The Bacillus subtilis Chemoreceptor McpC Senses Multiple Ligands Using Two Discrete Mechanisms

Bacillus subtilis can perform chemotaxis toward all 20 l-amino acids normally found in proteins. Loss of a single chemoreceptor, McpC, was previously found to reduce chemotaxis to 19 of these amino acids. In this study, we investigated the amino acid-sensing mechanism of McpC. We show that McpC alone can support chemotaxis to 17 of these amino acids to varying degrees. Eleven amino acids were found to directly bind the amino-terminal sensing domain of McpC in vitro. Sequence analysis indicates that the McpC sensing domain exhibits a dual Per-Arnt-Sim (PAS) domain structure. Using this structure as a guide, we were able to isolate mutants that suggest that four amino acids (arginine, glutamine, lysine, and methionine) are sensed by an indirect mechanism. We identified four candidate binding lipoproteins associated with amino acid transporters that may function in indirect sensing: ArtP, GlnH, MetQ, and YckB. ArtP was found to bind arginine and lysine; GlnH, glutamine; MetQ, methionine; and YckB, tryptophan. In addition, we found that ArtP, MetQ, and YckB bind the sensing domain of McpC, suggesting that the three participate in the indirect sensing of arginine, lysine, methionine, and possibly tryptophan as well. Taken together, these results further our understanding of amino acid chemotaxis in B. subtilis and gain insight into how a single chemoreceptor is able to sense many amino acids.     

Membrane Protein Rim21 Plays a Central Role in Sensing Ambient pH in Saccharomyces cerevisiae

External alkalization activates the Rim101 pathway in Saccharomyces cerevisiae. In this pathway, three integral membrane proteins, Rim21, Dfg16, and Rim9, are considered to be the components of the pH sensor machinery. However, how these proteins are involved in pH sensing is totally unknown. In this work, we investigated the localization, physical interaction, and interrelationship of Rim21, Dfg16, and Rim9. These proteins were found to form a complex and to localize to the plasma membrane in a patchy and mutually dependent manner. Their cellular level was also mutually dependent. In particular, the Rim21 level was significantly decreased in dfg16Δ and rim9Δ cells. Upon external alkalization, the proteins were internalized and degraded. We also demonstrate that the transient degradation of Rim21 completely suppressed the Rim101 pathway but that the degradation of Dfg16 or Rim9 did not. This finding strongly suggests that Rim21 is the pH sensor protein and that Dfg16 and Rim9 play auxiliary functions through maintaining the level of Rim21 and assisting in its plasma membrane localization. Even without external alkalization, the Rim101 pathway was activated in a Rim21-dependent manner by either protonophore treatment or depletion of phosphatidylserine in the inner leaflet of the plasma membrane, both of which caused plasma membrane depolarization like the external alkalization. Therefore, plasma membrane depolarization seems to be one of the key signals for the pH sensor molecule Rim21.     

Membrane Lipid Composition Regulates Tubulin Interaction with Mitochondrial Voltage-dependent Anion Channel

Elucidating molecular mechanisms by which lipids regulate protein function within biological membranes is critical for understanding the many cellular processes. Recently, we have found that dimeric αβ-tubulin, a subunit of microtubules, regulates mitochondrial respiration by blocking the voltage-dependent anion channel (VDAC) of mitochondrial outer membrane. Here, we show that the mechanism of VDAC blockage by tubulin involves tubulin interaction with the membrane as a critical step. The on-rate of the blockage varies up to 100-fold depending on the particular lipid composition used for bilayer formation in reconstitution experiments and increases with the increasing content of dioleoylphosphatidylethanolamine (DOPE) in dioleoylphosphatidylcholine (DOPC) bilayers. At physiologically low salt concentrations, the on-rate is decreased by the charged lipid. The off-rate of VDAC blockage by tubulin does not depend on the lipid composition. Using confocal fluorescence microscopy, we compared tubulin binding to the membranes of giant unilamellar vesicles (GUVs) made from DOPC and DOPC/DOPE mixtures. We found that detectable binding of the fluorescently labeled dimeric tubulin to GUV membranes requires the presence of DOPE. We propose that prior to the characteristic blockage of VDAC, tubulin first binds to the membrane in a lipid-dependent manner. We thus reveal a new potent regulatory role of the mitochondrial lipids in control of the mitochondrial outer membrane permeability and hence mitochondrial respiration through tuning VDAC sensitivity to blockage by tubulin. More generally, our findings give an example of the lipid-controlled protein-protein interaction where the choice of lipid species is able to change the equilibrium binding constant by orders of magnitude.     

Identification of a Chemoreceptor for Tricarboxylic Acid Cycle Intermediates: DIFFERENTIAL CHEMOTACTIC RESPONSE TOWARDS RECEPTOR LIGANDS*

We report the identification of McpS as the specific chemoreceptor for 6 tricarboxylic acid (TCA) cycle intermediates and butyrate in Pseudomonas putida. The analysis of the bacterial mutant deficient in mcpS and complementation assays demonstrate that McpS is the only chemoreceptor of TCA cycle intermediates in the strain under study. TCA cycle intermediates are abundantly present in root exudates, and taxis toward these compounds is proposed to facilitate the access to carbon sources. McpS has an unusually large ligand-binding domain (LBD) that is un-annotated in InterPro and is predicted to contain 6 helices. The ligand profile of McpS was determined by isothermal titration calorimetry of purified recombinant LBD (McpS-LBD). McpS recognizes TCA cycle intermediates but does not bind very close structural homologues and derivatives like maleate, aspartate, or tricarballylate. This implies that functional similarity of ligands, such as being part of the same pathway, and not structural similarity is the primary element, which has driven the evolution of receptor specificity. The magnitude of chemotactic responses toward these 7 chemoattractants, as determined by qualitative and quantitative chemotaxis assays, differed largely. Ligands that cause a strong chemotactic response (malate, succinate, and fumarate) were found by differential scanning calorimetry to increase significantly the midpoint of protein unfolding (T_m) and unfolding enthalpy (ΔH) of McpS-LBD. Equilibrium sedimentation studies show that malate, the chemoattractant that causes the strongest chemotactic response, stabilizes the dimeric state of McpS-LBD. In this respect clear parallels exist to the Tar receptor and other eukaryotic receptors, which are discussed.     

Myelin Membrane Structure and Composition Correlated: A Phylogenetic Study

We have correlated myelin membrane structure with biochemical composition in the CNS and PNS of a phylogenetic series of animals, including elasmobranchs, teleosts, amphibians, and mammals. X-ray diffraction patterns were recorded from freshly dissected, unfixed tissue and used to determine the thicknesses of the liquid bilayer and the widths of the spaces between membranes at their cytoplasmic and extracellular appositions. The lipid and protein compositions of myelinated tissue from selected animals were determined by TLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblotting, respectively. We found that (1) there were considerable differences in lipid (particularly glycolipid) composition, but no apparent phylogenetic trends; (2) the lipid composition did not seem to affect either the bilayer thickness, which was relatively constant, or the membrane separation; (3) the CNS of elasmobranch and teleost and the PNS of all four classes contained polypeptides that were recognized by antibodies against myelin P0 glycoprotein; (4) antibodies against proteolipid protein (PLP) were recognized only by amphibian and mammalian CNS; (5) wide extracellular spaces (ranging from 36 to 48 A) always correlated with the presence of P0-immunoreactive protein; (6) the narrowest extracellular spaces (approximately 31 A) were observed only in PLP-containing myelin; (7) the cytoplasmic space in PLP-containing myelin (approximately 31 A) averaged approximately 5 A less than that in P0-containing myelin; (8) even narrower cytoplasmic spaces (approximately 24 A) were measured when both P0 and 11-13-kilodalton basic protein were detected; (9) proteins immunoreactive to antibodies against myelin P2 basic protein were present in elasmobranch and teleost CNS and/or PNS, and in mammalian PNS, but not in amphibian tissues; and (10) among mammalian PNS myelins, the major difference in structure was a variation in membrane separation at the cytoplasmic apposition. These findings demonstrate which features of myelin structure have remained constant and which have become specifically altered as myelin composition changed during evolutionary development.     

The Influence of Natural Lipid Asymmetry upon the Conformation of a Membrane-inserted Protein (Perfringolysin O)

Eukaryotic membrane proteins generally reside in membrane bilayers that have lipid asymmetry. However, in vitro studies of the impact of lipids upon membrane proteins are generally carried out in model membrane vesicles that lack lipid asymmetry. Our recently developed method to prepare lipid vesicles with asymmetry similar to that in plasma membranes and with controlled amounts of cholesterol was used to investigate the influence of lipid composition and lipid asymmetry upon the conformational behavior of the pore-forming, cholesterol-dependent cytolysin perfringolysin O (PFO). PFO conformational behavior in asymmetric vesicles was found to be distinct both from that in symmetric vesicles with the same lipid composition as the asymmetric vesicles and from that in vesicles containing either only the inner leaflet lipids from the asymmetric vesicles or only the outer leaflet lipids from the asymmetric vesicles. The presence of phosphatidylcholine in the outer leaflet increased the cholesterol concentration required to induce PFO binding, whereas phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a novel deeply inserted conformation that does not form pores, even though it contains transmembrane segments. This conformation may represent an important intermediate stage in PFO pore formation. These studies show that lipid asymmetry can strongly influence the behavior of membrane-inserted proteins.     

Structure and Functional Analysis of LptC, a Conserved Membrane Protein Involved in the Lipopolysaccharide Export Pathway in Escherichia coli

LptC is a conserved bitopic inner membrane protein from Escherichia coli involved in the export of lipopolysaccharide from its site of synthesis in the cytoplasmic membrane to the outer membrane. LptC forms a complex with the ATP-binding cassette transporter, LptBFG, which is thought to facilitate the extraction of lipopolysaccharide from the inner membrane and release it into a translocation pathway that includes the putative periplasmic chaperone LptA. Cysteine modification experiments established that the catalytic domain of LptC is oriented toward the periplasm. The structure of the periplasmic domain is described at a resolution of 2.2-Å from x-ray crystallographic data. The periplasmic domain of LptC consists of a twisted boat structure with two β-sheets in apposition to each other. The β-sheets contain seven and eight antiparallel β-strands, respectively. This structure bears a high degree of resemblance to the crystal structure of LptA. Like LptA, LptC binds lipopolysaccharide in vitro. In vitro, LptA can displace lipopolysaccharide from LptC (but not vice versa), consistent with their locations and their proposed placement in a unidirectional export pathway.     

Interferon-induced Transmembrane Protein 3 Is a Type II Transmembrane Protein

The interferon-induced transmembrane (IFITM) proteins are a family of small membrane proteins that inhibit the cellular entry of several genera of viruses. These proteins had been predicted to adopt a two-pass, type III transmembrane topology with an intracellular loop, two transmembrane helices (TM1 and TM2), and extracellular N and C termini. Recent work, however, supports an intramembrane topology for the helices with cytosolic orientation of both termini. Here we determined the topology of murine Ifitm3. We found that the N terminus of Ifitm3 could be stained by antibodies at the cell surface but that this conformation was cell type-dependent and represented a minority of the total plasma membrane pool. In contrast, the C terminus was readily accessible to antibodies at the cell surface and extracellular C termini comprised most or all of those present at the plasma membrane. The addition of a C-terminal KDEL endoplasmic reticulum retention motif to Ifitm3 resulted in sequestration of Ifitm3 in the ER, demonstrating an ER-luminal orientation of the C terminus. C-terminal, but not N-terminal, epitope tags were also degraded within lysosomes, consistent with their luminal orientation. Furthermore, epitope-tagged Ifitm3 TM2 functioned as a signal anchor sequence when expressed in isolation. Collectively, our results demonstrate a type II transmembrane topology for Ifitm3 and will provide insight into its interaction with potential targets and cofactors.     

Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes

The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca²⁺-pump (PMCA) in the membrane region upon interaction with Ca²⁺, calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [¹²⁵I]TID-PC/16, measuring the shift of conformation E₂ to the auto-inhibited conformation E₁I and to the activated E₁A state, titrating the effect of Ca²⁺ under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca²⁺ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca²⁺ transport but does not modify the affinity for Ca²⁺ at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E₁I to the activated E₁A conformation and thus modulating the transport of Ca²⁺. This is reflected in the different apparent constants for Ca²⁺ in the absence of CaM (calculated by Ca²⁺-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca²⁺ site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca²⁺ and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca²⁺ binding and activation.     

Automating Single Subunit Counting of Membrane Proteins in Mammalian Cells

Elucidating subunit stoichiometry of neurotransmitter receptors is preferably carried out in a mammalian expression system where the rules of native protein assembly are strictly obeyed. Although successful in Xenopus oocytes, single subunit counting, manually counting photobleaching steps of GFP-tagged subunits, has been hindered in mammalian cells by high background fluorescence, poor control of expression, and low GFP maturation efficiency. Here, we present a fully automated single-molecule fluorescence counting method that separates tagged proteins on the plasma membrane from background fluorescence and contaminant proteins in the cytosol or the endoplasmic reticulum and determines the protein stoichiometry. Lower GFP maturation rates observed in cells cultured at 37 °C were partly offset using a monomeric version of superfolder GFP. We were able to correctly identify the stoichiometry of GluK2 and α1 glycine receptors. Our approach permits the elucidation of stoichiometry for a wide variety of plasma membrane proteins in mammalian cells with any commercially available TIRF microscope.     

Engineering a prokaryotic Cys-loop receptor with a third functional domain

Prokaryotic members of the Cys-loop receptor ligand-gated ion channel superfamily were recently identified. Previously, Cys-loop receptors were only known from multicellular organisms (metazoans). Contrary to the metazoan Cys-loop receptors, the prokaryotic ones consist of an extracellular (ECD) and a transmembrane domain (TMD), lacking the large intracellular domain (ICD) present in metazoa (between transmembrane segments M3 and M4). Using a chimera approach, we added the 115-amino acid ICD from mammalian serotonin type 3A receptors (5-HT(3A)) to the prokaryotic proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC). We created 12 GLIC-5-HT(3A)-ICD chimeras by replacing a variable number of amino acids in the short GLIC M3M4 linker with the entire 5-HT(3A)-ICD. Two-electrode voltage clamp recordings after expression in Xenopus laevis oocytes showed that only two chimeras were functional and produced currents upon acidification. The pH(50) was comparable with wild-type GLIC. 5-HT(3A) receptor expression can be inhibited by the chaperone protein RIC-3. We have shown previously that the 5-HT(3A)-ICD is required for the attenuation of 5-HT-induced currents when RIC-3 is co-expressed with 5-HT(3A) receptors in X. laevis oocytes. Expression of both functional 5-HT(3A) chimeras was inhibited by RIC-3 co-expression, indicating appropriate folding of the 5-HT(3A)-ICD in the chimeras. Our results indicate that the ICD can be considered a separate domain that can be removed from or added to the ECD and TMD while maintaining the overall structure and function of the ECD and TMD.     

Synip Arrests Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE)-dependent Membrane Fusion as a Selective Target Membrane SNARE-binding Inhibitor

The vesicle fusion reaction in regulated exocytosis requires the concerted action of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core fusion engine and a group of SNARE-binding regulatory factors. The regulatory mechanisms of vesicle fusion remain poorly understood in most exocytic pathways. Here, we reconstituted the SNARE-dependent vesicle fusion reaction of GLUT4 exocytosis in vitro using purified components. Using this defined fusion system, we discovered that the regulatory factor synip binds to GLUT4 exocytic SNAREs and inhibits the docking, lipid mixing, and content mixing of the fusion reaction. Synip arrests fusion by binding the target membrane SNARE (t-SNARE) complex and preventing the initiation of ternary SNARE complex assembly. Although synip also interacts with the syntaxin-4 monomer, it does not inhibit the pairing of syntaxin-4 with SNAP-23. Interestingly, synip selectively arrests the fusion reactions reconstituted with its cognate SNAREs, suggesting that the defined system recapitulates the biological functions of the vesicle fusion proteins. We further showed that the inhibitory function of synip is dominant over the stimulatory activity of Sec1/Munc18 proteins. Importantly, the inhibitory function of synip is distinct from how other fusion inhibitors arrest SNARE-dependent membrane fusion and therefore likely represents a novel regulatory mechanism of vesicle fusion.     

Characterization of the Blood-Brain Barrier: Protein Composition of the Capillary Endothelial Cell Membrane

Microvessels were isolated from canine cerebral cortex, and the composition of the endothelial cell membrane was investigated. Endothelial cell membranes were separated from the surrounding basement membrane, solubilized, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12% gels. Staining with Coomassie Blue revealed a characteristic banding pattern of at least 12 major proteins with apparent molecular weights between 14,000 and 250,000. When proteins from red blood cell ghosts were run simultaneously, no similarities were observed, except for proteins at apparent molecular weights of 43,000 (band 3) and 35,000 (band 4). These two proteins migrated exactly to the positions of the erythrocyte proteins actin and glyceraldehyde 3-phosphate dehydrogenase, respectively. Membrane glycoproteins in gels were also examined by the use of fluorescent lectins. Of the fluorescein isothiocyanate-conjugated (FITC) lectins tested, only FITC-concanavalin A had an affinity for any membrane components. Diazotized [125I]iodosulfanilic acid, a membrane-impermeable reagent, was used to label the internal (lumen) cell surface and the external (antilumen) cell surface. Autoradiography and determination of radioactivity levels in gel slices showed that several proteins were specifically labeled, and that major differences in radioactivity of proteins existed in internal and external labeling experiments. It is concluded that the protein composition of the luminal membrane is different from that of the antiluminal membrane.     

Dislocation by the m-AAA Protease Increases the Threshold Hydrophobicity for Retention of Transmembrane Helices in the Inner Membrane of Yeast Mitochondria

Sorting of mitochondrial inner membrane proteins is a complex process in which translocons and proteases function in a concerted way. Many inner membrane proteins insert into the membrane via the TIM23 translocon, and some are then further acted upon by the mitochondrial m-AAA protease, a molecular motor capable of dislocating proteins from the inner membrane. This raises the possibility that the threshold hydrophobicity for the retention of transmembrane segments in the inner membrane is different depending on whether they belong to membrane proteins that are m-AAA protease substrates or not. Here, using model transmembrane segments engineered into m-AAA protease-dependent proteins, we show that the threshold hydrophobicity for membrane retention measured in yeast cells in the absence of a functional m-AAA protease is markedly lower than that measured in its presence. Whether a given hydrophobic segment in a mitochondrial inner membrane protein will ultimately form a transmembrane helix may therefore depend on whether or not it will be exposed to the pulling force exerted by the m-AAA protease during biogenesis.     

Omp85 from the Thermophilic Cyanobacterium Thermosynechococcus elongatus Differs from Proteobacterial Omp85 in Structure and Domain Composition

Omp85 proteins are essential proteins located in the bacterial outer membrane. They are involved in outer membrane biogenesis and assist outer membrane protein insertion and folding by an unknown mechanism. Homologous proteins exist in eukaryotes, where they mediate outer membrane assembly in organelles of endosymbiotic origin, the mitochondria and chloroplasts. We set out to explore the homologous relationship between cyanobacteria and chloroplasts, studying the Omp85 protein from the thermophilic cyanobacterium Thermosynechococcus elongatus. Using state-of-the art sequence analysis and clustering methods, we show how this protein is more closely related to its chloroplast homologue Toc75 than to proteobacterial Omp85, a finding supported by single channel conductance measurements. We have solved the structure of the periplasmic part of the protein to 1.97 Å resolution, and we demonstrate that in contrast to Omp85 from Escherichia coli the protein has only three, not five, polypeptide transport-associated (POTRA) domains, which recognize substrates and generally interact with other proteins in bigger complexes. We model how these POTRA domains are attached to the outer membrane, based on the relationship of Omp85 to two-partner secretion system proteins, which we show and analyze. Finally, we discuss how Omp85 proteins with different numbers of POTRA domains evolved, and evolve to this day, to accomplish an increasing number of interactions with substrates and helper proteins.     

Differential Transmembrane Diffusion of Triiodothyronine and Thyroxine in Liposomes: Regulation by Lipid Composition

How thyroid hormones move across biological or model membranes is a subject of controversy. The passage of the 3,5,3′triiodo l-thyronine and 3,5,3′,5′ tetraiodo l-thyronine across model membranes was evaluated by the addition of the hormones to liposomes containing 2,4,6-trinitrobenzene sulfonic acid. Results indicate that hormones can react with an amino-reactive compound pre-encapsulated into phosphatidylcholine liposomes. The transversal motions of thyroid hormones were characterized by using physiological concentration levels of (¹²⁵I) 3,5,3′triiodo l-thyronine and (¹²⁵I) 3,5,3′,5′ tetraiodo l-thyronine. The hormone distribution between the two monolayers was time-dependent and kinetic data were fitted to a single exponential. Results obtained show that 3,5,3′ triiodo l-thyronine can permeate phospholipid membranes and the diffusion time increases in the gel and liquid-ordered phase. On the contrary, 3,5,3′, 5′ tetraiodo l-thyronine could not diffuse the liposomal membrane from dimyristoyl and dipalmitoyl phosphatidylcholine in gel phase and egg yolk phosphatidylcholine:cholesterol in the liquid-ordered phase. Our results in the liquid-ordered phase suggest that diffusion movement of thyroid hormones across cell membranes depends on the amount of cholesterol in the bilayer. Received: 1 June 1998/Revised: 14 October 1998     

Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis

Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca²⁺ response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)₂ or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcϵRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)₂ fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcϵRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family.     

Influence of a Transmembrane Protein on the Permeability of Small Molecules Across Lipid Membranes

The influence of the nonchannel conformation of the transmembrane protein gramicidin A on the permeability coefficients of neutral and ionized α-X-p-methyl-hippuric acid analogues (XMHA) (X = H, OCH₃, CN, OH, COOH, and CONH₂) across egg-lecithin membranes has been investigated in vesicle efflux experiments. Although 10 mol% gramicidin A increases lipid chain ordering, it enhances the transport of neutral XMHA analogues up to 8-fold, with more hydrophilic permeants exhibiting the greatest increase. Substituent contributions to the free energies of transfer of both neutral and anionic XMHA analogues from water into the bilayer barrier domain were calculated. Linear free-energy relationships were established between these values and those for solute partitioning from water into decadiene, chlorobutane, butyl ether, and octanol to assess barrier hydrophobicity. The barrier domain is similar for both neutral and ionized permeants and substantially more hydrophobic than octanol, thus establishing its location as being beyond the hydrated headgroup region and eliminating transient water pores as the transport pathway for these permeants, as the hydrated interface or water pores would be expected to be more hydrophilic than octanol. The addition of 10 mol% gramicidin A alters the barrier domain from a decadiene-like solvent to one possessing a greater hydrogen-bond accepting capacity. The permeability coefficients for ionized XMHAs increase with Na⁺ or K⁺ concentration, exhibiting saturability at high ion concentrations. This behavior can be quantitatively rationalized by Gouy-Chapman theory, though ion-pairing cannot be conclusively ruled out. The finding that transmembrane proteins alter barrier selectivity, favoring polar permeant transport, constitutes an important step toward understanding permeability in biomembranes. Received: 12 July 1999/Revised: 20 October 1999     

细菌趋化性的信号传导及调节机制研究进展

近年来,人们对细菌趋化性系统中的蛋白质生化和结构方面的认识逐渐加深,其调节趋化反应的信号传导系统在原核生物中较为保守,其中对大肠杆菌的趋化性研究得最透彻,为理解其他信号传导机制提供了有力的参考依据.详细介绍细菌趋化性的信号传导机制,并对包括趋化反应调节蛋白CheY的蛋白质结构以及两种修饰方式的趋化性调节机制最新进展进行了综述.     

Identification of a binding motif in the S5 helix that confers cholesterol sensitivity to the TRPV1 ion channel

The TRPV1 ion channel serves as an integrator of noxious stimuli with its activation linked to pain and neurogenic inflammation. Cholesterol, a major component of cell membranes, modifies the function of several types of ion channels. Here, using measurements of capsaicin-activated currents in excised patches from TRPV1-expressing HEK cells, we show that enrichment with cholesterol, but not its diastereoisomer epicholesterol, markedly decreased wild-type rat TRPV1 currents. Substitutions in the S5 helix, rTRPV1-R579D, and rTRPV1-F582Q, decreased this cholesterol response and rTRPV1-L585I was insensitive to cholesterol addition. Two human TRPV1 variants, with different amino acids at position 585, had different responses to cholesterol with hTRPV1-Ile(585) being insensitive to this molecule. However, hTRPV1-I585L was inhibited by cholesterol addition similar to rTRPV1 with the same S5 sequence. In the absence of capsaicin, cholesterol enrichment also inhibited TRPV1 currents induced by elevated temperature and voltage. These data suggest that there is a cholesterol-binding site in TRPV1 and that the functions of TRPV1 depend on the genetic variant and membrane cholesterol content.