Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy.
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 During the last decades, the use of high resolution electrophysiological techniques to study ion channels has provided a large amount of information on functional aspects of these important membrane proteins. Such a detailed information on channel function, however, has not been accompanied by structural knowledge until recently, when several structurally simpler homologues of mammalian ion channels found in extremophyle bacteria or Archaea and remarkably resistant to harsh experimental conditions, have been purified, crystallized and their structure solved at high resolution by x-ray diffraction methods (1-4). A K ϩ channel from the soil bacteria Streptomyces lividans named KcsA 4 (1), a homotetramer made up of identical 160-amino acid subunits, was the first of such structures to be solved (5, 6), and, although the x-ray structure corresponds to a closed channel conformation, it has contributed much to our current understanding of ion selectivity and permeation. Ironically, there was little or no functional information on KcsA by the time its structure was solved, and then several groups undertook the task of characterizing its single channel properties, which has been surrounded by controversy. For instance, Schrempf's group, discoverers of KcsA in S. lividans, reported a strong dependence of channel opening on acidic pH, multiple conductance states with opening probabilities near 0.5, and unusual permeabilities to Na ϩ , Li ϩ , Ca 2ϩ , or Mg 2ϩ , along with K ϩ (7-9). In contrast, Miller's group (10, 11) using purified KcsA reconstituted into planar lipid bilayers found a single conductance state with a much lower opening probability, as well as orthodox ion selectivity and other properties to validate KcsA as a bona fide K ϩ channel and as a faithful structural model for these molecules. The above discrepancies were never fully explained but, still, it became generally accepted that KcsA behaves as a moderately voltage-dependent, K ϩ -selective channel with a characteristic low opening probability and the peculiar property of opening only in response to very acidic pH condit...
2,2,2-Trifluoroethanol (TFE) effectively destabilizes the otherwise highly stable tetrameric structure of the potassium channel KcsA, a predominantly alpha-helical membrane protein [Valiyaveetil, F. I., Zhou, Y., and MacKinnon, R. (2002) Biochemistry 41, 10771-10777]. Here, we report that the effects on the protein structure of increasing concentrations of TFE in detergent solution include two successive protein concentration-dependent, cooperative transitions. In the first of such transitions, occurring at lower TFE concentrations, the tetrameric KcsA simultaneously increases the exposure of tryptophan residues to the solvent, partly loses its secondary structure, and dissociates into its constituent subunits. Under these conditions, simple dilution of the TFE permits a highly efficient refolding and tetramerization of the protein in the detergent solution. Moreover, following reconstitution into asolectin giant liposomes, the refolded protein exhibits nativelike potassium channel activity, as assessed by patch-clamp methods. Conversely, the second cooperative transition occurring at higher TFE concentrations results in the irreversible denaturation of the protein. These results are interpreted in terms of a protein and TFE concentration-dependent reversible equilibrium between the folded tetrameric protein and partly unfolded monomeric subunits, in which folding and oligomerization (or unfolding and dissociation in the other direction of the equilibrium process) are seemingly coupled processes. At higher TFE concentrations this is followed by the irreversible conversion of the unfolded monomers into a denatured protein form.
Binding of K+ and Na+ to the potassium channel KcsA has been characterized from the stabilization observed in the heat-induced denaturation of the protein as the ion concentration is increased. KcsA thermal denaturation is known to include (i) dissociation of the homotetrameric channel into its constituent subunits and (ii) protein unfolding. The ion concentration-dependent changes in the thermal stability of the protein, evaluated as the Tm value for thermal-induced denaturation of the protein, may suggest the existence of both high- and low-affinity K+ binding sites of KcsA, which lend support to the tenet that channel gating may be governed by K+ concentration-dependent transitions between different affinity states of the channel selectivity filter. We also found that Na+ binds to KcsA with a KD similar to that estimated electrophysiologically from channel blockade. Therefore, our findings on ion binding to KcsA partly account for K+ over Na+ selectivity and Na+ blockade and argue against the strict “snug fit” hypothesis used initially to explain ion selectivity from the X-ray channel structure. Furthermore, the remarkable effects of increasing the ion concentration, K+ in particular, on the Tm of the denaturation process evidence that synergistic effects of the metal-mediated intersubunit interactions at the channel selectivity filter are a major contributor to the stability of the tetrameric protein. This observation substantiates the notion of a role for ions as structural “effectors” of ion channels.
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 KcsA⅐K ؉ and KcsA⅐Na ؉ complexes of ϳ8 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 KcsA⅐K ؉ 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 KcsA⅐Na ؉ and KcsA⅐K ؉ complexes, which for the most part should arise from synergistic effects of the ion-mediated intersubunit interactions.
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