José M. González-Ros

Membranes: a meeting point for lipids, proteins and therapies

•  Introduction •  Membrane lipid composition •  Membrane lipid structure •  Membrane lipid organization ‐  Why so many different lipids? ‐  Lipid mixing and demixing ‐  Lateral pressure ‐  Surface electrostatics •  Role of lipids in cell functions •  Lipid influence in transmembrane protein function ‐  Prokaryotic potassium channel (KcsA) ‐  Mechanosensitive channels ‐  Voltage‐gated potassium channel (KvAP) ‐  Nicotinic acetylcholine receptor (nAcChR) ‐  G protein‐coupled receptors ‐  Other examples •  Non‐permanent proteins in membranes ‐  Proteins that interact reversibly with the bilayers ‐  Proteins that interact irreversibly with the bilayers ‐  Proteins that interact weakly with the membrane ‐  Proteins that interact strongly with the membrane ‐  G proteins and their interactions with membranes ‐  Small monomeric G proteins: the Ras and Ras‐like family ‐  Protein kinase C •  Membrane microdomains and lipid mediators in the control of heat‐shock protein response ‐  Stress sensing and signalling: the membrane sensor theory ‐  Hsp signalling in cancer and diabetes ‐  The role of membrane microdomains ‐  Lipid mediators of the stress response •  A subpopulation of Hsps can interact with and translocate through membranes ‐  Hsp90 in eukaryotic membranes ‐  Hsp70 in cell membranes ‐  Hsp27‐membrane interactions ‐  Secreted Hsps ‐  Representative cases where Hsps interact with membranes or release from the cells •  Concluding remarks

Altered Drug Membrane Permeability in a Multidrug- Resistant Leishmania tropica Line

We selected a Leishmania tropica cell line resistant to daunomycin (DNM) that presents a multidrug-resistant (MDR) phenotype characterized by overexpression of a P-glycoprotein of 150 kDa. The resistant line overexpressed an MDR-like gene, called ltrmdr1, located in an extrachromosomal circular DNA. DNM uptake experiments using laser flow cytometry showed a significant reduction in drug accumulation in the resistant parasites. The initial stages of the interaction of DNM with membranes from wild-type and DNM-resistant parasites were defined by a rapid kinetic stopped-flow procedure which can be described by two kinetic components. On the basis of a previous similar kinetic study with tumor cells, we ascribed the fast component to rapid interaction of DNM with membrane surface components and the slow component to passive diffusion of the drug across the membranes. The results reported here indicate that entrance of DNM into wild-type parasites was facilitated in respect to the resistant ones. We propose that resistance to DNM in L. tropica is a multifactorial event involving at least two complementary mechanisms. an altered drug membrane permeability and the overexpression of a protein related to P-glycoprotein that regulates drug efflux.

Clustering and Coupled Gating Modulate the Activity in KcsA, a Potassium Channel Model

Different patterns of channel activity have been detected by patch clamping excised membrane patches from reconstituted giant liposomes containing purified KcsA, a potassium channel from prokaryotes. The more frequent pattern has a characteristic low channel opening probability and exhibits many other features reported for KcsA reconstituted into planar lipid bilayers, including a moderate voltage dependence, blockade by Na+, and a strict dependence on acidic pH for channel opening. The predominant gating event in this low channel opening probability pattern corresponds to the positive coupling of two KcsA channels. However, other activity patterns have been detected as well, which are characterized by a high channel opening probability (HOP patterns), positive coupling of mostly five concerted channels, and profound changes in other KcsA features, including a different voltage dependence, channel opening at neutral pH, and lack of Na+ blockade. The above functional diversity occurs correlatively to the heterogeneous supramolecular assembly of KcsA into clusters. Clustering of KcsA depends on protein concentration and occurs both in detergent solution and more markedly in reconstituted membranes, including giant liposomes, where some of the clusters are large enough (up to micrometer size) to be observed by confocal microscopy. As in the allosteric conformational spread responses observed in receptor clustering (Bray, D. and Duke, T. (2004) Annu. Rev. Biophys. Biomol. Struct. 33, 53-73) our tenet is that physical clustering of KcsA channels is behind the observed multiple coupled gating and diverse functional responses.

Advances in modulating thermosensory TRP channels

Introduction: Thermosensory channels are a subfamily of the transient receptor potential (TRP) channel family that are activated by changes in the environmental temperature. These channels, known as thermoTRPs, cover the entire spectrum of temperatures, from noxious cold (< 15°C) to injurious heat (> 42°C). In addition, dysfunction of these channels contributes to the thermal hypersensitivity that accompanies painful conditions. Moreover, because of their wide tissue and cellular distribution, thermoTRPs are also involved in the pathophysiology of several diseases, from inflammation to cancer. Areas covered: Although the number of thermoTRPs is increasing with the identification of novel members such as TRPM3, we will cover the recent advances in the pharmacology of the classical thermosensory channels, namely TRPV1, TRPV2, TRPV3, TRPV4, TRPM8 and TRPA1. This review will focus on the therapeutic progress carried out for all these channels and will highlight the tenet that TRPV1, TRPM8 and TRPA1 are the most exploited channels, and that the interest on TRPV3 and TRPV4 is growing with the first TRPV3 antagonist that moves into Phase-II clinical trials. In contrast, the pharmacology of TRPV2 is yet in its infancy. Expert opinion: Despite the tremendous academic and industrial investment to develop therapeutic modulators of thermoTRPs, it apparently seems that we are still far from the first successful product, although hope is maintained high for all compounds currently in clinical trials. A major concern has been the appearance of side effects. A better knowledge of the thermosensory protein networks (signal-plexes), along with the application of system biology approaches may provide novel strategies to modulate thermoTRPs activity with improved therapeutic index. A case in point is TRPV1, where acting on interacting proteins is providing new therapeutic opportunities.

Susceptibility of multidrug resistance tumor cells to apoptosis induction by histone deacetylase inhibitors.

The main goal of our study has been to analyze the efficiency of new anticancer drugs, specifically histone deacetylase inhibitors, in tumor cells bearing a multidrug resistance phenotype. We report that the histone deacetylase inhibitors, Trichostatin A and Suberoylanilide Hydroxamic Acid (SAHA), dramatically reduce cell viability and promote apoptosis in different drug‐resistant cells, affecting in a much lesser extent to their parental drug‐sensitive counterparts. The differential effects induced by Trichostatin A and SAHA between drug‐sensitive and drug‐resistant cells are reflected on the main characteristics of the resistant phenotype. Thus, reverse transcription‐PCR and Western immunoblots confirm that both histone deacetylase inhibitors promote endogenous down‐regulation of P‐glycoprotein, which is overexpressed in the drug‐resistant cells. Transfection of drug‐sensitive cells with the P‐glycoprotein cDNA ruled out the a priori possible association between apoptosis and down‐regulation of P‐glycoprotein induced by the histone deacetylase inhibitors. The results suggest a therapeutic potential of histone deacetylase inhibitors in the treatment of cancers with acquired resistance.

Equilibrium binding of daunomycin and adriamycin to calf thymus DNA: Temperature and ionic strength dependence of thermodynamic parameters

Absorbance and fluorescence quenching monitoring of the binding of the anthracyclines adriamycin (ADM) and daunomycin (DNM) to calf thymus DNA, provides reproducible binding data only when moderate drug/DNA molar ratios are used in the assays. Under these conditions, the fraction of DNA-bound drug, in equilibrium with free anthracycline, which can be reliably detected, ranged from 40-60% to 80-95% of the total added drug, depending upon ionic strength and temperature. Use of the neighbour exclusion model adequately fits such data and predicts that (i) the affinity of ADM for binding to the DNA is always higher than that corresponding to DNM, under similar experimental conditions, (ii) the binding constant for both drugs exhibits a strong salt and temperature dependence, and (iii) the exclusion parameter, indicative of the size of the anthracycline binding sites on the DNA, equals 3.1 +/- 0.4 and 3.3 +/- 0.4 base pairs for ADM and DNM, respectively, and is independent of salt concentration. The salt and temperature dependence of the binding constant is used to estimate the thermodynamic parameters involved in the interaction of the drugs with the DNA. Binding of the drugs is an exothermic process and the binding free energy arises primarily from a large negative enthalpy which, as the entropy, strongly depends upon ionic strength, and is much larger than predicted by polyelectrolyte theory. The enthalpy and entropy changes observed, appear to compensate each other over the entire range of salt concentrations used, and may arise from a complex variety of contributions, including salt-induced changes in secondary structure of the DNA, as indicated by circular dichroism techniques.

Protein Self-Assembly and Lipid Binding in the Folding of the Potassium Channel KcsA†

Moderate concentrations of the alcohol 2,2,2-trifluoroethanol (TFE) cause the coupled unfolding and dissociation into subunits of the homotetrameric potassium channel KcsA, in a process that is partially irreversible when the protein is solubilized in plain dodecyl beta-d-maltoside (DDM) micelles [Barrera et al. (2005) Biochemistry 44, 14344-52]. Here we report that the transition from the folded tetramer to the unfolded monomer becomes completely reversible when KcsA is solubilized in mixed micelles composed of the detergent DDM and the lipids DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and DOPG (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]). This result suggests that lipids may act as effectors in the tetramerization of KcsA. The observed reversibility allowed the determination of the standard free energy of the folding reaction of KcsA: DeltaG = 30.5 +/- 3.1 kcal x mol-1. We also observed that, prior to the unfolding of the tetramer, the presence of lower TFE concentrations causes the disassembly of supramolecular clusters of KcsA into the individual tetrameric molecules. Within the limits of experimental resolution, this is also a reversible process, but unlike the tetramer to monomer transition from above, the level of clustering is not influenced by the presence of solubilized lipids. These observations suggest a distinct role of the lipids in the different in vitro assembly steps (folding/tetramerization and clustering) of KcsA.

Membrane-tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity

The transient receptor potential vanilloid 1 (TRPV1) channel is a thermosensory receptor implicated in diverse physiological and pathological processes. The TRP domain, a highly conserved region in the C terminus adjacent to the internal channel gate, is critical for subunit tetramerization and channel gating. Here, we show that cell‐penetrating, membrane‐anchored peptides patterned after this protein domain are moderate and selective TRPV1 antagonists both in vitro and in vivo, blocking receptor activity in intact rat primary sensory neurons and their peripheral axons with mean decline time of 30 min. The most potent lipopeptide, TRP‐p5, blocked all modes of TRPV1 gating with micromolar efficacy (IC50<10 µM), without significantly affecting other thermoTRP channels. In contrast, its retrosequence or the corresponding sequences of other TRPV channels did not alter TRPV1 channel activity (IC50»100 µM). TRP‐p5 did not affect the capsaicin sensitivity of the vanilloid receptor. Our data suggest that TRP‐p5 interferes with protein‐protein interactions at the level of the TRP domain that are essential for the “conformational” change that leads to gate opening. Therefore, these palmitoylated peptides, which we termed TRPducins, are noncompetitive, voltage‐independent, sequence‐specific TRPV1 blockers. Our findings indicate that TRPducin‐like peptides may embody a novel molecular strategy that can be exploited to generate a selective pharmacological arsenal for the TRP superfamily of ion channels.—Valente, P., Fernández‐Carvajal, A., Camprubí‐Robles, M., Gomis, A., Quirce, S., Viana, F., Fernández‐Ballester, G., González‐Ros, J. M., Belmonte, C., Planells‐Cases, R., Ferrer‐Montiel, A. Membrane‐tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity. FASEB J. 25, 1628–1640 (2011). www.fasebj.org

Partitioning of liquid-ordered/liquid-disordered membrane microdomains induced by the fluidifying effect of 2-hydroxylated fatty acid derivatives

Cellular functions are usually associated with the activity of proteins and nucleic acids. Recent studies have shown that lipids modulate the localization and activity of key membrane-associated signal transduction proteins, thus regulating the cells physiology. Membrane Lipid Therapy aims to reverse cell dysfunctions (i.e., diseases) by modulating the activity of membrane signaling proteins through regulation of the lipid bilayer structure. The present work shows the ability of a series of 2-hydroxyfatty acid (2OHFA) derivatives, varying in the acyl chain length and degree of unsaturation, to regulate the membrane lipid structure. These molecules have shown greater therapeutic potential than their natural non-hydroxylated counterparts. We demonstrated that both 2OHFA and natural FAs induced reorganization of lipid domains in model membranes of POPC:SM:PE:Cho, modulating the liquid-ordered/liquid-disordered structures ratio and the microdomain lipid composition. Fluorescence spectroscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential detergent solubilization experiments showed a destabilization of the membranes upon addition of the 2OHFAs and FAs which correlated with the observed disordering effect. The changes produced by these synthetic fatty acids on the lipid structure may constitute part of their mechanism of action, leading to changes in the localization/activity of membrane proteins involved in signaling cascades, and therefore modulating cell responses.

Role of the transient receptor potential vanilloid 1 in inflammation and sepsis

The transient receptor potential vanilloid 1 (TRPV1) is a thermoreceptor that responds to noxious temperatures, as well as to chemical agonists, such as vanilloids and protons. In addition, its channel activity is notably potentiated by proinflammatory mediators released upon tissue damage. The TRPV1 contribution to sensory neuron sensitization by proalgesic agents has signaled this receptor as a prime target for analgesic and anti-inflammatory drug intervention. However, TRPV1 antagonists have notably failed in clinical and preclinical studies because of their unwanted side effects. Recent reports have unveiled previously unrecognized anti-inflammatory and protective functions of TRPV1 in several diseases. For instance, this channel has been suggested to play an anti-inflammatory role in sepsis. Therefore, the use of potent TRPV1 antagonists as a general strategy to treat inflammation must be cautiously considered, given the deleterious effects that may arise from inhibiting the population of channels that have a protective function. The use of TRPV1 antagonists may be limited to treating those pathologies where enhanced receptor activity contributes to the inflamed state. Alternatively, therapeutic paradigms, such as reduction of inflammatory-mediated increase of receptor expression in the cell surface, may be a better strategy to prevent abrogation of the TRPV1 subpopulation involved in anti-inflammatory and protective processes.