We present a straightforward, accessible method for the fabrication of micropores with diameters from 2 to 800 micro m in films of amorphous Teflon (Teflon AF). Pores with diameters

Melittin, a toxin of bee venom, is a cationic polypeptide composed of 26 amino acids. The six residues of the C-terminal end are polar and 19 of the 20 residues of the N-terminal end are hydrophobic. Exposure of the lecithin bilayer to melittin results in the formation of channels that are more permeable to anions that to cations. Unilateral addition of melittin produces a voltage-dependent increase in membrane conductance when the side where the polypeptide is present in made positive but not when it is made negative. At a fixed voltage, the conductance increases with the fourth power of the melittin concentration in the aqueous phase. At a fixed peptide concentration, the conductance increases approximately e-fold per 6-mV increase in the electrical potential difference across the membrane. These results suggest that four melittin monomers are needed to form a channel and, furthermore, that a minimum of four equivalent electronic charges need to be displaced by the electrical field to explain the voltage dependence of the conductance.

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.

During entry into host cells, poliovirus undergoes a receptor-mediated conformational transition to form 135S particles with irreversible exposure of VP4 capsid sequences and VP1 N termini. To understand the role of VP4 during virus entry, the fate of VP4 during infection by site-specific mutants at threonine-28 of VP4 (4028T) was compared with that of the parental Mahoney type 1 virus. Three virus mutants were studied: the entry-defective, nonviable mutant 4028T.G and the viable mutants 4028T.S and 4028T.V, in which residue threonine-28 was changed to glycine, serine, and valine, respectively. We show that mutant and wild-type (WT) VP4 proteins are localized to cellular membranes after the 135S conformational transition. Both WT and viable 4028T mutant particles interact with lipid bilayers to form ion channels, whereas the entry-defective 4028T.G particles do not. In addition, the electrical properties of the channels induced by the mutant viruses are different from each other and from those of WT Mahoney and Sabin type 3 viruses. Finally, uncoating and/or cytoplasmic delivery of the viral genome is altered in the 4028T mutants: the 4028T.G lethal mutant does not release its genome into the cytoplasm, and genome delivery is slower during infection by mutant 4028T.V 135S particles than by mutant 4028T.S or WT 135S particles. The distinctive electrical characteristics of the different 4028T mutant channels indicate that VP4 sequences might form part of the channel structure. The different entry phenotypes of these VP4 mutants suggest that the ion channels may be related to VP4's role during genome uncoating and/or delivery.Poliovirus, a member of the Picornaviridae family, encapsidates its 7,400-nucleotide positive-sensed RNA genome within an icosahedrally symmetric protein shell that is formed by 60 copies of the four capsid proteins (VP1 to VP4). VP1, VP2, and VP3 form the surface of the virion, with VP1 located at each fivefold axis and VP2 and VP3 alternately positioned around each threefold axis. VP4 in its entirety as well as the amino termini of VP1, VP2, and VP3 are buried within the interior of the capsid, lying along the inner surface of the virion shell (12).Poliovirus entry into cells is initiated by binding to the poliovirus receptor (PVR) on the cell surface. PVR binding induces a conformational transition within the virus particle that leads to formation of altered particles (termed A particles) sedimenting at 135S (versus the 160S sedimentation value of the native particle) (references 8 and 23 and references therein). This conformational transition results in relocation of VP4 and the VP1 N termini from the particle interior to the virion exterior. The appearance of VP4 and VP1 domains on the particle surface is correlated with dramatic differences in the functional behavior of the native 160S and altered 135S particles. Functionally, these PVR-induced conformational rearrangements generate 135S particles that acquire the ability to bind to liposomes, to form ion channels in lipid bilayers, and ...

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.

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 ofanion-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 partides mediate the interaction between virus and the Upid bilayer of a cell membrane during penetration. In addition, the findings indicate that the "penetration proteins" ofsome enveloped and nonenveloped viruses share similarities in the way they interact with bilayers.

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 phosphatidylethanolamine 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.We have shown previously that certain L-amino acid peptides, which are long enough to traverse the membrane only once, can self-aggregate to form ion-permeable channels in bilayers (1-3). We have suggested that the capacity of peptides to assume amphipathic conformations allows them to self-aggregate with hydrophilic residues facing inward, to form the walls of the channel, and hydrophobic side chains facing outward, to interact with the hydrocarbon chains of the phospholipid molecules in the interior of the bilayer. Similar suggestions have been made about the probable conformation of putative intramembrane segments of channel-forming integral membrane proteins (4)(5)(6). Based on results of our work with melittin and its analogues, we have put forth the hypothesis that the voltage dependence of channels formed by peptides depends on the presence of positive charges on the side chains of some of the amino acid residues. Thus, when the primary sequence of the Nachannel protein was determined, our attention was drawn to the S4 segment, which is highly positively charged and potentially amphipathic. S4 is one of six segments (S1-S6) present in each of the four homologous domains of the Nachannel polypeptide. Moreover, it has been suggested that this segment is somehow involved in the voltage gating of the Na channel (4,7,8). We decided to synthesize this segment, which has the following structure: Arg-Val-Ile-Arg-Leu-AlaArg-Ile-Ala-Arg-Val-Leu-Arg-Leu-Ile-Arg-Ala-Ala-LysGly-Ile-Arg. Noticeable is the fact that this segment is highly polar; it contains seven arginine and one lysine residues, each located at every third position, and the intervening residues are nonpolar. Assuming that this segment forms a 310-helical structure, these charged residues would lie on a straight line parallel with the long axis on one side of the helix, making the segment strongly amphipathic. Moreover, since the distance per residue along the axis ofthe helix would be about 0.15 nm, the 22-amino acid segment would be -3.3 nm long, which is sufficient to traverse the hydrocarbon core of lipid bilayers. In what follows, we show that the peptide incorporates into lipid bilayers and forms cation-selective channels, which have voltage-dependent characteristics.MATERIALS AND METHODS Materials. Phosphatidylserine...

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.