Effects of palytoxin on cation occlusion and phosphorylation of the (Na+,K+)-ATPase.

Palytoxin (PTX) inhibits the (Na(+) + K+)-driven pump and simultaneously opens channels that are equally permeable to Na+ and K+ in red cells and other cell membranes. In an effort to understand the mechanism by which PTX induces these fluxes, we have studied the effects of PTX on: 1) K+ and Na+ occlusion by the pump protein; 2) phosphorylation and dephosphorylation of the enzyme when a phosphoenzyme is formed from ATP and from P(i); and 3) p-nitro phenyl phosphatase (p-NPPase) activity associated with the (Na+, K+)-ATPase. We have found that palytoxin 1) increases the rate of deocclusion of K+(Rb+) in a time- and concentration-dependent manner, whereas Na+ occluded in the presence of oligomycin is unaffected by the toxin; 2) makes phosphorylation from P(i) insensitive to K+, and 3) stimulates the p-NPPase activity. The results are consistent with the notion that PTX produces a conformation of the Na+, K(+)-pump that resembles the one observed when ATP is bound to its low-affinity binding site. Further, they suggest that the channels that are formed by PTX might arise as a consequence of a perturbation in the ATPase structure, leading to the loss of control of the outside "gate" of the enzyme and hence to an uncoupling of the ion transport from the catalytic function of the ATPase.

Ion channels formed by transcription factors recognize consensus DNA sequences.

Transcription factors (TFs) are proteins which bind to specific DNA sequences and thus participate in the regulation of the initiation of transcription. We report in this communication our observations that several of these proteins interact with lipid membranes and form ion-permeable channels. For each of the TFs that we studied, the single channel conductance was distinctively different, i.e. each TF had its own electrical signature. More importantly, we show for the first time that addition of cognate double-stranded DNA sequences leads to a specific response: an increase in the conductance of the TF-containing membrane. Strikingly, the effect of cognate DNA was observed when it was added to the trans-side of the membrane (opposite to where the TF was added), strongly suggesting that the TFs span the membrane and that the DNA-binding domain is trans-accessible. Alterations in the primary structure of the TF factors in their basic and DNA-binding regions change the characteristics of the conductance of the protein-containing membranes as well as the response to DNA addition, reinforcing the notion that the changes we measure are due to specific interactions.

Characterization of the ion channels formed by poliovirus in planar lipid membranes.

The steps in poliovirus infection leading to viral entry and uncoating are not well understood. Current evidence suggests that the virus first binds to a plasma membrane-bound receptor present in viable cells, leading to a conformational rearrangement of the viral proteins such that the virus crosses the membrane and releases the genomic RNA. The studies described in this report were undertaken to determine if poliovirus (160S) as well as one of the subviral particles (135S) could interact with membranes lacking poliovirus receptors in an effort to begin to understand the process of uncoating of the virus. We report that both forms of viral particles, 160S and 135S, interact with lipid membranes and induce the formation of ion-permeable channels in a manner that does not require acid pH. The channels induced by the viral particles 160S have a voltage-dependent conductance which depends on the ionic composition of the medium. Our findings raise the possibility that viral entry into cells may be mediated by direct interaction of viral surface proteins with membrane lipids.

The transient pore formed by homologous terminal complement complexes functions as a bidirectional route for the transport of autocrine and paracrine signals across human cell membranes.

BACKGROUND: We have previously shown that the membrane attack complex (MAC) of complement stimulates cell proliferation and that insertion of homologous MAC into the membranes of endothelial cells results in the release of potent mitogens, including basic fibroblast growth factor (bFGF). The mechanism of secretion of bFGF and other polypeptides devoid of signal peptides, such as interleukin 1 (IL-1) is still an open problem in cell biology. We have hypothesized that the homologous MAC pore itself could constitute a transient route for the diffusion of biologically active macromolecules in and out of the target cells. MATERIALS AND METHODS: Human red blood cell ghosts and artificial lipid vesicles were loaded with labeled growth factors, cytokines and IgG, and exposed to homologous MAC. The release of the 125I-macromolecules was followed as a function of time. The incorporation of labeled polypeptides and fluorescent dextran (MW: 10,000) was measured in MAC-impacted human red blood cells and human umbilical endothelial cells (HUVEC), respectively. RESULTS: Homologous MAC insertion into HUVEC resulted in the massive uptake of 10-kD dextran and induced the release of bFGF, in the absence of any measurable lysis. Red blood cell ghosts preloaded with bFGF, IL-1 beta, and the alpha-chain of interferon-gamma (IFN-gamma) released the polypeptides upon MAC insertion, but they did not release preloaded IgG. MAC-impacted ghosts took up radioactive IFN-gamma from the extracellular medium. Vesicles loaded with IL-I released the polypeptide when exposed to MAC. CONCLUSIONS: The homologous MAC pore in its nonlytic form allows for the export of cytosolic proteins devoid of signal peptides that are not secreted through the classical endoplasmic reticulum/Golgi exocytotic pathways. Our results suggest that the release, and perhaps the uptake, of biologically active macromolecules through the homologous MAC pore is a novel biological function of the complement system in mammals.

Interaction of palytoxin with red cells: structure-function studies.

Palytoxin (PTX), a potent toxin isolated from the marine soft coral Palythoa tuberculosa increases the cationic permeability of red cell membranes and inhibits the (Na+,K+)-activated ATPase, effects that are completely reversed by ouabain. It has also been shown to compete with ouabain for a binding site and it has been suggested that it binds to the Na+,K(+)-pump molecule in cells. In a search for analogues of PTX that would bind covalently and could thus be used to identify and characterize the binding site, we have used compounds which differed from PTX at one end or at both ends simultaneously. In order to determine whether these derivatives could be used to replace palytoxin, we tested their potency to induce an increased cation flux, complete with ouabain for its binding site, and inhibit the isolated, purified (Na+,K(+)-ATPase). The results obtained indicate that departures from the PTX structure at one end or at both ends simultaneously substantially decrease the ability of the compounds to increase the cationic permeability of red blood cells and to inhibit the (Na+,K(+)-ATPase). These actions were found to be completely reversed by ouabain, but the analogues are two to three orders of magnitude less potent than PTX in competing with ouabain for its binding site. These results suggest that both ends of the palytoxin molecule participate in the interactions of the toxin with its receptor and that modifications in these regions of the molecule produce significant alterations in its binding and subsequent action on red cell membranes.

Reconstitution of the influenza virus M2 ion channel in lipid bilayers.

M2, an integral membrane protein of influenza A virus, was purified from either influenza A virus-infected CV-1 cells or from Spodoptera frugiperda (Sf9) cells infected with a recombinant-M2 baculovirus. The purified protein, when incorporated into phospholipid bilayer membranes, produced ion-permeable channels with the following characteristics: (1) The channels appeared in bursts during which unit conductances of diverse magnitudes (25-500 pS) were observed. (2) The most probable open state was usually the lowest unit conductance (25-90 pS). (3) The channels were selective for cations; tNa = 0.75 when 150 mM NaCl bathed both sides of the membrane. (4) Amantadine reduced the probablity of opening of the high conductance state and also the conductance of the most probable state. (5) Reducing pH increased the mean current through the open channel as well as the conductance of the most probable state. (6) The sequence of selectivity for group IA monovalent cations was Rb > K > Cs approximately Na > Li. The pH activation, amantadine block and ion selectivity of the M2 protein ion channel in bilayers are consistent with those observed on expression of the M2 protein in oocytes of Xenopus laevis as well as for those predicted for the proposed role of an ion channel in the uncoating process of influenza virus. The finding that the M2 protein has intrinsic ion channel activity supports the hypothesis that it has ion channel activity in the influenza virus particle.

Inhibition of the actions of palytoxin on the K+ efflux from red cells and on the Na/K-ATPase activity by a monoclonal antibody.

Palytoxin (PTX) binds to the Na/K pump, inhibits the (Na/K)-ATPase and forms Na and K permeable channels in human red cells. Here, we report that a monoclonal antibody raised against a derivative of PTX (Bignami, G.S. et al. (1992) Toxicon 30, 687-700) inhibits these effects. The observations are consistent with a model in which (a) the antibody binds and, thus reduces the concentration of free PTX available to react with the Na/K pump, and, (b) the PTX-antibody complex also binds to the PTX receptor on the Na/K pump but in such a way that a cation permeable channel is not formed, probably by reducing the concentration of free PTX. Using this model, we estimate that the apparent dissociation constant for the binding of PTX to antibody is 0.2 nM.

Ion channels induced in lipid bilayers by subvirion particles of the nonenveloped mammalian reoviruses.

Mechanisms by which nonenveloped viruses penetrate cell membranes as an early step in infection are not well understood. Current ideas about the mode for cytosolic penetration by nonenveloped viruses include (i) formation of a membrane-spanning pore through which viral components enter the cell and (ii) local breakdown of the cellular membrane to provide direct access of infecting virus to the cell's interior. Here we report that of the three viral particles of nonenveloped mammalian reoviruses: virions, infectious subvirion particles, and cores (the last two forms generated from intact reovirus virions by proteolysis), only the infectious subvirion particles induced the formation of anion-selective, multisized channels in planar lipid bilayers under the experimental conditions used in this study. The value for the smallest size conductance varied depending on the lipid composition of the bilayer between 90 pS (Asolectin) and 300 pS (phosphatidylethanolamine:phosphatidylserine) and was found to be voltage independent. These findings are consistent with a proposal that the proteolytically activated infectious subviral particles mediate the interaction between virus and the lipid bilayer of a cell membrane during penetration. In addition, the findings indicate that the "penetration proteins" of some enveloped and nonenveloped viruses share similarities in the way they interact with bilayers.

Palytoxin induces an increase in the cation conductance of red cells.

Palytoxin (PTX), isolated from the marine soft coral Palythoa tuberculosa, increases the cation conductance of human red cell membranes. In the presence of 10(-10) M PTX and 10(-5) M DIDS, the membrane potential approximates the equilibrium potential for Na+ or K+ rather than Cl-. Even in the absence of DIDS, the Na+ and K+ conductances were greater than the Cl- conductance. The selectivity of the PTX-induced cation conductance is K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+ much greater than choline+ greater than TEA+ much greater than Mg2+. Measurements of K+ efflux revealed two apparent sites for activation by PTX, one with a Kal of 0.05 nM and a maximum flux, nu max1, of 1.4 mol/liter of cells per h and another with a Ka2 of 98 nM and a nu max2 of 24 mol/liter of cells per h. These effects of PTX are completely blocked by external ouabain (300 microM) and prevented by internal vanadate (100 microM). When the PTX channels are open, the Na,K pumps do not catalyze ATP hydrolysis. Upon thorough washout of cells exposed to about five molecules of PTX/pump, the Na,K pump of these cells operates normally. Blockage of the positively charged NH2 terminus of PTX with a p-bromobenzoyl group reduces the potency of the compound to induce Na and K fluxes by at least a factor of 100, and to compete with the binding of [3H]ouabain by at least a factor of 10. These data are consistent with the conclusion that PTX binds reversibly to the Na,K pumps in the red cell membrane and opens a (10-pS) channel equally permeable to Na and K at or near each pump site.

CD4+ lipid bilayers. A model for human immunodeficiency virus type 1 coat protein binding.

gp120, the coat glycoprotein of the human immunodeficiency virus type 1 (HIV1) binds to a molecule on the surface of a class of T-lymphocytes, CD4, which is also the receptor for major histocompatibility complex class II (MHCII). To study the events that follow the interaction of gp120 with CD4, we have incorporated CD4 into lipid bilayers and recorded the electrical changes which occur after the addition of gp120. Interaction of gp120 to CD4-containing bilayers induces multistate ion-permeable channels with a maximum conductance of 380-400 picosiemens. When CD4+ bilayers were preexposed to either MHCII or to OKT4A antibody, no channels were formed after the addition of gp120. These results indicate that CD(4+)-containing bilayers bind gp120, MHCII, and OKT4A, that binding of gp120 produces ion-permeable channels, and that CD4+ bilayers can be used to assay for gp120 in the solution bathing the bilayer.

Primary structure of peptides and ion channels. Role of amino acid side chains in voltage gating of melittin channels.

Melittin produces a voltage-dependent increase in the conductance of planar lipid bilayers. The conductance increases when the side of the membrane to which melittin has been added (cis-side) is made positive. This paper reports observations on the effect of modifying two positively charged amino acid residues within the NH2-terminal region of the molecule: lysine at position 7 (K7), and the NH2-terminal glycine (G1). We have synthesized melittin analogues in which K7 is replaced by asparagine (K7-N), G1 is blocked by a formyl group (G1-f), and in which both modifications of the parent compound were introduced (G1-f, K7-N). The time required to reach peak conductance during a constant voltage pulse was shorter in membranes exposed to the analogues than in membranes modified by melittin. The apparent number of monomers producing a conducting unit for [K7-N]-melittin and [G1-f]-melittin, eight, was found to be greater than the one for [G1-f], K7-N]-melittin and for melittin itself, four. The apparent gating charge per monomer was less for the analogues, 0.5-0.3 than for melittin, one. Essentially similar results were obtained with melittin analogues in which the charge on K7 or G1 or both was blocked by an uncharged N-linked spin label. These results show that the positive charges in the NH2-terminal region of melittin play a major but not exclusive role in the voltage gating of melittin channels in bilayers.

Voltage-activated cation transport in human erythrocytes.

We report here the effects of membrane potential on the permeability of the human erythrocyte to Na, K, and Ca. Membrane potential was changed either by varying the K concentration gradient in the presence of valinomycin or by varying the concentration gradient of the permeant anion nitrate in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. When the membrane potential was changed from inside negative (-10 mV) to inside positive (greater than 40 mV), influx, efflux, and net flux of Na and K increased. Marked net cation loss and cell shrinkage occurred in the absence of a chemical gradient for Na and K. This voltage-dependent increase in Na and K conductance is partially inhibited by 10 microM ruthenium red and persists when the membrane potential is returned to -10 mV after transient exposure to inside-positive potentials. A similar voltage-dependent behavior was found for Ca influx. The voltage-activated Ca influx is almost completely inhibited by 10 microM ruthenium red.

Voltage-gated channels formed in lipid bilayers by a positively charged segment of the Na-channel polypeptide.

The Na-channel polypeptide is responsible for the voltage-gated and time-dependent ionic permeability changes that give rise to the action potential in the membranes of nerve cells. We have synthesized a 22-amino acid peptide with a sequence identical to that of the segment named S4, repeat IV of the primary structure of the Na channel. We have found that this peptide induces a voltage- and time-dependent conductance in bilayers formed by a mixture of phosphatidyl-ethanolamine and phosphatidylserine. This conductance is activated when the cis side is made positive, with an apparent gating charge of 3. The results are consistent with the idea that this segment plays a role in determining the voltage sensitivity of the Na channel.

The synthetic precursor specific region of pre-pro-parathyroid hormone forms ion channels in lipid bilayers.

We have used the chemically synthesized sequence of pre-pro-parathyroid hormone and several of its analogues to test the notion that the capacity of amphipathic peptides to aggregate in membranes and form ion-permeable channels correlates with their ability to function as signal sequences for secreted proteins. We found that pre-pro-parathyroid hormone (the signal sequence and pro-region of parathyroid hormone (M], as well as some of its analogues, forms aggregates of monomers which are ion-permeable. The ion-permeable aggregates (2-3 monomers) formed by (M) are voltage-dependent and are more permeable for cations than for anions. The compounds which formed ion channels in bilayers also acted as potential signal sequences. We conclude that the ability of peptides to form ion-permeable pathways in bilayers may be correlated to their ability to function as signal peptides.

Solid-phase synthesis of melittin: purification and functional characterization.

The main component of the honey bee venom, melittin, is a cationic polypeptide containing 26 amino acids. Exposure of lipid bilayers to this peptide results in the formation of anion-selective channels with a variety of unit conductances. One of the possible causes for this heterogeneity in the conductance could be heterogeneity of the melittin preparation, and indeed, the existence of two prominent forms of naturally occurring melittin, differing only at the N-terminal amino group, has been documented. This paper describes the synthesis of the major form of melittin, using stepwise solid-phase methodology and the demonstration that the synthetic melittin, devoid of the minor component (N-formylmelittin) and other contaminants, interacts with lipid bilayers to form channels which are qualitatively indistinguishable from the ones formed by the naturally occurring toxin. This result indicates that the heterogeneity in the channels produced in bilayers by bee venom is not due to differences in the channel-forming properties of the formyl and non-formyl melittin but rather to differences in the number and orientation of melittin monomers of identical primary structure as they aggregate to form channels in the lipid bilayer.

Melittin lysis of red cells.

This paper describes experiments designed to explore interactions between human red blood cell membranes and melittin, the main component of bee venom. We found that melittin binds to human red cell membranes suspended in isotonic NaCl at room temperature, with an apparent dissociation constant of 3 X 10(-8) M and maximum binding capacity of 1.8 X 10(7) molecules/cell. When about 1% of the melittin binding sites are occupied, cell lysis can be observed, and progressive, further increases in the fraction of the total sites occupied lead to progressively greater lysis in a graded manner. 50% lysis occurs when there are about 2 X 10(6) molecules bound to the cell membrane. For any particular extent of melittin binding, lysis proceeds rapidly during the first few minutes but then slows and stops so that no further lysis occurs after one hour of exposure of cells to melittin. The graded lysis of erythrocytes by melittin is due to complete lysis of some of the cells, since both the density and the hemoglobin content of surviving, intact cells in a suspension that has undergone graded melittin lysis are similar to the values observed in the same cells prior to the addition of melittin. The cells surviving graded melittin lysis have an increased Na and reduced K, proportional to the extent of occupation of the melittin binding sites. Like lysis, Na accumulation and K loss proceed rapidly during the first few minutes of exposure to melittin but then stops so that Na, K and hemoglobin content of the cells remain constant after the first hour. These kinetic characteristics of both lysis and cation movements suggest that melittin modifies the permeability of the red cell membrane only for the first few minutes after the start of the interaction. Direct observation of cells by Nomarsky optics revealed that they crenate, become swollen and lyse within 10 to 30 sec after these changes in morphology are first seen. Taken together, these results are consistent with the idea that melittin produces lysis of human red cells at room temperature by a colloid osmotic mechanism.

Formation and properties of tetramers of band 3 protein from human erythrocyte membranes in planar lipid bilayers.

Lipid bilayer experiments were performed in the presence of solubilized band 3 protein from human red cell membranes. Band 3 protein increased the conductance of the lipid membranes by several orders of magnitude. Membrane conductance was found to be dependent on the fourth power of protein concentration. This shows that four band 3 subunits form an ion permeable pathway in the lipid bilayer membranes. It also shows that, in the membranes, the protein molecules undergo an association equilibrium which involves at least the monomer and the tetramer of the protein, relaxation towards equilibrium being rapid on the time scale of the experiment. The increase in bilayer conductance induced by the band 3 tetramer could be inhibited by pretreatment of the protein with several SH-reagents (pCMB, pCMBS, DTNB) which also inhibit water transport across the human red cell membrane. Other SH-reagents which do not influence water transport (iodoacetamide, N-ethylmaleimide) did not show any influence on the band 3 induced conductance increase. A band 3-mediated exchange of anions comparable to that in the erythrocyte membrane did not occur in the system studied by us. Our results suggest that, in the human erythrocyte membrane, a pore formed by the band 3 tetramer could be the pathway responsible for the protein-mediated part of water transport.