J. A. Ferragut

Association of daunomycin to membrane domains studied by fluorescence resonance energy transfer.

1,6-Diphenyl-1,3,5-hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene are fluorophores used to explore different hydrophobic domains of membrane bilayers (Andrich, M.P. and Vanderkooi, J.M. (1976) Biochemistry 15, 1257-1265; Prendergast, F.G., Haugland, R.P. and Callahan, P.J. (1981) Biochemistry 20, 7333-7338). Fluorescence resonance energy transfer between these fluorophores, acting as energy donors, and the anthracycline, daunomycin, as the acceptor, was used to analyze the interaction of the drug with natural membranes, and its relative location within the membrane bilayer. The transfer process was demonstrated by: (1) emission fluorescence of the acceptor when the samples were excited at the excitation maximum of the donor (360 nm); and (2) progressive quenching of the energy donor (at 428 nm) when in the presence of increasing acceptor concentration. Also, the disruption of the energy transfer by solubilization of the membrane with Triton X-100 evidences a role for the membrane in providing the appropriate site(s) for energy transfer to occur. At moderately low daunomycin/membrane lipid ratios, the different efficiencies of resonance energy transfer between the two donors and daunomycin predicts a preferential, but not exclusive, location of the drug at membrane surface domains, i.e., those regions of the bilayer explored by the 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene probe. In support of this observation, a large fraction (approx. 75%) of membrane-associated daunomycin was rapidly sequestered away from the membrane upon addition of excess DNA, which forms high-affinity complexes with daunomycin (Chaires, J.B., Dattagupta, n. and Crothers, D.M. (1982) Biochemistry 21, 3927-3932), thus acting as a drug sink. Also, a large fraction of drug was accessible to fluorescence quenching by iodide, a collisional water-soluble quencher. On the other hand, a smaller population of the membrane-associated daunomycin was characterized by slow sequestering by the added DNA and inaccessibility to quenching by iodide. We conclude that the daunomycin, which is only slowly sequestered, is located deep within the hydrophobic domains of the bilayer, likely to be those probed by 1,6-diphenyl-1,3,5-hexatriene.

Adoption of beta structure by the inactivating "ball" peptide of the Shaker B potassium channel

The conformation of the inactivating peptide of the Shaker B K+ channel (ShB peptide) and that of a noninactivating mutant (ShBL7E peptide) have been studied. Under all experimental conditions explored, the mutant peptide remains in a predominantly nonordered conformation. On the contrary, the inactivating ShB peptide has a great tendency to adopt a highly stable beta structure, particularly when challenged in vitro by anionic phospholipid vesicles. Because the putative peptide binding elements at the inner mouth of the channel comprise a ring of anionic residues and a hydrophobic pocket, we hypothesize that the conformational restrictions imposed on the ShB peptide by its interaction with the anionic lipid vesicles could partly imitate those imposed by the above ion channel elements. Thus, we propose that adoption of beta structure by the inactivating peptide may also occur during channel inactivation. Moreover, the difficulties encountered by the noninactivating ShBL7E peptide mutant to adopt beta structure and the observation that trypsin hydrolysis of the ShB peptide prevent both structure formation and channel inactivation lend further support to the hypothesis that adoption of beta structure by the inactivating peptide in a hydrophobic environment is important in determining channel blockade.

Protein orientation affects the efficiency of functional protein transplantation into the xenopus oocyte membrane.

Xenopus oocytes incorporate into their plasma membrane nicotinic acetylcholine receptors (nAChRs) after intracellular injection of lipid vesicles bearing this protein. The advantage of this approach over the classical oocyte expression system lies in the transplantation of native, fully processed proteins, although the efficiency of functional incorporation of nAChRs is low. We have now studied the incorporation into the oocyte membrane of the Torpedo chloride channel (ClC-0), a minor contaminant protein in some nAChR preparations. nAChR-injected oocytes incorporated functional ClC-0: i) in a higher number than functional nAChRs; ii) retaining their original properties; and iii) with a right-side-out orientation in the oocyte membrane. In an attempt to elucidate the reasons for the low efficiency in the functional incorporation of nAChRs into the oocyte membrane, we combined electrophysiological and [125I]alpha-bungarotoxin-binding experiments. Up to 3% of injected nAChRs were present in the oocyte plasma membrane at a given time. Thus, fusion of lipoproteosome vesicles to the oocyte plasma membrane is not the limiting factor for an efficient functional transplantation of foreign proteins. Accounting for the low rate of functional transplantation of nAChRs is their backward orientation in the oocyte membrane, since about 80% of them adopted an out-side-in orientation. Other factors, including differences in the susceptibility of the transplanted proteins to intracellular damage should also be considered.

Thermal stability of hepatitis B surface antigen S proteins

Thermal stability of hepatitis B surface antigen (HBsAg) has been studied by analyzing alterations in the native secondary structure and the antigenic activity. After heating for 19 h, circular dichrosim showed a cooperative transition with a midpoint at 49 degrees C. The conformational changes induced by temperature reduced the helical content of HBsAg S proteins from 49% at 23 degrees C to 26% at 60 degrees C and abolished the antigenic activity, as measured by binding to polyclonal antibodies. Furthermore, the six different antigenic determinants recognized by our panel of monoclonal antibodies were also shown to be dependent on the native structure of HBsAg proteins. Hence, it can be inferred that these epitopes are conformation-dependent. Binding of monoclonal antibodies to HBsAg protected the native structure of the corresponding antigenic determinant from thermal denaturation. In fact, binding of one of the monoclonals tested resulted not only in protection of the corresponding epitope, but also in a consistent increase of antibody binding with increasing temperature. Such an increase in antibody binding occurred simultaneously with an increase in the fluidity of surface lipid regions, as monitored by fluorescence depolarization of 1-(trimethylammoniophenyl)-6-phenyl-1,3,5-hexatriene. This correlation, along with the observation that lipids play an important role in maintaining the structure and antigenic activity of HBsAg (Gavilanes et al. (1990) Biochem. J. 265, 857-864), allow to speculate the certain epitopes of HBsAg which are close to the lipid-protein interface, are dependent on the fluidity of the surface lipid regions. Thus, any change in the physical state of the lipids could confer a different degree of exposure to the antigenic determinants.

Interaction between ion channel-inactivating peptides and anionic phospholipid vesicles as model targets

Studies of rapid (N-type) inactivation induced by different synthetic inactivating peptides in several voltage-dependent cation channels have concluded that the channel inactivation entrance (or receptor site for the inactivating peptide) consists of a hydrophobic vestibule within the internal mouth of the channel, separated from the cytoplasm by a region with a negative surface potential. These protein domains are conformed from alternative sequences in the different channels and thus are relatively unrestricted in terms of primary structure. We are reporting here on the interaction between the inactivating peptide of the Shaker B K+ channel (ShB peptide) or the noninactivating ShB-L7E mutant with anionic phospholipid vesicles, a model target that, as the channels inactivation entrance, contains a hydrophobic domain (the vesicle bilayer) separated from the aqueous media by a negatively charged vesicle surface. When challenged by the anionic phospholipid vesicles, the inactivating ShB peptide 1) binds to the vesicle surface with a relatively high affinity, 2) readily adopts a strongly hydrogen-bonded beta-structure, likely an intramolecular beta hairpin, and 3) becomes inserted into the hydrophobic bilayer by its folded N-terminal portion, leaving its positively charged C-terminal end exposed to the extravesicular aqueous medium. Similar experiments carried out with the noninactivating, L7E-ShB mutant peptide show that this peptide 1) binds also to the anionic vesicles, although with a lower affinity than does the ShB peptide, 2) adopts only occasionally the characteristic beta-structure, and 3) has completely lost the ability to traverse the anionic interphase at the vesicle surface and to insert into the hydrophobic vesicle bilayer. Because the negatively charged surface and the hydrophobic domains in the model target may partly imitate those conformed at the inactivation entrance of the channel proteins, we propose that channel inactivation likely includes molecular events similar to those observed in the interaction of the ShB peptide with the phospholipid vesicles, i.e., binding of the peptide to the region of negative surface potential, folding of the bound peptide as a beta-structure, and its insertion into the channels hydrophobic vestibule. Likewise, we relate the lack of channel inactivation seen with the mutant ShB-L7E peptide to the lack of ability shown by this peptide to cross through the anionic interphase and insert into the hydrophobic domains of the model vesicle target.

The Surface Charge of Membranes Modulates the Interaction with the Anthracycline Daunomycin a

The cytotoxic effects observed for nonpenetrating, polymerimmobilized anthracycline strongly suggest that the interaction of the drugs with the plasma membrane of tumor cells may be important in the molecular mechanisms of drug cytotoxicity. In the present study, we have used positively (PCL) and negatively (NCL) charged liposomes (Avanti Polar Lipids) as model systems to determine how the surface charge of the membrane influences its interaction with ionized drugs such as daunomycin (DNM), an anthracycline antibiotic that contains an ionizable amino group (pK = 7.6-8.2) at the daunosamine sugar moiety. Equilibrium binding of DNM to liposomes was carried out by using ultracentrifugation and fluorescence anisotropy techniques to distinguish between free and liposome-bound drug (FIG. 1). pH values ranging from 6 to 8.3 were chosen for the studies to produce different ionization states of the drug without changing the net charge of the stearylamine and the dicetyl phosphate groups present in the PCL and NCL, respectively. Binding parameters were obtained from Klotz plots, which gave straight lines with a slope equal to 1 / n , where n is the maximum number of binding sites per phospholipid molecule, and a y-intercept of l / ( n . Kapp), where Kapp is the apparent binding constant and ( n Kapp) equals the overall binding constant, K,. FIGURE 1 shows direct binding data corresponding to the interaction between DNM and NCL (A) or PCL (B) at different pH. It should be noted that the abscissa in B needs to include much higher liposome concentrations (in terms of phospholipid contents) to begin to evidence saturation. The results clearly suggest that the interaction between the drug and the vesicles is mainly governed by the presence of negative charges at the liposome surface, while the ionization state of the drug, as evidenced from the results obtained at different pH, is less important in altering drug binding. Nonetheless, drug-binding parameters, as determined from Klotz plots, were sufficiently sensitive to the pH as to discriminate between binding of neutral and cationic species of the drug (TABLE 1). For either NCL or PCL, high pH values favoring the presence of un-ionized drug species result in larger K, values due to an increased binding stoichiometry. Furthermore, fluorescence resonance energy transfer studies similar to those reported previously indicate that the

Effects of pH on the kinetics of the interaction between anthracyclines and lipid bilayers

Abstract In the present study we have analyzed the kinetics of the initial steps (first 10 seconds) of the interaction between the anthracycline daunomycin (DNM) and artificial lipid vesicles bearing opposite surface charge. The process can be monitored by measuring the fluorescence increase of the drug accompanying its association with the lipid bilayers. The results indicated that DNM consistently interacts to a larger extent with the liposomes having negative surface charge than with those having positive surface charge, suggesting the involvement of electrostatic components in the interaction. In contrast, DNM associates with the vesicles bearing positive surface charge 2 – 3 times faster (in terms of the apparent rate constants describing the process of interaction) than with those having negative surface charge, an observation probably related to the more fluid physical state of the former. Regarding the rate of access of DNM to the vesicles, rather than depending on the surface charge of the vesicles, this is critically affected by the ionization state of the drug, i. e. by the pH. Thus, the rate at which the interaction proceeds is increased nearly 15-fold when the pH of the medium increases from 7.0 to 8.3, regardless of the surface charge of the liposomes. On this basis, and taking into account the fact that the anthracyclines enter the cells by passive diffusion, possible effects of pH on the transport of these drugs through the membranes of tumor cells are discussed.

Interaction of the Nicotinic Acetylcholine Receptor with Ligands and Membrane Lipids Studied by Fourier-Transform Infrared Spectroscopy and Photoaffinity Labeling

Monitoring of the amide I band by Fourier-transform infrared spectroscopy (FT-IR) is a valid and flexible approach to monitor changes in the secondary structure of reconstituted Acetylcholine Receptor (AcChR). The continuous exposure of the AcChR to a cholinergic agonist (carbamylcholine), which drives the AcChR into the desensitized state, produces only minor changes in AcChR secondary structure. Nevertheless, carbamylcholine alters the AcChR tertiary or quaternary structure, as indicated by the increased thermal stability of the protein assessed from the temperature-dependence of the infrared spectrum. On the contrary, presence of a competitive cholinergic antagonist (d-tubocurarine) produces no detectable effects on AcChR structure. Cholesterol or the neut-al lipids present in asolectin extracts produce an ordering of the AcChR secondary structure observed by FT-IR and also enable the protein to increase its thermal stability in response to carbamylcholine. These effects of cholesterol or asolectin neutral lipids seem mediated by a direct interaction of all the AcChR subunits with the lipids, as suggested by labeling of the AcChR by a photoactivatable cholesterol analogue. The interaction between the AcChR and the cholesterol analogue is sensitive to AcChR desensitization since the presence of carbamylcholine during photolysis decreases the extent of labeling and alters the labeling stoichiometry in the AcChR subunits. This suggests the occurrence of an agonist-induced change in the arrangement of the transmembrane portion of the desensitized protein, which is consistent with the agonist-induced alteration of the AcChR protein tertiary or quaternary structure detected by FT-IR.

Role of Membrane Components on Anthracycline Cytotoxicity

The plasma membrane of tumour cells is receiving increasing attention in regard to cellular multidrug resistance. We have studied plasma membranes from wild P388 murine leukemia cells and from stable multidrug resistant sublines with primary resistance to daunomycin, which do not express P-glycoproteins. The results obtained with the isolated plasma membranes suggest that there is a role for certain lipids, namely phospatidylserine and cholesterol, whose relative abundance is different in membranes from drug-sensitive or -resistant cells, in determining (i) the extent of daunomycin binding to the membrane and (ii) the location of daunomycin within the membrane bilayer. Furthermore, calorimetric studies on the interaction between model lipid vesicles with daunomycin and/or verapamil, the best known resistance-reverting agent, indicate that verapamil prevents, in a concentration-dependent manner, the alterations in the phospholipid phase transition expected from the presence of daunomycin in the bilayer.