Asia M. Fernández

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

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-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.

Labeling of the nicotinic acetylcholine receptor by a photoactivatable steroid probe : effects of cholesterol and cholinergic ligands

A photoactivatable steroid, p-azidophenacyl 3 alpha-hydroxy-5 beta-cholan-24- ate (APL), has been synthesized and used instead of cholesterol to functionally reconstitute purified acetylcholine receptor (AcChR) into vesicles made of asolectin phospholipids. Upon irradiation, the extent of AcChR photolabeling by APL is directly proportional to the amount of APL incorporated into the reconstituted vesicles and the maximum stoichiometry observed corresponds to approx. 50 mol of APL bound per mol of AcChR. Furthermore, all four subunits of the AcChR become labeled by APL and the observed labeling pattern resembles the 2:1:1:1 stoichiometry characteristic of these subunits within the AcChR complex. The presence of either cholesterol or neutral lipids from asolectin in the reconstituted bilayer decreases both, the incorporation of APl into the vesicles and the covalent labeling of the AcChR upon irradiation, without altering the stoichiometry of labeling in AcChR subunits stated above. This suggests that the potential interaction sites for the photoactivatable probe in the reconstituted AcChR are mostly those normally occupied by the natural neutral lipids. Carbamylcholine, a cholinergic agonist, also reduces the extent of APL photolabeling of the AcChR in a dose-dependent manner but, in contrast to the effects of cholesterol, the presence of carbamylcholine alters the stoichiometry of labeling in the AcChR subunits. This, along with the observation that such a decrease in the extent of APL photolabeling caused by carbamylcholine can be blocked by preincubation with alpha-bungarotoxin, suggest that AcChR desensitization induced by prolonged exposure to cholinergic agonists encompasses a rearrangement of transmembrane portions of the AcChR protein, which can be sensed by the photoactivatable probe. Conversely, presence of (+)-tubocurarine, a competitive cholinergic antagonist, has no effects on altering either the extent of APL photolabeling of the AcChR or the distribution of the labeling among AcChR subunits.

Protein structural effects of agonist binding to the nicotinic acetylcholine receptor

The effects on the protein structure produced by binding of cholinergic agonists to purified acetylcholine receptor (AcChR) reconstituted into lipid vesicles, has been studied by Fourier‐transform infrared spectroscopy and differential scanning calorimetry. Spectral changes in the conformationally sensitive amide 1 infrared band indicates that the exposure of the AcChR to the agonist carbamylcholine, under conditions which drive the AcChR into the desensitized state, produces alterations in the protein secondary structure. Quantitative estimation of these agonist‐induced alterations by band‐fitting analysis of the amide 1 spectral band reveals no appreciable changes in the percent of α‐helix, but a decrease in β‐sheet structure, concomitant with an increase in less ordered structures. Additionally, agonist binding results in a concentration‐dependent increase in the protein thermal stability, as indicated by the temperature dependence of the protein infrared spectrum and by calorimetric analysis, which further suggest that AcChR desensitization induced by the cholinerpic agonist implies significant rearrangements in the protein structure.

Structural stabilization of botulinum neurotoxins by tyrosine phosphorylation.

Tyrosine phosphorylation of botulinum neurotoxins augments their proteolytic activity and thermal stability, suggesting a substantial modification of the global protein conformation. We used Fourier‐transform infrared (FTIR) spectroscopy to study changes of secondary structure and thermostability of tyrosine phosphorylated botulinum neurotoxins A (BoNT A) and E (BoNT E). Changes in the conformationally‐sensitive amide I band upon phosphorylation indicated an increase of the α‐helical content with a concomitant decrease of less ordered structures such as turns and random coils, and without changes in β‐sheet content. These changes in secondary structure were accompanied by an increase in the residual amide II absorbance band remaining upon H‐D exchange, consistent with a tighter packing of the phosphorylated proteins. FTIR and differential scanning calorimetry (DSC) analyses of the denaturation process show that phosphorylated neurotoxins denature at temperatures higher than those required by non‐phosphorylated species. These findings indicate that tyrosine phosphorylation induced a transition to higher order and that the more compact structure presumably imparts to the phosphorylated neurotoxins the higher catalytic activity and thermostability.

Cholesterol stabilizes the structure of the nicotinic acetylcholine receptor reconstituted in lipid vesicles

Abstract A technique of heat inactivation of α-bungarotoxin binding sites, has been used to probe structural alteration of the nicotinic acetylcholine receptor when reconstituted into soybean lipid vesicles containing different amounts of added cholesterol. The profiles of heat inactivation of α-bungarotoxin binding sites are gradually shifted to higher temperatures, as the cholesterol/phospholipid molar ratio in the reconstituted vesicles is increased from 0 to 0.4, thus, indicating that presence of cholesterol within the lipid matrix produces a structural stabilization of the reconstituted acetylcholine receptor protein. The observed stabilization of receptor structure induced by cholesterol is such that, depending upon the different conditions used to form the reconstituted vesicles by detergent dialysis procedures, the profiles of heat inactivation for the reconsutituted receptor vesicles at a cholesterol/phospholipid molar ratio of 0.4 become undistinguishable from that exhibited by native acetylcholine receptor membranes isolated from the electric organ of Torpedo . Increasing the cholesterol concentration in the reconstituted vesicles also induces a decrease in the apparent ‘fluidity’ of the membrane, which correlates very closely with the observed stabilization of the receptor protein. Such a correlation, however, does not necessarily imply that changes in receptor structure are caused by pertubations of the membrane ‘fluidity’. This conclusion is based on experiments using local anesthetics, well known to cause alteration of membrane lipid dynamics, but unable to modify the characteristic heat-inactivation profiles from native acetylcholine receptor membranes. As a possible alternative to the above observations, it is suggested that the effects of cholesterol on receptor structure could be exerted through direct interaction with the receptor protein. Also, since similarly high concentrations of cholesterol have been reported to be required for optimal cation-gating activity of reconstituted acetylcholine receptor, we interpret our data as indicative of a correlation between structural and functional alterations of the acetylcholine receptor induced by the presence of cholesterol within the membrane bilayer.

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.

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.