayers and combine with other subunits to form channel oligomers (11). The 37–39 pS conductances of intact PS⫹SP, reconstituted PS⫹SP, and

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directly incorporated SPs resemble the 40 pS conductance of other ␣-helical proteins forming cation channels as pentameric clusters (12). The SP-C ␣-helix is 37 Å long with a central hydrophobic region 23 Å long (13). This structure provides sufficient length to span the hydrophobic domain of bilayers (14). It has been observed by FTIR spectroscopy that upon interaction with bilayers, the SP-C molecule inserts by spanning the bilayer in an ␣-helical configuration (15). Coupled with the observation that SP-C spans bilayers with a 24° tilt (16), SP-C appears well suited to compose part of an oligomeric channel complex (15). By contrast, its propensity to exist in monomeric state within lipid bilayer (17) limits its suitability for channel formation in a pure form. In this regard, SP-C might complex with SP-B to form a channel oligomer. The structure of SP-B is homologous with that of other saposin-like proteins including the Entamoeba histolytica pore-forming peptide (18). SP-B is larger than SP-C with approximately half of the molecule in ␣-helical configuration. However, in contrast to the transmembrane orientation of SP-C in lipid bilayer, FTIR spectroscopy indicates that SP-B interacts with bilayers by shallow anchoring at the membrane surface (19), thus aligning parallel with the bilayer axis and limiting its suitability as a complete channel protein. The possibility that channel activities observed in these studies were caused by an unidentified contaminant in the employed buffer solutions, intact PS⫹SP preparation, casting bilayer lipids, or reconstituting lipids cannot be fully excluded. However, the likelihood of this possibility is very low for the following reasons. Channel activity was not observed in the absence of SPs presented as either intact PS⫹SP or extracted SPs. Experiments employed several different batches of the supplied intact PS⫹SP. Analyses of the intact PS⫹SP by others and of the extracted SPs by others and by us have failed to identify components other than the identified lipids and hydrophobic proteins (7,10). These observations combined with repeated demonstration of uniform channel properties over several months limit the probability of a contaminating channel protein. The identification of channel insertion by SPs in lipid bilayers offers new understanding about the mechanisms of pulmonary surfactant in health and disease. We speculate that insertion of channels explains the promotional effect of surfactant upon fluid clearance in alveoli (4) and the inhibitory effects

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upon microorganism growth (3), and upon neutrophil (5) and monocyte activations (20). The identification of surfactant-like particles at epithelia of the middle ear and small bowel supports the additional possibility that insertion of cation channels may contribute to the salt-water physiology or immunomodulatory cascades of epithelial tissues other than lung (21). In this regard, it has been suggested that alterations of middle ear surfactant may predispose to middle ear disease (22). Continued investigation of channel insertion by SPs may refine current therapeutic uses of pulmonary surfactant in the lung while stimulating novel applications to nonpulmonary diseases.

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Vaccaro AM, Salvioli R, Tatti M, Ciaffoni F. Saposins and their interaction with lipids. Neurochem Res 24:307–314, 1999.

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Vandenbussche G, Clercx A, Clercx M, Curstedt T, Johansson J, Jornvall H, Ruysschaert JM. Secondary structure and orientation of the surfactant protein SP-B in a lipid environment. Biochemistry 31:9169 –9176, 1992.

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Pinot F, Walti H, Haagsman HP, Polla BS, Bachelet M. Curosurf modulates cAMP accumulation through a membrane-controlled mechanism. Am J Physiol Lung Cell Mol Physiol 278:L99 –L104, 2000.

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Mira E, Benazzo M, DePaoli F, Casasco A, Calligaro A. Surfactants of the airways. Critical review and personal research. Acta Otorhinolaryngol Ital 17:3–16, 1997.

ACKNOWLEDGMENT This work was supported in part by National Institutes of Health Grant HL-02292.

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