J.M. González-Ros

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.

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.

Protein orientation affects the efficiency of functional protein transplantation into the xenopus oocyte membrane.

Xenopus oocytes incorporate into their plasma membrane nicotinic acetylcholine receptors (nAChRs) after intracellular injection of lipid vesicles bearing this protein. The advantage of this approach over the classical oocyte expression system lies in the transplantation of native, fully processed proteins, although the efficiency of functional incorporation of nAChRs is low. We have now studied the incorporation into the oocyte membrane of the Torpedo chloride channel (ClC-0), a minor contaminant protein in some nAChR preparations. nAChR-injected oocytes incorporated functional ClC-0: i) in a higher number than functional nAChRs; ii) retaining their original properties; and iii) with a right-side-out orientation in the oocyte membrane. In an attempt to elucidate the reasons for the low efficiency in the functional incorporation of nAChRs into the oocyte membrane, we combined electrophysiological and [125I]alpha-bungarotoxin-binding experiments. Up to 3% of injected nAChRs were present in the oocyte plasma membrane at a given time. Thus, fusion of lipoproteosome vesicles to the oocyte plasma membrane is not the limiting factor for an efficient functional transplantation of foreign proteins. Accounting for the low rate of functional transplantation of nAChRs is their backward orientation in the oocyte membrane, since about 80% of them adopted an out-side-in orientation. Other factors, including differences in the susceptibility of the transplanted proteins to intracellular damage should also be considered.

Thermal stability of hepatitis B surface antigen S proteins

Thermal stability of hepatitis B surface antigen (HBsAg) has been studied by analyzing alterations in the native secondary structure and the antigenic activity. After heating for 19 h, circular dichrosim showed a cooperative transition with a midpoint at 49 degrees C. The conformational changes induced by temperature reduced the helical content of HBsAg S proteins from 49% at 23 degrees C to 26% at 60 degrees C and abolished the antigenic activity, as measured by binding to polyclonal antibodies. Furthermore, the six different antigenic determinants recognized by our panel of monoclonal antibodies were also shown to be dependent on the native structure of HBsAg proteins. Hence, it can be inferred that these epitopes are conformation-dependent. Binding of monoclonal antibodies to HBsAg protected the native structure of the corresponding antigenic determinant from thermal denaturation. In fact, binding of one of the monoclonals tested resulted not only in protection of the corresponding epitope, but also in a consistent increase of antibody binding with increasing temperature. Such an increase in antibody binding occurred simultaneously with an increase in the fluidity of surface lipid regions, as monitored by fluorescence depolarization of 1-(trimethylammoniophenyl)-6-phenyl-1,3,5-hexatriene. This correlation, along with the observation that lipids play an important role in maintaining the structure and antigenic activity of HBsAg (Gavilanes et al. (1990) Biochem. J. 265, 857-864), allow to speculate the certain epitopes of HBsAg which are close to the lipid-protein interface, are dependent on the fluidity of the surface lipid regions. Thus, any change in the physical state of the lipids could confer a different degree of exposure to the antigenic determinants.

Lipid-Protein Interactions in Reconstituted Membranes Containing Nicotinic Acetylcholine Receptor

The nicotinic acetylcholine receptor (AcChR) from Torpedo is a large transmembrane glycoprotein composed of four different polypeptide subunits (α, β, γ and δ) in a 2:1:1:1 stoichiometry. Binding of cholinergic agonists to extracellular domains on the a subunits, causes the formation of a transient cation channel within the protein, responsible for the initiation of postsynaptic membrane depolarization.

Interaction of the Nicotinic Acetylcholine Receptor with Ligands and Membrane Lipids Studied by Fourier-Transform Infrared Spectroscopy and Photoaffinity Labeling

Monitoring of the amide I band by Fourier-transform infrared spectroscopy (FT-IR) is a valid and flexible approach to monitor changes in the secondary structure of reconstituted Acetylcholine Receptor (AcChR). The continuous exposure of the AcChR to a cholinergic agonist (carbamylcholine), which drives the AcChR into the desensitized state, produces only minor changes in AcChR secondary structure. Nevertheless, carbamylcholine alters the AcChR tertiary or quaternary structure, as indicated by the increased thermal stability of the protein assessed from the temperature-dependence of the infrared spectrum. On the contrary, presence of a competitive cholinergic antagonist (d-tubocurarine) produces no detectable effects on AcChR structure. Cholesterol or the neut-al lipids present in asolectin extracts produce an ordering of the AcChR secondary structure observed by FT-IR and also enable the protein to increase its thermal stability in response to carbamylcholine. These effects of cholesterol or asolectin neutral lipids seem mediated by a direct interaction of all the AcChR subunits with the lipids, as suggested by labeling of the AcChR by a photoactivatable cholesterol analogue. The interaction between the AcChR and the cholesterol analogue is sensitive to AcChR desensitization since the presence of carbamylcholine during photolysis decreases the extent of labeling and alters the labeling stoichiometry in the AcChR subunits. This suggests the occurrence of an agonist-induced change in the arrangement of the transmembrane portion of the desensitized protein, which is consistent with the agonist-induced alteration of the AcChR protein tertiary or quaternary structure detected by FT-IR.

Role of Membrane Components on Anthracycline Cytotoxicity

The plasma membrane of tumour cells is receiving increasing attention in regard to cellular multidrug resistance. We have studied plasma membranes from wild P388 murine leukemia cells and from stable multidrug resistant sublines with primary resistance to daunomycin, which do not express P-glycoproteins. The results obtained with the isolated plasma membranes suggest that there is a role for certain lipids, namely phospatidylserine and cholesterol, whose relative abundance is different in membranes from drug-sensitive or -resistant cells, in determining (i) the extent of daunomycin binding to the membrane and (ii) the location of daunomycin within the membrane bilayer. Furthermore, calorimetric studies on the interaction between model lipid vesicles with daunomycin and/or verapamil, the best known resistance-reverting agent, indicate that verapamil prevents, in a concentration-dependent manner, the alterations in the phospholipid phase transition expected from the presence of daunomycin in the bilayer.