José A. Poveda

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.

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.

Protein-promoted membrane domains

The current notion of biological membranes encompasses a very complex structure, made of dynamically changing compartments or domains where different membrane components partition. These domains have been related to important cellular functions such as membrane sorting, signal transduction, membrane fusion, neuronal maturation, and protein activation. Many reviews have dealt with membrane domains where lipid-lipid interactions direct their formation, especially in the case of raft domains, so in this review we considered domains induced by integral membrane proteins. The nature of the interactions involved and the different mechanisms through which membrane proteins segregate lipid domains are presented, in particular with regard to those induced by the nAChR. It may be concluded that coupling of favourable lipid-lipid and lipid-protein interactions is a general condition for this phenomenon to occur.

Lipid modulation of ion channels through specific binding sites.

Ion channel conformational changes within the lipid membrane are a key requirement to control ion passage. Thus, it seems reasonable to assume that lipid composition should modulate ion channel function. There is increasing evidence that this implicates not just an indirect consequence of the lipid influence on the physical properties of the membrane, but also specific binding of selected lipids to certain protein domains. The result is that channel function and its consequences on excitability, contractility, intracellular signaling or any other process mediated by such channel proteins, could be subjected to modulation by membrane lipids. From this it follows that development, age, diet or diseases that alter lipid composition should also have an influence on those cellular properties. The wealth of data on the non-annular lipid binding sites in potassium channel from Streptomyces lividans (KcsA) makes this protein a good model to study the modulation of ion channel structure and function by lipids. The fact that this protein is able to assemble into clusters through the same non-annular sites, resulting in large changes in channel activity, makes these sites even more interesting as a potential target to develop lead compounds able to disrupt such interactions and hopefully, to modulate ion channel function. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cells Physiology, Pathology and Therapy.

Experimental Resistance to Drug Combinations in Leishmania donovani: Metabolic and Phenotypic Adaptations

ABSTRACT Together with vector control, chemotherapy is an essential tool for the control of visceral leishmaniasis (VL), but its efficacy is jeopardized by growing resistance and treatment failure against first-line drugs. To delay the emergence of resistance, the use of drug combinations of existing antileishmanial agents has been tested systematically in clinical trials for the treatment of visceral leishmaniasis (VL). In vitro, Leishmania donovani promastigotes are able to develop experimental resistance to several combinations of different antileishmanial drugs after 10 weeks of drug pressure. Using an untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics approach, we identified metabolic changes in lines that were experimentally resistant to drug combinations and their respective single-resistant lines. This highlighted both collective metabolic changes (found in all combination therapy-resistant [CTR] lines) and specific ones (found in certain CTR lines). We demonstrated that single-resistant and CTR parasite cell lines show distinct metabolic adaptations, which all converge on the same defensive mechanisms that were experimentally validated: protection against drug-induced and external oxidative stress and changes in membrane fluidity. The membrane fluidity changes were accompanied by changes in drug uptake only in the lines that were resistant against drug combinations with antimonials, and surprisingly, drug accumulation was higher in these lines. Together, these results highlight the importance and the central role of protection against oxidative stress in the different resistant lines. Ultimately, these phenotypic changes might interfere with the mode of action of all drugs that are currently used for the treatment of VL and should be taken into account in drug development.

Antifungal effects and mechanism of action of viscotoxin A3.

Viscotoxins are cationic proteins, isolated from different mistletoe species, that belong to the group of thionins, a group of basic cysteine‐rich peptides of ≈ 5 kDa. They have been shown to be cytotoxic to different types of cell, including animal, bacterial and fungal. The aim of this study was to obtain information on the cell targets and the mechanism of action of viscotoxin isoform A3 (VtA3). We describe a detailed study of viscotoxin interaction with fungal‐derived model membranes, its location inside spores of Fusarium solani, as well as their induced spore death. We show that VtA3 induces the appearance of ion‐channel‐like activity, the generation of H2O2, and an increase in cytoplasmic free Ca2+. Moreover, we show that Ca2+ is involved in VtA3‐induced spore death and increased H2O2 concentration. The data presented here strongly support the notion that the antifungal activity of VtA3 is due to membrane binding and channel formation, leading to destabilization and disruption of the plasma membrane, thereby supporting a direct role for viscotoxins in the plant defence mechanism.

Effects of Conducting and Blocking Ions on the Structure and Stability of the Potassium Channel KcsA

This article reports on the interaction of conducting (K+) and blocking (Na+) monovalent metal ions with detergent-solubilized and lipid-reconstituted forms of the K+ channel KcsA. Monitoring of the protein intrinsic fluorescence reveals that the two ions bind competitively to KcsA with distinct affinities (dissociation constants for the KcsA·K+ and KcsA·Na+ complexes of ∼8 and 190 mm, respectively) and induce different conformations of the ion-bound protein. The differences in binding affinity as well as the higher K+ concentration bathing the intracellular mouth of the channel, through which the cations gain access to the protein binding sites, should favor that only KcsA·K+ complexes are formed under physiological-like conditions. Nevertheless, despite such prediction, it was also found that concentrations of Na+ well below its dissociation constant and even in the presence of higher K+ concentrations, cause a remarkable decrease in the protein thermal stability and facilitate thermal dissociation into subunits of the tetrameric KcsA, as concluded from the temperature dependence of the protein infrared spectra and from gel electrophoresis, respectively. These latter observations cannot be explained based on the occupancy of the binding sites from above and suggest that there must be additional ion binding sites, whose occupancy could not be detected by fluorescence and in which the affinity for Na+ must be higher or at least similar to that of K+. Moreover, cation binding as reported by means of fluorescence does not suffice to explain the large differences in free energy of stabilization involved in the formation of the KcsA·Na+ and KcsA·K+ complexes, which for the most part should arise from synergistic effects of the ion-mediated intersubunit interactions.

The influence of a membrane environment on the structure and stability of a prokaryotic potassium channel, KcsA

The lack of a membrane environment in membrane protein crystals is considered one of the major limiting factors to fully imply X‐ray structural data to explain functional properties of ion channels [Gulbis, J.M. and Doyle, D. (2004) Curr. Opin. Struct. Biol. 14, 440–446]. Here, we provide infrared spectroscopic evidence that the structure and stability of the potassium channel KcsA and its chymotryptic derivative 1–125 KcsA reconstituted into native‐like membranes differ from those exhibited by these proteins in detergent solution, the latter taken as an approximation of the mixed detergent‐protein crystal conditions.

Biophysical and functional characterization of an ion channel peptide confined in a sol-gel matrix.

Immobilization of zwitterionic lipid membranes in sol-gel matrices induces irreversible alterations of the bilayer fluidity, which can limit the use of these systems for practical applications. Recently, we have reported that electrostatic interactions between phospholipids polar heads and the negative-charged silica surface of the porous matrix should be the cause of such behavior. In the present work, we analyze the effect of these interactions on the biophysical and functional properties of the ion-channel peptide gramicidin, entrapped in a sol-gel matrix, to get more insight on the ability of these inorganic materials to immobilize ion channels and other membrane-bound proteins. Gramicidin was reconstituted in anionic and zwitterionic liposomes and the effects of sol-gel immobilization on the biophysical properties of gramicidin were determined from changes in the photophysical properties of its tryptophan residues. In addition, the physical state of the immobilized lipid membrane containing gramicidin was analyzed by measuring the spectral shift of the fluorescent probe Laurdan. Finally, the ion-channel activity of the peptide was monitored upon sol-gel immobilization through a fluorescence quenching assay using the fluorescent dye pyrene-1,3,6,8-tetrasulfonic acid (PTSA). Results show that the channel properties of the immobilized gramicidin are preserved in both zwitterionic and anionic liposomes, even though the zwitterionic polar heads interact with the porous surface of the host matrix.

N-type Inactivation of the Potassium Channel KcsA by the Shaker B “Ball” Peptide MAPPING THE INACTIVATING PEPTIDE-BINDING EPITOPE

The effects of the inactivating peptide from the eukaryotic Shaker BK+ channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturation transfer difference (STD) NMR methods to map the molecular interactions between the inactivating peptide and its channel target. The results indicate that binding of the ShB peptide to KcsA involves the ortho and meta protons of Tyr8, which exhibit the strongest STD effects; the C4H in the imidazole ring of His16; the methyl protons of Val4, Leu7, and Leu10 and the side chain amine protons of one, if not both, the Lys18 and Lys19 residues. When a noninactivating ShB-L7E mutant is used in the studies, binding to KcsA is still observed but involves different amino acids. Thus, the strongest STD effects are now seen on the methyl protons of Val4 and Leu10, whereas His16 seems similarly affected as before. Conversely, STD effects on Tyr8 are strongly diminished, and those on Lys18 and/or Lys19 are abolished. Additionally, Fourier transform infrared spectroscopy of KcsA in presence of 13C-labeled peptide derivatives suggests that the ShB peptide, but not the ShB-L7E mutant, adopts a β-hairpin structure when bound to the KcsA channel. Indeed, docking such a β-hairpin structure into an open pore model for K+ channels to simulate the inactivating peptide/channel complex predicts interactions well in agreement with the experimental observations.