Alamethicin, a Transmembrane Channel

Peptides possessing antibiotic activity, isolated from microbial sources, have been the subject of intensive structural and biological investigation over the past two decades.

Effects of membrane channel-forming polypeptides on mitochondrial oxidative phosphorylation. A comparison of alamethicin, gramicidin A, melittin and tetraacetyl melittin

Transmembrane channel-forming polypeptides can function as uncouplers of mitochondrial oxidative phosphorylation. The observed effects are dependent on the phosphate ion (Pi) concentration in the medium. At low Pi (2.5 mM) the order of uncoupling efficiencies is gramicidin A much greater than alamethicin greater than tetraacetyl melittin greater than melittin. The remarkably high activity of gramicidin A suggests insertion of preformed channel dimers into the membrane. It is also suggested that lipid phase association of peptides is necessary in the other cases. At Pi = 100 mM inhibitory effects are observed for alamethicin and tetraacetyl melittin. Less pronounced inhibition is seen for melittin, while no such effect is noted for gramicidin A. The site of inhibition is shown to be complex IV, and the differences in the behavior of the peptides are rationalized in terms of channel structures.

Effects of membrane channel-forming polypeptides on mitochondrial oxidative phosphorylation. A comparison of alamethicin, gramicidin A, melittin and tetraacetyl melittin.

Transmembrane channel-forming polypeptides can function as uncouplers of mitochondrial oxidative phosphorylation. The observed effects are dependent on the phosphate ion (Pi) concentration in the medium. At low Pi (2.5 mM) the order of uncoupling efficiencies is gramicidin A much greater than alamethicin greater than tetraacetyl melittin greater than melittin. The remarkably high activity of gramicidin A suggests insertion of preformed channel dimers into the membrane. It is also suggested that lipid phase association of peptides is necessary in the other cases. At Pi = 100 mM inhibitory effects are observed for alamethicin and tetraacetyl melittin. Less pronounced inhibition is seen for melittin, while no such effect is noted for gramicidin A. The site of inhibition is shown to be complex IV, and the differences in the behavior of the peptides are rationalized in terms of channel structures.

Electrochemical study of gramicidin D forming ion-permeable channels in the bilayer lipid membranes

Gramicidin within the lipid bilayer matrix is a well-known channel-forming polypeptide, but the mechanism of the ions across the membrane induced by gramicidin is not well understood. We found that at very low concentration of gramicidin in a bilayer lipid membrane, the channel behavior was controlled by the voltage applied across the membrane. When the voltage is higher than 75 mV, the channel is closing, while lower than 75 mV, the channel is opening. But when the concentration of the gramicidin in the BLMs is high, the channel behavior is changed into voltage-independent. (C) 1998 Elsevier Science Ltd. All rights reserved.

Ca2+-independent phospholipase A2-dependent gating of TRPM8 by lysophospholipids

TRPM8 represents an ion channel activated by cold temperatures and cooling agents, such as menthol, that underlies the cold-induced excitation of sensory neurons. Interestingly, the only human tissue outside the peripheral nervous system, in which the expression of TRPM8 transcripts has been detected at high levels, is the prostate, a tissue not exposed to any essential temperature variations. Here we show that the TRPM8 cloned from human prostate and heterologously expressed in HEK-293 cells is regulated by the Ca(2+)-independent phospholipase A(2) (iPLA(2)) signaling pathway with its end products, lysophospholipids (LPLs), acting as its endogenous ligands. LPLs induce prominent prolongation of TRPM8 channel openings that are hardly detectable with other stimuli (e.g. cold, menthol, and depolarization) and that account for more than 90% of the total channel open time. Down-regulation of iPLA(2) resulted in a strong inhibition of TRPM8-mediated functional responses and abolished channel activation. The action of LPLs on TRPM8 channels involved either changes in the local lipid bilayer tension or interaction with the critical determinant(s) in the transmembrane channel core. Based on this, we propose a novel concept of TRPM8 regulation with the involvement of iPLA(2) stimulation. This mechanism employs chemical rather than physical (temperature change) signaling and thus may be the main regulator of TRPM8 activation in organs not exposed to any essential temperature variations, as in the prostate gland.

Approches transcriptomiques dans l’étude de la fibrose kystique

Les avancées en biotechnologie ont permis l’identification d’un grand nombre de mécanismes moléculaires, soulignant également la complexité de la régulation génique. Néanmoins, avoir une vision globale de l’homéostasie cellulaire, nous est pour l’instant inaccessible et nous ne sommes en mesure que d’en avoir qu’une vue fractionnée. Étant donné l’avancement des connaissances des dysfonctionnements moléculaires observés dans les maladies génétiques telles que la fibrose kystique, il est encore difficile de produire des thérapies efficaces. La fibrose kystique est causée par la mutation de gène CFTR (cystic fibrosis transmembrane conductance regulator), qui code pour un canal chlorique transmembranaire. La mutation la plus fréquente (ΔF508) induit un repliement incorrect de la protéine et sa rétention dans le réticulum endoplasmique. L’absence de CFTR fonctionnel à la membrane a un impact sur l’homéostasie ionique et sur l’hydratation de la muqueuse respiratoire. Ceci a pour conséquence un défaut dans la clairance mucocilliaire, induisant infection chronique et inflammation excessive, deux facteurs fondamentaux de la physiopathologie. L’inflammation joue un rôle très important dans l’évolution de la maladie et malgré le nombre important d’études sur le sujet, la régulation du processus inflammatoire est encore très mal comprise et la place qu’y occupe le CFTR n’est pas établie. Toutefois, plusieurs autres facteurs, tels que le stress oxydatif participent à la physiopathologie de la maladie, et considérer leurs impacts est important pour permettre une vision globale des acteurs impliqués. Dans notre étude, nous exploitons la technologie des puces à ADN, pour évaluer l’état transcriptionnel d’une cellule épithéliale pulmonaire humaine fibro-kystique. Dans un premier temps, l’analyse de notre expérience identifie 128 gènes inflammatoires sur-exprimés dans les cellules FK par rapport aux cellules non FK où apparaissent plusieurs familles de gènes inflammatoires comme les cytokines ou les calgranulines. L’analyse de la littérature et des annotations suggèrent que la modulation de ces transcripts dépend de la cascade de NF-κB et/ou des voies de signalisation associées aux interférons (IFN). En outre, leurs modulations pourraient être associées à des modifications épigénétiques de leurs loci chromosomiques. Dans un second temps, nous étudions l’activité transcriptionnelle d’une cellule épithéliale pulmonaire humaine FK en présence de DMNQ, une molécule cytotoxique. Notre but est d’identifier les processus biologiques perturbés par la mutation du gène CFTR en présence du stress oxydatif. Fondé sur une analyse canonique de redondance, nous identifions 60 gènes associés à la mort cellulaire et leur variance, observée dans notre expérience, s’explique par un effet conjoint de la mutation et du stress oxydatif. La mesure de l’activité des caspases 3/7, des effecteurs de l’apoptose (la mort cellulaire programmée), montre que les cellules porteuses de la mutation ΔF508, dans des conditions de stress oxydatif, seraient moins apoptotiques que les cellules saines. Nos données transcriptomiques suggèrent que la sous-activité de la cascade des MAPK et la sur-expression des gènes anti-apoptotiques pourraient être impliquées dans le déséquilibre de la balance apoptotique.

Protein-Protein- und Protein-Lipid-Wechselwirkungen beeinflussen die Oligomerisierung und Funktion des E. coli Aquaglyceroporins GlpF

Aquaporine sind hochselektive Transmembrankanäle, die in allen Lebensformen den Fluss von Wasser und kleinen, polaren Molekülen wie Glycerol über Lipidmembranen ermöglichen. Obwohl die Kanalpore für den Substratfluss im Monomer lokalisiert ist, liegen Aquaporine innerhalb biologischer Membranen als Homotetramere vor. Im Rahmen dieser Arbeit wurden proteinbezogene und lipidmembranassoziierte Einflüsse auf die Oligomerisierung und Funktion des bakteriellen Aquaglyceroporins GlpF sowohl in vitro als auch in vivo untersucht. rnDie erhöhte Stabilität der Aquaporinpore sowie Interaktion zwischen den GlpF-Monomeren sind Triebkräfte der Aquaporin-Tetramerisierung. Ferner erfordern die GlpF-Tetramerisierung und -Aktivität bei Abschirmung der Ladung anionischer Lipide und einer minimalen Membrandicke von 27 Å keine spezielle Lipidumgebung. Da anionische Lipide die GlpF-Funktion jedoch störten, kann die GlpF-Aktivität in vivo möglicherweise durch die selektive Anreicherung von anionischen Lipiden in der unmittelbaren Proteinumgebung reguliert werden. Ungünstige Lipid-GlpF-Interaktionen können jedoch in Lipidumgebungen mit hoher Ordnung in der Acylkettenregion entstehen, die zu einer Aggregation der GlpF-Tetramere und reduzierten Aktivität führen. rnFerner wurde die Auswirkung der nephrogenen Diabetes insipidus verursachenden Aquaporin 2-Punktmutation V71M auf die Oligomerisierung und Funktion des homologen, bakteriellen Aquaglyceroporins GlpF untersucht. Da weder die Oligomierisierung noch die Aktivität des homologen, bakteriellen Aquaglyceroporins eingeschränkt sind, beruht der Krankheitsmechanismus der Aquaporin 2-Mutante V71M vermutlich auf einem defekten Transportmechansimus im Menschen. rn

AMPA RECEPTOR ALLOSTERISM: MEASUREMENT OF THE CONFORMATIONAL CHANGES IN THE LIGAND BINDING DOMAIN OF A FUNCTIONAL RECEPTOR

Ionotropic glutamate receptors are important excitatory neurotransmitter receptors in the mammalian central nervous system that have been implicated in a number of neuropathologies such as epilepsy, ischemia, and amyotrophic lateral sclerosis. Glutamate binding to an extracellular ligand binding domain initiates a series of structural changes that leads to the formation of a cation selective transmembrane channel, which consequently closes due to desensitization of the receptor. The crystal structures of the AMPA subtype of the glutamate receptor have been particularly useful in providing initial insight into the conformational changes in the ligand binding domain; however, these structures are limited by crystallographic constraint. To gain a clear picture of how agonist binding is coupled to channel activation and desensitization, it is essential to study changes in the ligand binding domain in a dynamic, physiological state. In this dissertation, a technique called Luminescence Resonance Energy Transfer was used to determine the conformational changes associated with activation and desensitization in a functional AMPA receptor (ÄN*-AMPA) that contains the ligand binding domain and transmembrane segments; ÄN*-AMPA has been modified such that fluorophores can be introduced at specific sites to serve as a readout of cleft closure or to establish intersubunit distances. Previous structural studies of cleft closure of the isolated ligand binding domain in conjunction with functional studies of the full receptor suggest that extent of cleft closure correlates with extent of activation. Here, LRET has been used to show that a similar relationship between cleft closure and activation is observed in the “full length” receptor showing that the isolated ligand binding domain is a good model of the domain in the full length receptor for changes within a subunit. Similar LRET investigations were used to study intersubunit distances specifically to probe conformational changes between subunits within a dimer in the tetrameric receptor. These studies show that the dimer interface is coupled in the open state, and decoupled in the desensitized state, similar to the isolated ligand binding domain crystal structure studies. However, we show that the apo state dimer interface is not pre-formed as in the crystal structure, hence suggesting a mechanism for functional transitions within the receptor based on LRET distances obtained.

Intrinsic characteristics of the proton pump in the luminal membrane of a tight urinary epithelium. The relation between transport rate and delta mu H.

A number of tight urinary epithelia, as exemplified by the turtle bladder, acidify the luminal solution by active transport of H+ across the luminal cell membrane. The rate of active H+ transport (JH) decreases as the electrochemical potential difference for H+ [delta mu H = mu H(lumen) - mu H(serosa)] across the epithelium is increased. The luminal cell membrane has a low permeability for H+ equivalents and a high electrical resistance compared with the basolateral cell membrane. Changes in JH thus reflect changes in active H+ transport across the luminal membrane. To examine the control of JH by delta mu H in the turtle bladder, transepithelial electrical potential differences (delta psi) were imposed at constant acid-base conditions or the luminal pH was varied at delta psi = 0 and constant serosal PCO2 and pH. When the luminal compartment was acidified from pH 7 to 4 or was made electrically positive, JH decreased as a linear function of delta mu H as previously described. When the luminal compartment was made alkaline from pH 7 to 9 or was made electrically negative, JH reached a maximal value, which was the same whether the delta mu H was imposed as a delta pH or a delta psi. The nonlinear JH vs. delta mu H relation does not result from changes in the number of pumps in the luminal membrane or from changes in the intracellular pH, but is a characteristic of the H+ pumps themselves. We propose a general scheme, which, because of its structural features, can account for the nonlinearity of the JH vs. delta mu H relations and, more specifically, for the kinetic equivalence of the effects of the chemical and electrical components of delta mu H. According to this model, the pump complex consists of two components: a catalytic unit at the cytoplasmic side of the luminal membrane, which mediates the ATP-driven H+ translocation, and a transmembrane channel, which mediates the transfer of H+ from the catalytic unit to the luminal solution. These two components may be linked through a buffer compartment for H+ (an antechamber).

Determination of the orientational distribution and orientation factor for transfer between membrane-bound fluorophores using a confocal microscope

Orientational fluorophores have been a useful tool in physical chemistry, biochemistry, and more recently structural biology due to the polarized nature of the light they emit and that fact that energy can be transferred between them. We present a practical scheme in which measurements of the intensity of emitted fluorescence can be used to determine limits on the mean and distribution of orientation of the absorption transition moment of membrane-bound. uorophores. We demonstrate how information about the orientation of. uorophores can be used to calculate the orientation factor k(2) required for use in FRET spectroscopy. We illustrate the method using images of AlexaFluor probes bound to MscL mechanosensitive transmembrane channel proteins in spherical liposomes.

A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease

Central core disease is a rare, nonprogressive myopathy that is characterized by hypotonia and proximal muscle weakness. In a large Mexican kindred with an unusually severe and highly penetrant form of the disorder, DNA sequencing identified an I4898T mutation in the C-terminal transmembrane/luminal region of the RyR1 protein that constitutes the skeletal muscle ryanodine receptor. All previously reported RYR1 mutations are located either in the cytoplasmic N terminus or in a central cytoplasmic region of the 5,038-aa protein. The I4898T mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. The response of the mutant RyR1 Ca2+ channel to the agonists halothane and caffeine in a Ca2+ photometry assay was completely abolished. Coexpression of normal and mutant RYR1 cDNAs in a 1:1 ratio, however, produced RyR1 channels with normal halothane and caffeine sensitivities, but maximal levels of Ca2+ release were reduced by 67%. [3H]Ryanodine binding indicated that the heterozygous channel is activated by Ca2+ concentrations 4-fold lower than normal. Single-cell analysis of cotransfected cells showed a significantly increased resting cytoplasmic Ca2+ level and a significantly reduced luminal Ca2+ level. These data are indicative of a leaky channel, possibly caused by a reduction in the Ca2+ concentration required for channel activation. Comparison with two other coexpressed mutant/normal channels suggests that the I4898T mutation produces one of the most abnormal RyR1 channels yet investigated, and this level of abnormality is reflected in the severe and penetrant phenotype of affected central core disease individuals.

Transmembrane topology of a CLC chloride channel

CLC chloride channels form a large and conserved gene family unrelated to other channel proteins. Knowledge of the transmembrane topology of these channels is important for understanding the effects of mutations found in human myotonia and inherited hypercalciuric kidney stone diseases and for the interpretation of structure–function studies. We now systematically study the topology of human ClC-1, a prototype CLC channel that is defective in human myotonia. Using a combination of in vitro glycosylation scanning and protease protection assays, we show that both N and C termini face the cytoplasm and demonstrate the presence of 10 (or less likely 12) transmembrane spans. Difficult regions were additionally tested by inserting cysteines and probing the effect of cysteine-modifying reagents on ClC-1 currents. The results show that D3 crosses the membrane and D4 does not, and that L549 between D11 and D12 is accessible from the outside. Further, since the modification of cysteines introduced between D11 and D12 and at the extracellular end of D3 strongly affect ClC-1 currents, these regions are suggested to be important for ion permeation.