Regulation Of Membrane Proteins Research Articles

How Lipids Regulate Membrane Protein Function

To what extent can our understanding of how water molecules interact with a water-soluble protein help us to understand how lipid molecules interact with a membrane protein? A first shell of water molecules is found covering the surface of a water-soluble protein, and water molecules are also found buried within the structure. Interactions of these water molecules with the protein help define its structure, and thus its function. Similarly, the surface of the transmembrane region of a membrane protein is covered with a first-shell of perturbed lipid molecules, referred to as the lipid annulus. Binding constants of lipids to these annular sites can be determined using a fluorescence quenching method, studying the quenching of the fluorescence of Trp residues in the protein by lipids with bromine-containing chains. Such studies show that the lipid annulus is heterogeneous - the mechanosensitive channel MscL, for example, contains a ‘hot-spot’ where anionic lipids bind with high affinity. Binding of anionic lipids to this hot-spot has a large effect on the flux through the MscL channel. Lipid molecules can also be found buried within the structure of a membrane protein, for example, at protein-protein interfaces in multimeric proteins. An example is provided by the homotetrameric potassium channel KcsA. The crystal structure of KcsA by MacKinnon shows an anionic lipid molecule bound at each monomer-monomer interface. Occupation of these sites by anionic lipid molecules is not required for tetramer formation, but is important for function. The open probability of the channel increases markedly with increasing anionic lipid content in the membrane, three of the four inter-subunit binding sites having to be occupied by anionic lipid for the channel to be open.

Hermansky-Pudlak Syndrome Protein Complexes Associate with Phosphatidylinositol 4-Kinase Type II α in Neuronal and Non-neuronal Cells

The Hermansky-Pudlak syndrome is a disorder affecting endosome sorting. Disease is triggered by defects in any of 15 mouse gene products, which are part of five distinct cytosolic molecular complexes: AP-3, homotypic fusion and vacuole protein sorting, and BLOC-1, -2, and -3. To identify molecular associations of these complexes, we used in vivo cross-linking followed by purification of cross-linked AP-3 complexes and mass spectrometric identification of associated proteins. AP-3 was co-isolated with BLOC-1, BLOC-2, and homotypic fusion and vacuole protein sorting complex subunits; clathrin; and phosphatidylinositol-4-kinase type II alpha (PI4KIIalpha). We previously reported that this membrane-anchored enzyme is a regulator of AP-3 recruitment to membranes and a cargo of AP-3 ( Craige, B., Salazar, G., and Faundez, V. (2008) Mol. Biol. Cell 19, 1415-1426 ). Using cells deficient in different Hermansky-Pudlak syndrome complexes, we identified that BLOC-1, but not BLOC-2 or BLOC-3, deficiencies affect PI4KIIalpha inclusion into AP-3 complexes. BLOC-1, PI4KIIalpha, and AP-3 belong to a tripartite complex, and down-regulation of either PI4KIIalpha, BLOC-1, or AP-3 complexes led to similar LAMP1 phenotypes. Our analysis indicates that BLOC-1 complex modulates the association of PI4KIIalpha with AP-3. These results suggest that AP-3 and BLOC-1 act, either in concert or sequentially, to specify sorting of PI4KIIalpha along the endocytic route.

Insulator Elements in Erythrocyte Membrane Genes

Insulators are DNA sequences and associated binding proteins that establish and/or maintain the boundaries between euchromatin and heterochromatin. One type of insulator establishes chromatin domains to separate enhancers and promoters and prevent their interaction (enhancer-blocking insulators) whereas another type creates a barrier to protect against heterochromatin-mediated gene silencing (barrier insulators). In the well-characterized chicken beta-globin LCR 5′HS4 insulator, binding of CTCF mediates enhancer-blocking functions and USF proteins mediate barrier activity. Because varying transcripts of erythrocyte membrane protein genes are expressed in both erythroid and nonerythroid cells, we hypothesized that that insulator elements that bind USF and CTCF participate in their regulation. Advances in technology have permitted rapid identification of DNA sequences bound by transcription factors and other DNA-associated proteins on a genome-wide scale. Coupling chromatin immunoprecipitation to microarrays that contain genomic regions (ChIP-chip) is a high resolution technique available for mapping protein-DNA interactions in vivo. We used ChIP-chip to identify CTCF and USF factor binding sites with potential insulator function that regulate membrane protein gene expression in erythroid cells. ChIP was performed with K562 and HeLa cells using antibodies against CTCF, USF1, and USF2. DNA obtained from these IPs was hybridized to a custom NimbleGen high-density human genomic DNA microarray. Chip probes were ~50bp in length, Tm ≥76°C, tiled ~65bp apart. Regions of repetitive DNA excluded. The chip included 15 erythrocyte membrane protein genes, most encoding complex loci with multiple tissue-, cell-, and developmental stage-specific transcripts, including alpha spectrin, beta spectrin, ankyrin, spectrin, band 3, ƒnalpha adducin, beta adducin, gamma adducin, ICAM4, erythroid associated membrane protein, protein 4.1R, protein 4.2, dematin, beta actin, tropomodulin, and tropomyosin. Each gene plus 50 to 100kb of flanking DNA were included on the chip. Binding sites on the custom DNA array were identified using the Tamalpais peak calling algorithm using L1–L3 level of stringency (Genom Res 16:595, 2006) or Tilescope using a signal cutoff of 0.9 (Genome Biol 8:R81, 2007). In K562 chromatin, 117 sites of CTCF occupancy were identified. There were 49 sites in 5′ flanking DNA, 3 in promoters, 9 in coding sequence, 20 in introns, 1 in 3′ untranslated region, and 35 in 3′ flanking DNA. Sites of USF1 and USF2 occupancy were identified in 7/15 and 9/15 genes, respectively. 15 USF1 sites were identified; 10 in 5′ flanking DNA, 1 in a promoter (<500bp from transcription start site), and 4 in 3′ flanking DNA. 22 sites of USF2 occupancy were identified; 9 in 5′ flanking DNA, 1 in a promoter, 4 in introns, and 8 in 3′ flanking DNA. USF1 and USF2 frequently heterodimerize and 10 sites bound both USF1 and USF2. To validate ChIP-chip results, a site binding USF2 near the ankyrin gene erythroid promoter was further analyzed. This site was chosen because the USF2-associated sequence directs uniform, copy-number dependent expression of a linked reporter gene in transgenic mice, the erythroid ankyrin promoter is positioned between 2 alternate, nonerythroid ankyrin promoters, and DNaseI mapping revealed that this region is contained within an erythroid-specific chromatin domain. Quantitative ChIP confirmed USF2 occupancy, as well as USF1 occupancy, in this region. For functional analysis, we used a gene silencing/position-effect variegation (PEV) assay to determine whether this region possess barrier element function. Using a HS2-beta globin promoter-GFP cassette as negative control and a cHS4-HS2-beta globin promoter-GFP-cHS4 cassette as positive control, the ankyrin promoter region fragment (p<0.001) functioned as barrier elements in this PEV assay. These data demonstrate that there are numerous binding sites for insulator-associated DNA binding proteins in erythrocyte membrane protein genes and indicate that these elements likely play a significant role in the regulation of tissue-specific expression of many genes expressed in erythroid cells.

Multiplex Kinase Signaling Modifies Cardiac Function at the Level of Sarcomeric Proteins

The transient change in tension development and length in a working cardiac myocyte during the heart beat reflects the integrated effects of kinases in signaling cascades regulating mechanisms controlling the dynamics and intensity of a transient increase in cytoplasmic Ca2+ as well as the responsiveness of the sarcomeres to Ca2+. Kinases modifying regulatory membrane proteins represent a major mechanism for controlling the coupling of transmembrane voltage to the release of Ca2+ into the cytoplasm that triggers contraction by binding to TnC2 and for regulating dynamics of the return of Ca2+ to the diastolic state by membrane transporters and exchangers (1). Binding of Ca2+ to TnC triggers a strong reaction of sarcomere thick filament cross-bridges with the thin filament actins and the promotion of force development and shortening (2, 3). Sarcomeres are not passive responders to these transient changes in cytoplasmic Ca2+. Protein-protein interactions downstream of Ca2+-TnC are subject to functionally significant modifications by signaling cascades that modify the number and kinetics of actin-cross-bridge reactions (Fig. 1). FIGURE 1. Kinases affecting sarcomeric proteins. Major substrates for these kinases are illustrated in a region of overlap between thin actin-containing and thick myosin-containing filaments. Also shown is a portion of the network of proteins at the Z-disk, ... I focus here on control mechanisms at the level of the sarcomere and on kinases immediately upstream of sarcomeric protein substrates. Major substrates are (i) thin filament proteins TnI, TnT, and Tm, which are important in transducing the Ca2+-TnC signal (4, 5); (ii) MyBP-C (6) and RLC (7), which control the radial movement of cross-bridges from the thick filament backbone; and (iii) titin, a giant third filament controlling diastolic tension as well as length-dependent radial movement of cross-bridges (8, 9). Detailed discussion of how phosphorylation modifies the function of these proteins has been reviewed elsewhere (2, 4–9). In general, phosphorylation of thin filament proteins controls sarcomere Ca2+ sensitivity, kinetics of Ca2+ binding to TnC (related to dynamics of relaxation), and the number and kinetics of cross-bridges reacting with the thin filament (related to levels and rates of rise and fall of tension). Phosphorylations of MyBP-C and RLC control Ca2+ sensitivity and rates of contraction/relaxation by modifying the local concentration of cross-bridges at the interface with actins. MyBP-C may also interact with and affect thin filament activation. Cardiac but not skeletal isoforms of titin contain phosphorylation sites within a unique sequence, located in the elastic segment. Phosphorylation of a unique cardiac titin reduces passive tension (8). To appreciate the potential role of how kinases modify sarcomeric function, it is important to consider the working cardiac myocyte operating in an environment influenced by immediate prevailing mechanical (load and length), neural, endocrine, autocrine, and paracrine control mechanisms and by the short- and long-term history of this environment. Beat-to-beat control mechanisms, which occur, for example, as hemodynamic load rises with exercise or falls with rest, are related to the immediate prevailing regulatory mechanisms. Mechanisms occurring over the time scale of hours, days, and longer are related to growth and remodeling in response to chronic changes in load or chemical environment as occur with sustained bouts of exercise, hypertension, or ischemia. Kinases and phosphorylations play a significant role in compensation and adaptation to beat-to-beat and chronic changes in hemodynamic load. However, maladaptive kinase activation may induce remodeling and phosphorylations of sarcomeric proteins with cardiovascular disorders, leading to heart failure (10, 11).

Membrane Protein Crry Maintains Homeostasis of the Complement System

Complement activation is tightly regulated to avoid excessive inflammatory and immune responses. Crry(-/-) is an embryonic lethal phenotype secondary to the maternal complement alternative pathway (AP) attacking a placenta deficient in this inhibitor. In this study, we demonstrate that Crry(-/-) mice could be rescued on a partial as well as on a complete factor B (fB)- or C3-deficient maternal background. The C3 and fB protein concentrations in Crry(-/-)C3(+/-) and Crry(-/-)fB(+/-) mice were substantially reduced for gene dosage secondary to enhanced AP turnover. Based on these observations, a breeding strategy featuring reduced maternal AP-activating capacity rescued the lethal phenotype. It led to a novel, stable line of Crry SKO mice carrying normal alleles for C3 and fB. Crry SKO mice also had accelerated C3 and fB turnover and therefore reduced AP- activating potential. These instructive results represent an example of a membrane regulatory protein being responsible for homeostasis of the complement system. They imply that there is constant turnover on cells of the AP pathway which functions as an immune surveillance system for pathogens and altered self.

The Ubiquitin-Protein Ligase Nedd4-2 Differentially Interacts with and Regulates Members of the Tweety Family of Chloride Ion Channels

The Tweety proteins comprise a family of chloride ion channels with three members identified in humans (TTYH1-3) and orthologues in fly and murine species. In humans, increased TTYH2 expression is associated with cancer progression, whereas fly Tweety is associated with developmental processes. Structurally, Tweety proteins are characterized by five membrane-spanning domains and N-glycan modifications important for trafficking to the plasma membrane, where these proteins are oriented with the amino terminus located extracellularly and the carboxyl terminus cytoplasmically. In addition to N-glycosylation, ubiquitination mediated by the HECT type E3 ubiquitin ligase Nedd4-2 is a post-translation modification important in regulating membrane proteins. In the present study, we performed a comprehensive analysis of the ability of each of TTYH1-3 to interact with Nedd4-2 and to be ubiquitinated and regulated by this ligase. Our data indicate that Nedd4-2 binds to two family members, TTYH2 and TTYH3, which contain consensus PY ((L/P)PXY) binding sites for HECT type E3 ubiquitin ligases, but not to TTYH1, which lacks this motif. Consistently, Nedd4-2 ubiquitinates both TTYH2 and TTYH3. Importantly, we have shown that endogenous TTYH2 and Nedd4-2 are binding partners and demonstrated that the TTYH2 PY motif is essential for these interactions. We have also shown that Nedd4-2-mediated ubiquitination of TTYH2 is a critical regulator of cell surface and total cellular levels of this protein. These data, indicating that Nedd4-2 differentially interacts with and regulates TTYH1-3, will be important for understanding mechanisms controlling Tweety proteins in physiology and disease.

Complement activation by malignant B cells from patients with chronic lymphocytic leukaemia (CLL).

It has previously been reported that the expression of the complement receptors CR1 (CD35) and CR2 (CD21) on malignant B cells in CLL is reduced compared with the expression on normal B cells, while deposition of complement C3 fragments, as a consequence of alternative pathway (AP) activation of complement, is observed on mononuclear cells from patients with B CLL. Following our demonstration that normal B cells are capable of activating the AP of complement in a CR2-dependent fashion, we have chosen to re-examine the complement-activating ability of B CLL cells in relation to their altered phenotype with respect to CR2 and the complement regulatory membrane proteins, CR1, decay accelerating factor (DAF) (CD55) and membrane cofactor protein (MCP) (CD46). Flow cytometry was used to measure expression of complement receptors and regulatory proteins on CD5+ B cells from CLL patients, as well as the deposition of C3 fragments occurring both in vivo and after in vitro AP activation. We have confirmed the reduced expression of CR1 and CR2 on CLL cells and have shown that AP activation in the presence of homologous, normal serum was reduced on B CLL cells compared with normal B cells. The degree of AP activation correlated directly with CR2 expression. In addition, we observed that CLL cells bear in vivo-deposited C3d,g, although at a significantly lower level than normal B cells.

Ezrin, Radixin, and Moesin (ERM) proteins function as pleiotropic regulators of human immunodeficiency virus type 1 infection

Ezrin, radixin, and moesin (ERM) proteins supply functional linkage between integral membrane proteins and cytoskeleton in mammalian cells to regulate membrane protein dynamisms and cytoskeleton rearrangement. To assess potential role of the ERM proteins in HIV-1 lifecycle, we examined if suppression of ERM function in human cells expressing HIV-1 infection receptors influences HIV-1 envelope (Env)-mediated HIV-1-vector transduction and cell–cell fusion. Expression of an ezrin dominant negative mutant or knockdown of ezrin, radixin, or moesin with siRNA uniformly decreased transduction titers of HIV-1 vectors having X4-tropic Env. In contrast, transduction titers of R5-tropic Env HIV-1 vectors were decreased only by radixin knockdown: ezrin knockdown had no detectable effects and moesin knockdown rather increased transduction titer. Each of the ERM suppressions had no detectable effects on cell surface expression of CD4, CCR5, and CXCR4 or VSV-Env-mediated HIV-1 vector transductions. Finally, the individual knockdown of ERM mRNAs uniformly decreased efficiency of cell–cell fusion mediated by X4- or R5-tropic Env and HIV-1 infection receptors. These results suggest that (i) the ERM proteins function as positive regulators of infection by X4-tropic HIV-1, (ii) moesin additionally functions as a negative regulator of R5-tropic HIV-1 virus infection at the early step(s) after the membrane fusion, and (iii) receptor protein dynamisms are regulated differently in R5- and X4-tropic HIV-1 infections.

SynechoNET: integrated protein-protein interaction database of a model cyanobacterium Synechocystis sp. PCC 6803

BackgroundCyanobacteria are model organisms for studying photosynthesis, carbon and nitrogen assimilation, evolution of plant plastids, and adaptability to environmental stresses. Despite many studies on cyanobacteria, there is no web-based database of their regulatory and signaling protein-protein interaction networks to date.DescriptionWe report a database and website SynechoNET that provides predicted protein-protein interactions. SynechoNET shows cyanobacterial domain-domain interactions as well as their protein-level interactions using the model cyanobacterium, Synechocystis sp. PCC 6803. It predicts the protein-protein interactions using public interaction databases that contain mutually complementary and redundant data. Furthermore, SynechoNET provides information on transmembrane topology, signal peptide, and domain structure in order to support the analysis of regulatory membrane proteins. Such biological information can be queried and visualized in user-friendly web interfaces that include the interactive network viewer and search pages by keyword and functional category.ConclusionSynechoNET is an integrated protein-protein interaction database designed to analyze regulatory membrane proteins in cyanobacteria. It provides a platform for biologists to extend the genomic data of cyanobacteria by predicting interaction partners, membrane association, and membrane topology of Synechocystis proteins. SynechoNET is freely available at or directly at .

Regulation of ion transport proteins by membrane phosphoinositides

Over the past decade, there has been an explosion in the number of membrane transport proteins that have been shown to be sensitive to the abundance of phosphoinositides in the plasma membrane. These proteins include voltage-gated potassium and calcium channels, ion channels that mediate sensory and nociceptive responses, epithelial transport proteins and ionic exchangers. Each of the regulatory lipids is also under multifaceted regulatory control. Phosphoinositide modulation of membrane proteins in neurons often has a dramatic effect on neuronal excitability and synaptic transmitter release. The repertoire of lipid signalling mechanisms that regulate membrane proteins is intriguingly complex and provides a rich array of topics for neuroscience research.

Membrane orientation of the Na,K-ATPase regulatory membrane protein CHIF determined by solid-state NMR

Corticosteroid hormone-induced factor (CHIF) is a major regulatory subunit of the Na,K-ATPase, and a member of an evolutionarily conserved family of membrane proteins that regulate the function of the enzyme complex in a tissue-specific and physiological-state-specific manner. Here we present the structure of CHIF oriented in the membrane, determined by solid-state NMR orientation-dependent restraints. Because CHIF adopts a similar structure in lipid micelles and bilayers, it is possible to assign the solid-state NMR spectrum measured for (15)N-labeled CHIF in oriented bilayers from the structure determined in micelles, to obtain the global orientation of the protein in the membrane.

Concentrative Export from the Endoplasmic Reticulum of the γ-Aminobutyric Acid Transporter 1 Requires Binding to SEC24D

Re-uptake of gamma-aminobutyric acid (GABA) into presynaptic specializations is mediated by the GABA transporter 1 (GAT1), a member of the SLC6 gene family. Here, we show that a motif in the COOH terminus of GAT1 ((566)RL(567)), which is conserved in SLC6 family members, is a binding site for the COPII coat component Sec24D. We also identified residues in Sec24D ((733)DD(734)) that are required to support the interaction with GAT1 and two additional family members, i.e. the transporters for serotonin and dopamine. We used three strategies to prevent recruitment of Sec24D to GAT1: knock-down of Sec24D by RNA interference, overexpression of Sec24D-VN (replacement of (733)DD(734) by (733)VN(734)), and mutation of (566)RL(567) to (566)AS(567) (GAT1-RL/AS). In each instance, endoplasmic reticulum (ER) export of GAT1 was impaired: in the absence of Sec24D or upon coexpression of dominant negative Sec24D-VN, GAT1 failed to undergo concentrative ER export; GAT1-RL/AS also accumulated in the ER and exerted a dominant negative effect on cell surface targeting of wild type GAT1. Our observations show that concentrative ER-export is contingent on a direct interaction of GAT1 with Sec24D; this also provides a mechanistic explanation for the finding that oligomeric assembly of transporters is required for their ER export: transporter oligomerization supports efficient recruitment of COPII components.

Palmitoylation-dependent protein sorting

S-palmitoylation is a posttranslational modification that regulates membrane–protein interactions. However, palmitate is more than just a hydrophobic membrane anchor, as many different types of protein are palmitoylated, including transmembrane proteins. Indeed, there is now compelling evidence that palmitoylation plays a key role in regulating various aspects of protein sorting within the cell.

GABAAReceptor Function is Regulated by Lipid Bilayer Elasticity

Docosahexaenoic acid (DHA) and other polyunsaturated fatty acids (PUFAs) promote GABA(A) receptor [(3)H]-muscimol binding, and DHA increases the rate of GABA(A) receptor desensitization. Triton X-100, a structurally unrelated amphiphile, similarly promotes [(3)H]-muscimol binding. The mechanism(s) underlying these effects are poorly understood. DHA and Triton X-100, at concentrations that affect GABA(A) receptor function, increase the elasticity of lipid bilayers measured as decreased bilayer stiffness using gramicidin channels as molecular force transducers. We have previously shown that membrane protein function can be regulated by amphiphile-induced changes in bilayer elasticity and hypothesized that GABA(A) receptors could be similarly regulated. We therefore studied the effects of four structurally unrelated amphiphiles that decrease bilayer stiffness (Triton X-100, octyl-beta-glucoside, capsaicin, and DHA) on GABA(A) receptor function in mammalian cells. All the compounds promoted GABA(A) receptor [(3)H]-muscimol binding by increasing the binding capacity of high-affinity binding without affecting the associated equilibrium binding constant. A semiquantitative analysis found a similar quantitative relation between the effects on bilayer stiffness and [(3)H]-muscimol binding. Membrane cholesterol depletion, which also decreases bilayer stiffness, similarly promoted [(3)H]-muscimol binding. In whole-cell voltage-clamp experiments, Triton X-100, octyl-beta-glucoside, capsaicin, and DHA all reduced the peak amplitude of the GABA-induced currents and increased the rate of receptor desensitization. The effects of the amphiphiles did not correlate with the expected changes in monolayer spontaneous curvature. We conclude that GABA(A) receptor function is regulated by lipid bilayer elasticity. PUFAs may generally regulate membrane protein function by affecting the elasticity of the host lipid bilayer.

Thioredoxin Target Proteins in Chloroplast Thylakoid Membranes

In recent years, impressive progress has been made in the identification of thioredoxin-linked proteins. However, due to technical difficulties inherent in working with hydrophobic proteins, identifications so far have been restricted to proteins in the soluble fraction. Thus, our knowledge of redox regulated membrane proteins is quite limited. To gain information in this area, the authors have applied an adaptation of the approach based on the fluorescent thiol probe monobromobimane (mBBr) to identify redox-linked proteins of chloroplast thylakoids. By application of this procedure, 14 potential membrane-bound thioredoxin target proteins were identified, including seven new candidates functional in processes associated with photosynthetic electron flow, ATP synthesis, and Photosystem II/Photosystem I state transitions.

The SIRP family of receptors and immune regulation

The immune system must be highly regulated to obtain optimal immune responses for the elimination of pathogens without causing undue side effects. This tight regulation involves complex interactions between membrane proteins on leukocytes. Members of the signal-regulatory protein (SIRP) family, which are expressed mainly by myeloid cells, provide one example of these regulatory membrane proteins. There are three SIRP-family genes that encode proteins that have similar extracellular regions but different signalling potentials, and are therefore known as 'paired receptors'. In this Review, we describe recent studies defining the ligands of the SIRP-family members, with particular emphasis on relating the molecular interactions of these proteins to their role in immune-cell regulation.

Distinct modes of regulated receptor insertion to the somatodendritic plasma membrane

Many neural signaling receptors are regulated by endocytosis, but little is known about receptor insertion into the plasma membrane. Time-lapse imaging of the beta2 adrenergic receptor expressed in cultured rat hippocampal neurons, using pH-sensitive green fluorescent protein tagging and total internal reflection fluorescence microscopy, resolved distinct vesicular fusion events mediating receptor insertion into the somatodendritic plasma membrane. A 'transient' insertion mode resulted in rapid lateral dispersion of receptors immediately after insertion. A 'persistent' insertion mode resulted in the retention of inserted receptors in surface-accessible domains, which were relatively immobile for a prolonged 'wait' period before dispersing laterally. Distinct insertion modes were oppositely regulated by receptor activation and by mechanisms differing in their dependence on the signaling effector cyclic AMP-dependent protein kinase. These results reveal a new mechanism for homeostatic regulation of postsynaptic signaling and a 'kiss-and-wait' mode of regulated membrane protein insertion in neurons.

Allosteric receptors after 30 years

The history of the allosteric concept is traced from its origins in studies on regulatory enzymes, repressors, membrane proteins, and hemoglobin to its recent applications concerning ionotropic and metabotropic membrane receptors. The principles common to all systems are reviewed, including conformational transitions between symmetric (or pseudo-symmetric) states. Novel features unanticipated by the 1965 Monod-Wyman-Changeux (MWC) model involve (a) ligand binding sites at interfaces between subunits, as found in several classes of allosteric enzymes and pentameric ligand-gated receptors; and (b) effector molecules bound asymmetrically along an axis of symmetry, as in the case of the binding of 2,3-diphosphoglyerate to hemoglobin or the binding of various channel blockers to ionotropic receptors. Special attention is given to the nicotinic receptor, which been extensively characterized with respect to both structure and function. In particular, the roles of natural mutations (congenital myasthenic syndromes and autosomal dominant nocturnal frontal lobe epilepsies) and site-directed mutations in studying the mechanisms of activation and desensitization are reviewed, including the insights derived from constitutively active receptors and gain-of-function mutations. The extents to which these phenomena are consistent with the MWC-type models are considered and compared with «intermediate-state» models.

Phospholemman

See related article, pages 558–565 The control of intracellular Na+ levels has long been known to be a crucial part of the regulation of cardiac contractility and the treatment of heart failure. Cardiac glycosides have been used to improve the symptoms of heart failure since William Withering published trials of a foxglove extract obtained from a gypsy woman in the late 1700’s. The active ingredients, digitalis and digoxin, were found to target sodium pump activity in the 1950’s, and the sodium pump was identified as Na,K-ATPase by Nobel Laureate Jens Skou in the 1970’s. Recent studies have established that digitalis does not improve survival in heart failure, but American College of Cardiology/American Heart Association guidelines recommend its use in combination with ACE inhibitors and β-adrenergic blockade in patients with symptomatic left ventricular systolic dysfunction. A modest level of inhibition of Na,K-ATPase by digitalis slightly raises cardiac intracellular Na+ concentrations, which in turn decreases the driving force for Ca2+ extrusion via NCX1, the Na+:Ca2+ exchanger. In the therapeutic range, the extra cytoplasmic Ca2+ will be loaded into the sarcoplasmic reticulum, resulting in normal diastolic Ca2+ concentrations but improved systolic Ca2+ release, and thus improved contractility. β-adrenergic stimulation, on the other hand, improves the loading of the sarcoplasmic reticulum (SR) by acting on the Ca2+-ATPase, SERCA. β-adrenergic stimulation improves SR loading by phosphorylating a small regulatory membrane protein, phospholamban, which otherwise acts to reduce the …

Capsaicin Regulates Voltage-Dependent Sodium Channels by Altering Lipid Bilayer Elasticity

At submicromolar concentrations, capsaicin specifically activates the TRPV1 receptor involved in nociception. At micro- to millimolar concentrations, commonly used in clinical and in vitro studies, capsaicin also modulates the function of a large number of seemingly unrelated membrane proteins, many of which are similarly modulated by the capsaicin antagonist capsazepine. The mechanism(s) underlying this widespread regulation of protein function are not understood. We investigated whether capsaicin could regulate membrane protein function by changing the elasticity of the host lipid bilayer. This was done by studying capsaicin's effects on lipid bilayer stiffness, measured using gramicidin A (gA) channels as molecular force-transducers, and on voltage-dependent sodium channels (VDSC) known to be regulated by bilayer elasticity. Capsaicin and capsazepine (10-100 microM) increase gA channel appearance rate and lifetime without measurably altering bilayer thickness or channel conductance, meaning that the changes in bilayer elasticity are sufficient to alter the conformation of an embedded protein. Capsaicin and capsazepine promote VDSC inactivation, similar to other amphiphiles that decrease bilayer stiffness, producing use-dependent current inhibition. For capsaicin, the quantitative relation between the decrease in bilayer stiffness and the hyperpolarizing shift in inactivation conforms to that previously found for other amphiphiles. Capsaicin's effects on gA channels and VDSC are similar to those of Triton X-100, although these amphiphiles promote opposite lipid monolayer curvature. We conclude that capsaicin can regulate VDSC function by altering bilayer elasticity. This mechanism may underlie the promiscuous regulation of membrane protein function by capsaicin and capsazepine-and by amphiphilic drugs generally.