Evidence for bidirectional transverse diffusion of spin-labeled phospholipids in the plasma membrane of guinea pig blood cells

The distribution and transverse diffusion kinetics of four spin-labeled phospholipid analogues (two with choline heads: phosphatidylcholine (PC) and sphingomyelin (SM); two with amino heads: phosphatidylserine (PS) and phosphatidylethanolamine (PE) were studied in the plasma membrane of guinea pig blood cells: erythrocytes, reticulocytes, and leukemic lymphocytes. Nitroxide reduction by the internal content of the cells was used as an indicator to determine the phospholipids that penetrated the cells. The reduction rates were in the order, PS greater than PE greater than PC greater than SM in all cells. Reoxidation of phospholipids extracted by serum albumin revealed the distribution of the phospholipids at a given time. In all cells, the distribution equilibrium was reached in less than 2 h and the amounts left in the external leaflet were in the following proportional order: PS less than PE less than PC less than SM. In the erythrocytes and especially in the reticulocytes, the shape change induced by adding phospholipids relaxed partially or completely at a lower speed but kept the same proportional order as at equilibrium. All the results were analyzed quantitatively with a simple kinetic model including the rates of transverse diffusion (flip and flop), the exchange between plasma membrane and internal membranes, and the reduction rate of free radicals (determined in either the internal or external membrane leaflet). The calculated rate constants of transverse diffusion varied from 2 x 10(-3) to 1.2 x 10(-1) min-1 for the flip and from 4 x 10(-3) to 1.2 x 10(-1) for the flop, depending on the polar head and the cell type. Possible interpretations of the external phospholipid reduction mechanism and cell deformation are discussed.     

AMPA receptor subunits expressed by single Purkinje cells.

Several subunits of the glutamate receptor of the AMPA subtype have been cloned recently. These subunits, named GluR1, GluR2, GluR3, and GluR4, exist as two splicing variants (flip and flop). We have determined the subset of AMPA receptor subunits expressed by single cerebellar Purkinje cells in culture. This was achieved by combining whole-cell patch-clamp recordings and a molecular analysis, based on the polymerase chain reaction, of the messenger RNAs harvested into the patch pipette at the end of each recording. We found that each single cell expresses the messenger RNAs encoding the following five subunits: the flip and flop versions of GluR1 and GluR2 as well as GluR3flip, GluR2 being the most abundant. In addition, GluR3flop and GluR4flip were scarcely expressed in half of these neurons, and GluR4flop was never detected.     

ERG6 and PDR5 regulate small lipophilic drug accumulation in yeast cells via distinct mechanisms

Diagnosis and circumvention of multi-drug resistance requires an understanding of the underlying cellular mechanisms. In the model organism Saccharomyces cerevisiae, deletions of PDR5 or ERG6 increase sensitivity to many small lipophilic drugs. Pdr5p is a plasma membrane ATP-binding cassette transporter that actively exports drugs, thereby lowering their intracellular levels. The mechanism by which ERG6, an enzyme in sterol biosynthesis, affects drug accumulation is less clear. We show here that ERG6 limits the rate of passive drug diffusion across the membrane, without affecting Pdr5p-mediated drug export. Consistent with their action by distinct mechanisms, PDR5 and ERG6 effects on drug accumulation are additive.     

ASF/SF2 and SC35 regulate the glutamate receptor subunit 2 alternative flip/flop splicing

The properties of the glutamate receptor subunits 1-4 (GluR1-4) are influenced by the alternative splicing of two homologous and mutually exclusive exons flip and flop. The flip form is most abundant during early development, while the flop form is dominant in adults. From transfections with a GluR2 mini-gene we show that flip is the preferred splice form in all tested cell lines, but coexpression of the SR-proteins ASF/SF2 and SC35 increases the flop to flip splice ratio. The increased flop incorporation depends on ASF/SF2- and SC35-dependent enhancer elements located in the flop exon, which stimulate the splicing between the flop exon and the preceding exon 13.     

Lipid‐Bilayer Permeation of Drug‐Like Compounds

Lipid‐bilayer permeation is determinant for the disposition of xenobiotics in the body. It controls the pharmacokinetic behavior of drugs and is, in many cases, a prerequisite for intracellular targeting. Permeation of in vivo barriers is in general predicted from lipophilicity and related parameters. This article goes beyond the empirical correlations, and elucidates the processes and their interplay determining bilayer permeation. A flip‐flop model for bilayer permeation, which considers the partitioning rate constants beside the translocation rate constants, is compared with the diffusion model based on Fick's first law. According to the flip‐flop model, the ratios of aqueous volumes to barrier area can determine whether partitioning or translocation is rate‐limiting. The flip‐flop model allows permeation of anions and cations, and expands our understanding of pH‐dependent permeation kinetics. Some experimental evidences for ion‐controlled permeation at pH 7 are also included in this work.     

Stable Expression of Recombinant AMPA Receptor Subunits: Binding Affinities and Effects of Allosteric Modulators

Abstract: Homomeric AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors (GluRs) were stably expressed in kidney cells from cDNAs encoding GluR1 flop, GluR2 flip, GluR2 flop, and GluR3 flop subunits. The recombinant receptors were of the expected size and showed functional properties in whole-cell recording as previously reported. [³H]AMPA binding to all subunits was increased to a similar extent by the chaotropic ion thiocyanate (SCN^?). Significant differences were found in the Scatchard plots, however, which were linear and of high affinity for GluR1 and -3 receptors (K_D values of 33 and 52 nM, respectively) but showed curvature for GluR2 receptors, indicating the presence of two components with distinct affinities. As with brain AMPA receptors, solubilization of GluR2 receptors reduced the number of lower-affinity sites and correspondingly increased the number of higher-affinity sites. The sulfhydryl reagent p-chloromercuriphenylsulfonic acid, which increases binding to brain receptors, produced only minor changes except in the case of GluR2 flip. These results indicate that GluR2, among the subunits examined here, most closely resembles the native AMPA receptors in brain membranes. [³H]AMPA binding was inhibited in a noncompetitive manner by two drugs that change the desensitization kinetics of the AMPA receptor. In agreement with physiological observations, the apparent affinity of cyclothiazide for GluR2 flip (EC₅₀ = 7 µM) was higher than that for receptors made of flop subunits (49–130 µM). In contrast, BDP-37, a member of the benzamide family of drugs, exhibited a lower potency for GluR2 flip (58 µM) than for any of the flop isoforms (18–40 µM). These results predict that the action of centrally active AMPA-receptor modulators varies across brain regions depending on their flip/flop composition.     

Membrane-spanning peptides induce phospholipid flop: a model for phospholipid translocation across the inner membrane of E. coli

The mechanism by which phospholipids translocate (flop) across the E. coli inner membrane remains to be elucidated. We tested the hypothesis that the membrane-spanning domains of proteins catalyze phospholipid flop by their mere presence in the membrane. As a model, peptides mimicking the transmembrane stretches of proteins, with the amino acid sequence GXXL(AL)(n)XXA (with X = K, H, or W and n = 8 or 12), were incorporated in large unilamellar vesicles composed of E. coli phospholipids. Phospholipid flop was measured by assaying the increase in accessibility to dithionite of a 2,6-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminocaproyl (C(6)NBD)-labeled phospholipid analogue, initially exclusively present in the inner leaflet of the vesicle membrane. Fast flop of C(6)NBD-phosphatidylglycerol (C(6)NBD-PG) was observed in vesicles in which GKKL(AL)(12)KKA was incorporated, with the apparent first-order flop rate constant (K(flop)) linearly increasing with peptide:phospholipid molar ratios, reaching a translocation half-time of approximately 10 min at a 1:250 peptide:phospholipid molar ratio at 25 degrees C. The peptides of the series GXXL(AL)(8)XXA also induced flop of C(6)NBD-PG, supporting the hypothesis that transmembrane parts of proteins mediate phospholipid translocation. In this series, K(flop) decreased in the order X = K > H > W, indicating that peptide-lipid interactions in the interfacial region of the membrane modulate the efficiency of a peptide to cause flop. For the peptides tested, flop of C(6)NBD-phosphatidylethanolamine (C(6)NBD-PE) was substantially slower than that of C(6)NBD-PG. In vesicles without peptide, flop was negligible both for C(6)NBD-PG and for C(6)NBD-PE. A model for peptide-induced flop is proposed, which takes into account the observed peptide and lipid specificity.     

A model for the asymmetric lipid distribution in the human erythrocyte membrane

The asymmetric transverse distribution of phospholipids in the human erythrocyte membrane can be explained by differences between the rate constants of flip and flop motion of the lipids. A selective interaction between aminophospholipids and spectrin does not need to be assumed for creating and maintaining the asymmetric localization of these lipids. Shape transformation of red cells could be caused by alterations of the flip-flop rate constants leading to a change of the lipid distribution and, consequently, to a differential area expansion of the outer and inner membrane leaflet.     

Gramicidin-induced enhancement of transbilayer reorientation of lipids in the erythrocyte membrane

Incorporation of the channel-forming antibiotic gramicidin into the membrane of human erythrocytes highly (up to 30-fold) enhances rates of reorientation (flip) of lysophosphatidylcholine and palmitoylcarnitine to the inner membrane layer after their primary incorporation into the outer layer. Despite the high increase of flip rates by gramicidin, the asymmetric orientation of the inner membrane layer phospholipids phosphatidylethanolamine and phosphatidylserine is stable as demonstrated by the lack of accessibility of these lipids toward cleavage by exogenous phospholipase A2. On the other hand, gramicidin enhances the rate of cleavage of outer membrane layer phosphatidylcholine by phospholipase A2, which indicates changes in the packing of phosphatidylcholine following gramicidin binding. The increase of flip becomes detectable when about 10(5) copies of gramicidin per cell have been bound (gramicidin to membrane phospholipid ratio of 1:2000). This is a 1000-fold higher concentration than that required for an increase of K+ permeability mediated by the gramicidin channel. Acceleration of flip is thus not simply correlated with channel formation. The enhancement of flip is markedly dependent on structural details of gramicidin. Formylation of its four tryptophan residues abolishes the effect. Even at high concentrations of formylated gramicidin at which the extents of binding of native and of formylated gramicidin to the membrane are comparable, no flip acceleration is produced. Enhancement of flip by gramicidin occurs after a temperature-dependent lag phase. At 37 degrees C, flip rates begin to increase within a few minutes and at 25 degrees C, only after 3 h. This lag phase is most likely not due to limitations by the rate of binding of gramicidin to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane disorder and phospholipid scrambling in electropermeabilized and viable cells

Membrane electropermeabilization relies on the transient permeabilization of the plasma membrane of cells submitted to electric pulses. This method is widely used in cell biology and medicine due to its efficiency to transfer molecules while limiting loss of cell viability. However, very little is known about the consequences of membrane electropermeabilization at the molecular and cellular levels. Progress in the knowledge of the involved mechanisms is a biophysical challenge. As a transient loss of membrane cohesion is associated with membrane permeabilization, our main objective was to detect and visualize at the single-cell level the incidence of phospholipid scrambling and changes in membrane order. We performed studies using fluorescence microscopy with C6-NBD-PC and FM1-43 to monitor phospholipid scrambling and membrane order of mammalian cells. Millisecond permeabilizing pulses induced membrane disorganization by increasing the translocation of phosphatidylcholines according to an ATP-independent process. The pulses induced the formation of long-lived permeant structures that were present during membrane resealing, but were not associated with phosphatidylcholine internalization. These pulses resulted in a rapid phospholipid flip/flop within less than 1 s and were exclusively restricted to the regions of the permeabilized membrane. Under such electrical conditions, phosphatidylserine externalization was not detected. Moreover, this electrically-mediated membrane disorganization was not correlated with loss of cell viability. Our results could support the existence of direct interactions between the movement of membrane zwitterionic phospholipids and the electric field.     

PDR16-mediated azole resistance in Candida albicans

Many Candida albicans azole-resistant (AR) clinical isolates overexpress the CDR1 and CDR2 genes encoding homologous multidrug transporters of the ATP-binding cassette family. We show here that these strains also overexpress the PDR16 gene, the orthologue of Saccharomyces cerevisiae PDR16 encoding a phosphatidylinositol transfer protein of the Sec14p family. It has been reported that S. cerevisiae pdr16Delta mutants are hypersusceptible to azoles, suggesting that C. albicans PDR16 may contribute to azole resistance in these isolates. To address this question, we deleted both alleles of PDR16 in an AR clinical strain overexpressing the three genes, using the mycophenolic acid resistance flipper strategy. Our results show that the homozygous pdr16Delta/pdr16Delta mutant is approximately twofold less resistant to azoles than the parental strain whereas reintroducing a copy of PDR16 in the mutant restored azole resistance, demonstrating that this gene contributes to the AR phenotype of the cells. In addition, overexpression of PDR16 in azole-susceptible (AS) C. albicans and S. cerevisiae strains increased azole resistance by about twofold, indicating that an increased dosage of Pdr16p can confer low levels of azole resistance in the absence of additional molecular alterations. Taken together, these results demonstrate that PDR16 plays a role in C. albicans azole resistance.     

GluR3 flip and flop: differences in channel opening kinetics

Ample evidence from earlier studies of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, GluR3 included, suggests that alternative splicing not only enriches AMPA receptor diversity but also, more importantly, creates receptor variants that are functionally different. However, it is not known whether alternative splicing affects the receptor channel opening that occurs in the microsecond time domain. Using a laser-pulse photolysis technique combined with whole-cell recording, we characterized the channel opening rate process for two alternatively spliced variants of GluR3, i.e., GluR3flip and GluR3flop. We show that the alternative splicing that generates flip and flop variants of GluR3 receptors regulates the channel opening process by controlling the rate of channel closing but not the rate of channel opening or the glutamate binding affinity. Specifically, the flop variant closes its channel almost 4-fold faster than the flip variant. We therefore propose that the function of the flip-flop sequence module in the channel opening process of AMPA receptors is to stabilize the open channel conformation, presumably by its pivotal structural location. Furthermore, a comparison of the flip isoform among all AMPA receptor subunits, based on the magnitude of the channel opening rate constant, suggests that GluR3 is kinetically more similar to GluR2 and GluR4 than to GluR1.     

Arabidopsis PEN3/PDR8, an ATP binding cassette transporter, contributes to nonhost resistance to inappropriate pathogens that enter by direct penetration

Arabidopsis thaliana is a host to the powdery mildew Erysiphe cichoracearum and nonhost to Blumeria graminis f. sp hordei, the powdery mildew pathogenic on barley (Hordeum vulgare). Screening for Arabidopsis mutants deficient in resistance to barley powdery mildew identified PENETRATION3 (PEN3). pen3 plants permitted both increased invasion into epidermal cells and initiation of hyphae by B. g. hordei, suggesting that PEN3 contributes to defenses at the cell wall and intracellularly. pen3 mutants were compromised in resistance to the necrotroph Plectosphaerella cucumerina and to two additional inappropriate biotrophs, pea powdery mildew (Erysiphe pisi) and potato late blight (Phytophthora infestans). Unexpectedly, pen3 mutants were resistant to E. cichoracearum. This resistance was salicylic acid-dependent and correlated with chlorotic patches. Consistent with this observation, salicylic acid pathway genes were hyperinduced in pen3 relative to the wild type. The phenotypes conferred by pen3 result from the loss of function of PLEIOTROPIC DRUG RESISTANCE8 (PDR8), a highly expressed putative ATP binding cassette transporter. PEN3/PDR8 tagged with green fluorescent protein localized to the plasma membrane in uninfected cells. In infected leaves, the protein concentrated at infection sites. PEN3/PDR8 may be involved in exporting toxic materials to attempted invasion sites, and intracellular accumulation of these toxins in pen3 may secondarily activate the salicylic acid pathway.