PB1-F2, an influenza A virus-encoded proapoptotic mitochondrial protein, creates variably sized pores in planar lipid membranes.

A frameshifted region of the influenza A virus PB1 gene encodes a novel protein, termed PB1-F2, a mitochondrial protein that can induce cell death. Many proapoptotic proteins are believed to act at the mitochondrial outer membrane to form an apoptotic pore with lipids. We studied the interaction of isolated, synthetic PB1-F2 (sPB1-F2) peptide with planar phospholipid bilayer membranes. The presence of nanomolar concentrations of peptide in the bathing solution induced a transmembrane conductance that increased in a potential-dependent manner. Positive potential on the side of protein addition resulted in a severalfold increase in the rate of change of membrane conductance. sPB1-F2-treated membranes became permeable to monovalent cations, chloride, and to a lesser extent, divalent ions. Despite various experimental conditions, we did not detect the distinctive conductance levels typical of large, stable pores, protein channels, or even pores that are partially proteinaceous. Rather, membrane conductance induced by sPB1-F2 fluctuated and visited almost all conductance values. sPB1-F2 also dramatically decreased bilayer stability in an electric field, consistent with a decrease in the line tension of a lipidic pore. Since similar membrane-destabilizing profiles are seen with proapoptotic proteins (e.g., Bax) and the cytoplasmic helix of human immunodeficiency virus gp41, we suggest that the basis for sPB1-F2-induced cell death may be the permeabilization and destabilization of mitochondrial membranes, leading to macromolecular leakage and apoptosis.

Fast two dimensional computer simulation of bilayer hemifusion.

Computer simulation of model membranes was used to evaluate the possible mechanism of lipid bilayer fusion. The simplified two dimensional model of the membrane cross section was used as an analog of three dimensional reality. Lipid molecules were represented by rod-like structures, and forces between them were limited to attraction/repulsion interactions described by a simple energy function with a minimum; 300-400 molecules were modeled in every simulation. Using the energy minimization procedure, it was possible to obtain stable linear or circular bilayer structures (two dimensional analogs of planar membranes and liposomes). In response to changes in attraction/repulsion equilibrium between molecules these bilayers were able to reorganize via cooperative process. By increasing the headgroup attraction parameter for contacting monolayers, it was possible to induce formation of a zone of hemifusion in the area of bilayer contact. The possible correlation between cooperative bilayer rearrangement in the model and in real bilayers is discussed.

Probing the structure-function relationship of alpha-latrotoxin-formed channels with antibodies and pronase.

The major toxic component of black widow spider (Latrodectus mactans tredecimguttatus) venom, alpha-latrotoxin, is known to form ionic channels in different membranes. In order to probe the extramembrane domains of alpha-latrotoxin molecule, alpha-latrotoxin channels in planar lipid membrane were treated with antibodies to latrotoxin or with pronase added to different sides of the membrane. It was found that antibody addition to the same side as the toxin (cis) decreased channel conductance only at positive potentials across the membrane. In contrast, trans side addition of antibodies changed the channel conductance at both positive and negative potentials: at positive potential conductance first slightly increased then decreased by more then 50%; at negative potential it decreased much more quickly, to only about 20% of the initial value. No dependence on membrane potential was found for pronase treatment of incorporated channels. For both cis and trans application of pronase, channel selectivity for Ca2+, Mg2+, Ba2+ and K+, Na+, Li+ ions did not change significantly but Cd2+ block was decreased. Trans pronase treatment also resulted in some rectification of I/V curves and an increase in channel conductance. We interpret these findings as evidence that alpha-latrotoxin channel has protruding parts on both sides of the membrane and that its conformation in the membrane depends on membrane potential.

Filter switching device for dual-wavelength videoimaging.

An inexpensive, dual-wavelength, videoimaging system that can be used for parallel observation of two fluorescent dyes is described. All four filters, two for excitation and two for emission, are placed on the same oscillating holder. Filters are coupled with a single dichroic mirror having two spectral windows. A coil driven by an electronic circuit connected to photosensors, which determine the position of the holder, moves the magnet that shifts the position of the filters. Since the filter holder is placed between two springs, it oscillates with the frequency of mechanical resonance. As a result the filter switching did not require much power and did not produce significant vibrations of the base. Switching frequencies up to 4.5 s(-1) were reached with the first experimental device. System performance was tested using phospholipid vesicles loaded with water-soluble and membrane dyes. It has been demonstrated that the device can be used successfully in experiments on membrane fusion with rhodamine- and calcein-labeled liposomes.

An amphipathic peptide from the C-terminal region of the human immunodeficiency virus envelope glycoprotein causes pore formation in membranes.

The peptide fragment of the carboxy-terminal region of the human immunodeficiency virus (HIV) transmembrane protein (gp41) has been implicated in T-cell death. This positively charged, amphipathic helix (amino acids 828 to 848) of the envelope protein is located within virions or cytoplasm. We studied the interaction of the isolated, synthetic amphipathic helix of gp41 with planar phospholipid bilayer membranes and with Sf9 cells using voltage clamp, potentiodynamic, and single-cell recording techniques. We found that the peptide binds strongly to planar membranes, especially to the negatively charged phosphatidylserine bilayer. In the presence of micromolar concentrations of peptide sufficient to make its surface densities comparable with those of envelope glycoprotein molecules in HIV virions, an increase in bilayer conductance and a decrease in bilayer stability were observed, showing pore formation in the planar lipid bilayers. These pores were permeable to both monovalent and divalent cations, as well as to chloride. The exposure of the inner leaflet of cell membranes to even 25 nM peptide increased membrane conductance. We suggest that the carboxy-terminal fragment of the HIV type 1 envelope protein may interact with the cell membrane of infected T cells to create lipidic pores which increase membrane permeability, leading to sodium and calcium flux into cells, osmotic swelling, and T-cell necrosis or apoptosis.

Correlations between changes in membrane capacitance induced by changes in ionic environment and the conductance of channels incorporated into bilayer lipid membranes.

The action of metal polycations and pH on ionic channels produced in bilayer lipid membranes (BLM) by three different toxins was studied by measuring membrane capacitance and channel conductance. Here, we show that critical concentrations of Cd2+, La3+ or Tb3+ induce complex changes in membrane capacitance. The time course of capacitance changes is similar to the time course of channel blocking by these ions at low concentration. No changes in BLM capacitance or conductance were observed in the range of pH 5.8-9.0. A pH shift from 7.4 to 3-4 or 11-12 induced large changes in BLM capacitance and channel conductance. For all studied channel-forming proteins, the initial capacitance increase preceded the conductance decrease caused by addition of polycations or by a change in pH. A close relationship between membrane lipid packing and ion channel protein is suggested.

The ion-conducting properties of Ca2+ ATPase in rabbit skeletal muscle sarcoplasmic reticulum.

Ca2+ ATPase was isolated from rabbit skeletal muscle sarcoplasmic reticulum and used to form structures resembling potential-dependent calcium channels within the membrane lipid bilayer of liposomes. The orientation of these structures in the bilayer was dependent on the conditions used for enzyme incorporation. The results obtained indicate that Ca2+ ATPase may be involved in the passive transport of calcium ions from the sarcoplasmic reticulum which may be regulated by the membrane potential. The membrane potential within the reticulum is probably positive at the moment of calcium ion release.

Detection of transient capacitance increase associated with channel formation in lipid bilayers.

The insertion of alpha- and beta-latrotoxins and sea anemone (Radianthus macrodactilus) toxin into bilayer lipid membranes (BLMs) was investigated using the method of simultaneous conductance/capacitance measurement. All the toxins investigated induced capacitance changes which preceded toxin-induced conductance increases. The processes that may underlie the observed effect are discussed.

Channels produced by spider venoms in bilayer lipid membrane: mechanisms of ion transport and toxic action.

The selectivity of ion channels produced by latrotoxin obtained from a black widow spider venom and by venom from the spider Steatoda paykulliana in bilayer phospholipid membrane was studied. Experimental current-voltage curves of these channels were used for the estimation of parameters of a two barrier model of their energy profiles. Selectivities of both types of channels are similar. Alkaline earth cations are permeable, the permeability increasing in the order Mg2+ less than Ca2+ less than Sr2+ less than Ba2+. In contrast transition metal cations block the channel, their efficiency decreases in the order: Cd2+ greater than or equal to Ni2+ greater than Zn2+ greater than Co2+ greater than Mn2+ (Steatoda paykulliana spider venom) and Cd2+ greater than Co2+ greater than Ni2+ greater than Zn2+ greater than Mn2+ (latrotoxin). Amplitudes of current carried by corresponding ions are mainly determined by the depth of the potential well for this ion, i.e., by its affinity to the cation binding site in the channel. The channels are also permeable to monovalent cations but they do not bind them. Selectivity for monovalent cations depends on Ca2+ concentration at the cis-side of membrane in the micromolar range. However, the addition of Ca2+ to the trans-side up to 10 mM does not affect currents carried by monovalent ions. It is suggested that venom-induced calcium channels have two conformational states with different selectivities which interconvert upon binding one calcium ion. Possible general schemes for the organisation of calcium channels in excitable membranes are also discussed. Finally, using a mathematical model of synaptic transmission, possible mechanisms of toxic action of spider venoms are considered.