Lipid Bilayer Interactions Research Articles

Method to measure strong protein–protein interactions in lipid bilayers using a steric trap

Measuring high affinity protein-protein interactions in membranes is extremely challenging because there are limitations to how far the interacting components can be diluted in bilayers. Here we show that a steric trap can be employed for stable membrane interactions. We couple dissociation to a competitive binding event so that dissociation can be driven by increasing the affinity or concentration of the competitor. The steric trap design used here links monovalent streptavidin binding to dissociation of biotinylated partners. Application of the steric trap method to the well-characterized glycophorin A transmembrane helix (GpATM) reveals a dimer that is dramatically stabilized by 4-5 kcal/mol in palmitoyloleoylphosphatidylcholine bilayers compared to detergent. We also find larger effects of mutations at the dimer interface in bilayers compared to detergent suggesting that the dimer is more organized in a membrane environment. The high affinity we measure for GpATM in bilayers indicates that a membrane vesicle many orders of magnitude larger than a bacterial cell would be required to measure the dissociation constant using traditional dilution methods. Thus, steric trapping can open new biological systems to experimental scrutiny in natural bilayer environments.

Ethanol effects on binary and ternary supported lipid bilayers with gel/fluid domains and lipid rafts

Ethanol–lipid bilayer interactions have been a recurrent theme in membrane biophysics, due to their contribution to the understanding of membrane structure and dynamics. The main purpose of this study was to assess the interplay between membrane lateral heterogeneity and ethanol effects. This was achieved by in situ atomic force microscopy, following the changes induced by sequential ethanol additions on supported lipid bilayers formed in the absence of alcohol. Binary phospholipid mixtures with a single gel phase, dipalmitoylphosphatidylcholine (DPPC)/cholesterol, gel/fluid phase coexistence DPPC/dioleoylphosphatidylcholine (DOPC), and ternary lipid mixtures containing cholesterol, mimicking lipid rafts (DOPC/DPPC/cholesterol and DOPC/sphingomyelin/cholesterol), i.e., with liquid ordered/liquid disordered (ld/lo) phase separation, were investigated. For all compositions studied, and in two different solid supports, mica and silicon, domain formation or rearrangement accompanied by lipid bilayer thinning and expansion was observed. In the case of gel/fluid coexistence, low ethanol concentrations lead to a marked thinning of the fluid but not of the gel domains. In the case of ld/lo all the bilayer thins simultaneously by a similar extent. In both cases, only the more disordered phase expanded significantly, indicating that ethanol increases the proportion of disordered domains. Water/bilayer interfacial tension variation and freezing point depression, inducing acyl chain disordering (including opening and looping), tilting, and interdigitation, are probably the main cause for the observed changes. The results presented herein demonstrate that ethanol influences the bilayer properties according to membrane lateral organization.

Using Microcantilevers to Study the Interactions of Lipid Bilayers with Solid Surfaces

We report the use of free-standing microcantilever beams, which have been used as an ultrasensitive method for measuring the surface free energy changes on a substrate induced by the adsorption of thin films, to probe the interactions between a solid surface and a phospholipid bilayer. We relate the observed deflection of a cantilever to the changes in the surface free energy of the solid surface which supports the phospholipid bilayer. We observe that the deflection is influenced by electrostatic and intermolecular interactions of the bilayer with the substrate. Increasing the surface charge density in the supported lipid bilayer (SLB), by increasing the ratio of cationic to zwitterionic lipids in bilayer, resulted in an increase in cantilever deflection. The surface free energy changes due to lipid transfer between anionic unilamellar vesicles and a cationic supported bilayer were also observed using microcantilevers. Finally, the adsorption free energy of a mixed lipid and cholesterol bilayer was measured demonstrating a detectable decrease in affinity between the phospholipid bilayer and the solid surface as a result of the addition of cholesterol. Our results reveal a new technique to probe the adsorption free energy of a SLB system as a function of the interactions governing the structure of supported lipid membranes.

TARP Phosphorylation Regulates Synaptic AMPA Receptors through Lipid Bilayers

Neurons use neurotransmitters to communicate across synapses, constructing neural circuits in the brain. AMPA-type glutamate receptors are the predominant excitatory neurotransmitter receptors mediating fast synaptic transmission. AMPA receptors localize at synapses by forming protein complexes with transmembrane AMPA receptor regulatory proteins (TARPs) and PSD-95-like membrane-associated guanylate kinases. Among the three classes of ionotropic glutamate receptors (AMPA, NMDA, and kainate type), AMPA receptor activity is most regulatable by neuronal activity to adjust synaptic strength. Here, we mutated the prototypical TARP, stargazin, and found that TARP phosphorylation regulates synaptic AMPA receptor activity in vivo. We also found that stargazin interacts with negatively charged lipid bilayers in a phosphorylation-dependent manner and that the lipid interaction inhibited stargazin binding to PSD-95. Cationic lipids dissociated stargazin from lipid bilayers and enhanced synaptic AMPA receptor activity in a stargazin phosphorylation-dependent manner. Thus, TARP phosphorylation plays a critical role in regulating AMPA receptor-mediated synaptic transmission via a lipid bilayer interaction.

Effects of cholesterol on thermal stability of discoidal high density lipoproteins

Reverse cholesterol transport in plasma involves variations in HDL cholesterol concentration. To understand physicochemical and functional implications of such variations, we analyzed stability of reconstituted HDL containing human apolipoproteins (apoA-I, apoA-II, or apoC-I), phosphatidylcholines varying in chain length (12-18 carbons) and unsaturation (0 or 1), and 0-35 mol% cholesterol. Lipoprotein heat denaturation was monitored by circular dichroism for protein unfolding/dissociation and by light scattering for particle fusion. We found that cholesterol stabilizes relatively unstable complexes; for example, incorporation of 10-30 mol% cholesterol in apoC-I:dimyristoyl phosphatidylcholine complexes increased their kinetic stability by deltaDeltaG* congruent with 1 kcal/mol. In more stable complexes containing larger proteins and/or longer-chain lipids, incorporation of 10% cholesterol did not significantly alter the disk stability; however, 15% or more cholesterol destabilized the apoA-I-containing complexes and led to vesicle formation. Thus, cholesterol tends to stabilize less stable lipoproteins, apparently by enhancing favorable packing interactions, but in more stable lipoproteins, where such interactions are already highly optimized, the stabilizing effect of cholesterol decreases and, eventually, becomes destabilizing. These results help uncouple the functional roles of particle stability and chain fluidity and suggest that structural disorder in HDL surface, rather than chain fluidity, is an important physicochemical determinant of HDL function.

Measuring the Thermodynamic Stability of Strong Protein-Protein Interactions in Lipid Bilayers Using a Steric Trap

Protein-protein interactions within cell membranes play crucial roles in the assembly of membrane proteins and cell signaling. Although thermodynamic analysis of binding affinity is essential for understanding the stability, specificity, and function of membrane proteins, these measurements can be difficult to make for high affinity interactions in lipid bilayers. To address this problem, we have developed a steric trap method, which couples the dissociation of a membrane protein complex to another measurable binding event. The method postulates that a concomitant binding of two bulky monovalent streptavidins (mSA) to a doubly biotinylated protein complex occur only when the protein is dissociated due to the steric hindrance. This leads to an attenuated binding affinity of the second mSA, which is directly correlated to the stability of a target interaction. We tested the method using a glycophorinA transmembrane domain fusion to staphylococcal nuclease (SNGpA), which forms a stable dimer in various lipid environments. Equilibrium binding of mSA to the enzymatically biotinylated SNGpA exhibited two distinctive phases, which corresponds to the tight first mSA binding and the weaker second binding in decyl maltoside (DM) micelles and palmitoyloleoyl phophatidylcholine (POPC) bilayers. The stability of GpA dimer extracted from the second binding event at different micellar concentrations yielded the dissociation constants (Kd) of 10−8∼10−7 M, which agree well with the previous results. The stability of GpA dimer is enhanced in POPC bilayers by ∼4 orders of magnitude at comparable mole fractions. The difference free energies between wild-type and destabilized mutants in both systems correlate with the equilibrium sedimentation data measured in C8E5 micelles. Our results suggest that the steric trap method provides a powerful tool to study the strong protein-protein interactions in lipid bilayers.

Cholesterol-Rich Fluid Membranes Solubilize Ceramide Gel Domains. Implications for the Organization of Mammalian Membranes

A uniquely sensitive method for ceramide-domain detection allowed us to study in detail cholesterol-ceramide interactions in lipid bilayers with low (physiological) ceramide (Cer) concentrations, and ranging from low or no cholesterol (Chol) (a situation similar to intracellular membranes, such as endoplasmic reticulum) to high Chol, (similar to mammalian plasma membrane). Fluorescence spectroscopy and microscopy experiments were conducted showing that for low Chol amounts Cer segregates into gel domains that disappear upon increasing Chol levels. This was observed in raft (sphingomyelin/Chol-containing) and non-raft (sphingomyelin-absent) membranes, i.e. mimicking different types of cell membranes. Chol-Cer interactions have been described mainly as raft sphingomyelin-dependent. In this work, sphingomyelin independence is demonstrated. Moreover, we show that Cer-rich domains re-appear when either Chol is converted by cholesterol oxidase to cholestenone, or temperature is decreased. The inability of cholestenone-rich membranes to dissolve Cer-gel domains shows that the cholesterol ordering and packing properties are fundamental to the mixing process. Cer solubility is dependent on the average gel-fluid transition temperature of the remaining membrane lipids, and is higher in Chol-rich fluid membranes than in Chol-poor ones. We also show that the solubility of Chol in Cer domains is low. The results are rationalized by a ternary phospholipid/ ceramide/ cholesterol phase diagram, providing the framework for a better understanding of biochemical phenomena modulated by Chol-Cer interactions such as cholesterol oxidase activity, lipoprotein metabolism and lipid targeting in cancer therapy. It also suggests that the lipid compositions of different organelles are such that ceramide gel domains are not formed, unless a stress or pathological situation occurs.Further details in Castro, BM, Silva, LC, Fedorov, A, de Almeida, RFM, and Prieto, M (2009). J. Biol. Chem.284 (5), 22978-22987.FCT (Portugal) is acknowledged for research grants and scholarships.

Multi-Scale Dynamics of Rhodopsin Activation as a Paradigm for GPCR Function

G-protein coupled receptors (GPCRs) are membrane proteins that act as signaling cascade initiators responsible for a myriad of cellular processes. Ligand binding causes GPCR conformational changes that allow the receptor to interact with its cognate G-protein. Several techniques have structurally characterized rhodopsin photointermediates, but none have directly revealed the protein dynamics.Here we report a model whereby ps-ns ligand dynamics are coupled to the μs-ms protein motions during activation. These protein motions represent activated conformational substates on a hierarchy of time scales. Previous models propose that rhodopsin activation is a simple switch whereby retinal isomerizes from an 11-cis to an all-trans conformer, transforming from an inverse agonist to an agonist. In contrast, our model is motivated by FTIR and UV-visible results showing thermodynamic coupling to several substates in rhodopsin activation (metaI, metaIIa, and metaIIb) [1]. Furthermore, new 2H NMR data from selectively labeled retinal ligands bound to rhodopsin are able to show that each retinylidene methyl group, especially the C9-methyl, acts as a dynamical hotspot in the activation pathway [2]. Relaxation times are fitted to three-fold jump and continuous diffusion models and correlated to methyl rotation rates in the different rhodopsin activation states, revealing distinct site-specific characteristics for each photointermediate. Recent solid-state NMR [3] and EPR [4] studies showed appreciable protein movements in the photointermediate pathway, further supporting our data. An activation mechanism emerges whereby conformational substates depend on a multivariate energy landscape encompassing retinal and protein dynamics as well as lipid bilayer interactions. [1] M. Mahlingam et al. (2008) PNAS105, 17795-17800. [2] M.F. Brown et al. (2009) BBA, in press. [3] S. Ahuja et al. (2009) J. Biol. Chem.284, 10190-10201. [4] C. Altenbach et al. (2008) PNAS105, 7439-7444.

Surface Electrostatics and Lipid-Substrate Interactions of Nanopore-Confined Lipid Bilayers

Substrate-supported lipid bilayers serve many purposes: from acting as versatile models of cellular membranes to biotechnological applications including substrate functionalization and stabilizing membrane proteins in functional conformations. While adsorption and subsequent reorganization of phospholipid vesicles on solid substrates were studied in the past, the exact nature of physicochemical interactions between the lipids and substrate surfaces remain largely unknown. Here we employed recently synthesized pH-sensitive spin-labeled phospholipids - derivatives of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (PTE) with pH-reporting nitroxides that are covalently attached to the lipid's headgroup - to investigate surface electrostatics of nanotubular lipid bilayers confined in cylindrical nanopores. The lipid nanotubes were formed by self-assembling phospholipids inside ordered nanochannels of anodic aluminum oxide with pore diameters from 60 to 170 nm and diameter-to-pore length ratio of up to 1:1000. 31P NMR confirmed formation of macroscopically aligned lipid nanotubes with just 1-2° mosaic spread from zwitterionic DMPC, anionic DMPG, and their mixtures. Interfacial potentials were measured by carrying out titration experiments and observing the protonation state of the nitroxide tag by EPR. For nanopore-confined DMPC:DMPG (1:1) bilayers the protonation equilibrium was shifted to more acidic values: when the single lipid bilayer was deposited per nanopore the pKa of the nitroxide probe was shifted by (−0.91±0.05) pH units but only by (−0.34±0.05) when three bilayers per nanopore were present. Notably, the nitrogen hyperfine coupling constant for non-protonated nitroxides remained the same in all the samples indicating essentially the same interfacial dielectric environment. Thus, these shifts in pKa must come from changes in the lipid bialyer surface potential that was estimated to increase by 52±3 mV. EPR data on the lipid-substrate interface were combined with differential scanning calorimetry to elucidate effects of pore curvature, surface modification, and binding of antibacterial peptides on lipid-substrate interactions. Supported by DE-FG02-02ER15354.

Combined QCM-D and EIS study of supported lipid bilayer formation and interaction with pore-forming peptides

A novel set-up combining the quartz crystal microbalance with dissipation monitoring technique (QCM-D) and electrochemical impedance spectroscopy (EIS) under flow conditions was successfully used to follow supported lipid bilayer (SLB) formation on SiO(2). This study demonstrates the simultaneous detection, in real time, of both the electrical and the structural properties of the SLB. The combination of the two techniques provided novel insights regarding the mechanism of SLB formation: we found indications for an annealing process of the lipid alkyl chains after the mass corresponding to complete bilayer coverage had been deposited. Moreover, the interaction of the SLB with the pore-forming toxin, gramicidin D (grD) was studied for grD concentrations ranging from 0.05 to 40 mg L(-1). Membrane properties were altered depending on the toxin concentration. For low grD concentrations, the electrical properties of the SLB changed upon insertion of active ion channels. For higher concentrations, the QCM-D data showed dramatic changes in the viscoelastic properties of the membrane while the EIS spectra did not change. AFM confirmed significant structural changes of the membrane at higher grD concentrations. Thus, the application of combined QCM-D and EIS detection provides complementary information about the system under study. This information will be particularly important for the continued detailed investigation of interactions at model membrane surfaces.

Membrane Curvature Modeling and Lipid Organization in Supported Lipid Bilayers

Membranes in the cell exist in a wide range of shapes and provide for compartmentalization and transport throughout the cell. Curvature plays an important role in this cellular organization and even the organization of lipids within the membrane itself. Supported lipid bilayers (SLB) continue to be an important means of measuring the thermodynamic and mechanical properties of phospholipid membranes, but on some supports, the proximity of the solid surface may modify the behavior of the adsorbed bilayer. To overcome this problem, we use a technique for spin coating lipids on the substrate that creates multilamellar stacks of membranes[1] where the influence of the substrate on upper layers is weakened. The substrates we have used are nanoscopically patterned with steps and these features induce curvature in the membranes that appear to be step-height dependent. This provides a platform for adhesion, mobility and organizational studies. We show that multilamellar SLB on patterned substrates exhibit curvature induced phase separated domain organization and increased lateral lipid mobility. Molecular dynamics of coarse-grained supported lipid bilayers[2] are used to simulate membranes supported on corrugated surfaces and we discuss and compare the behavior with experimental systems. We show that substrate corrugation height, adhesion energy, and mechanical moduli can be controlled to predict adsorbed membrane curvature. Furthermore we model lipid mixtures in the regions of substrate induced curvature to show the relationship between bending energies and phase separation. [1] M.H. Jensen, E.J. Morris, A.C. Simonsen, Domain Shapes, Coarsening, and Random Patterns in Ternary Membranes, Langmuir 23 (2007) 8135-8141. [2] M.I. Hoopes, M. Deserno, M.L. Longo, R. Faller, Coarse-grained modeling of interactions of lipid bilayers with supports, The Journal of Chemical Physics 129 (2008) 175102-175107.

Cholesterol-rich Fluid Membranes Solubilize Ceramide Domains: IMPLICATIONS FOR THE STRUCTURE AND DYNAMICS OF MAMMALIAN INTRACELLULAR AND PLASMA MEMBRANES

A uniquely sensitive method for ceramide domain detection allowed us to study in detail cholesterol-ceramide interactions in lipid bilayers with low (physiological) ceramide concentrations, ranging from low or no cholesterol (a situation similar to intracellular membranes, such as endoplasmic reticulum) to high cholesterol (similar to mammalian plasma membrane). Diverse fluorescence spectroscopy and microscopy experiments were conducted showing that for low cholesterol amounts ceramide segregates into gel domains that disappear upon increasing cholesterol levels. This was observed in different raft (sphingomyelin/cholesterol-containing) and non-raft (sphingomyelin-absent) membranes, i.e. mimicking different types of cell membranes. Cholesterol-ceramide interactions have been described mainly as raft sphingomyelin-dependent. Here sphingomyelin independence is demonstrated. In addition, ceramide-rich domains re-appear when either cholesterol is converted by cholesterol oxidase to cholestenone or the temperature is decreased. Ceramide is more soluble in cholesterol-rich fluid membranes than in cholesterol-poor ones, thereby increasing the chemical potential of cholesterol. Ceramide solubility depends on the average gel-fluid transition temperature of the remaining membrane lipids. The inability of cholestenone-rich membranes to dissolve ceramide gel domains shows that the cholesterol ordering and packing properties are fundamental to the mixing process. We also show that the solubility of cholesterol in ceramide domains is low. The results are rationalized by a ternary phospholipid/ceramide/cholesterol phase diagram, providing the framework for the better understanding of biochemical phenomena modulated by cholesterol-ceramide interactions such as cholesterol oxidase activity, lipoprotein metabolism, and lipid targeting in cancer therapy. It also suggests that the lipid compositions of different organelles are such that ceramide gel domains are not formed unless a stress or pathological situation occurs.

Characterization of the Dynamic Structural Changes of Melittin - Lipid Bilayer Interactions

Melittin, the soluble peptide of bee venom, has been demonstrated to induce lysis of phospholipid liposomes. We have previously explored the dependence of lysis on liposome composition and now explore the interaction in more structural detail at the level of the lipid bilayer. Supported Lipid Bilayers are probed optically using the waveguide technique Dual Polarization Interferometry (DPI) to obtain data on the mass and birefringence of the lipid bilayer structures. The birefringence is a measure of the degree of alignment and compression of the lipid tails and is highly sensitive to changes of ordering within the membrane. The interaction of the bilayers with Melittin is probed as a function of peptide concentration and the resultant mass and birefringence changes related to the interaction mechanism. For the zwitterionic phosphatidyl choline the lytic ability of melittin is dependent on the degree of acyl chain mobility, with melittin able to induce lysis of liposomes in the liquid crystalline state, whilst those in the gel state show strong resistance to lysis. Thus the interaction of Melittin with Dimyristoyl-Glycero-Phospho-choline (DMPC) lipid bilayers is probed both above and below its transition temperature.

Orientational Order and Raft Interactions in Lipid Bilayers

Model and reconstituted membranes undergo a mixing-demixing phase transition, and, at low temperatures, the membrane is often a two-dimensional emulsion of liquid ordered and liquid disordered phases. This phase separation may be important for many functions of cellular membranes, including vesicle trafficking and signaling, and it has been implicated in a number of human diseases. We describe here the effects of orientational order (tilt, nematic, or hexatic) in one of the low-temperature phases on the behavior of the two-dimensional emulsion. We found that the orientational order in the continuous component of the emulsion can lead to the formation of companion singularities in the order parameter around the inclusions of isotropic liquid disordered phase. The orientational order parameter and strong anchoring boundary conditions also give rise to long-range interactions between the inclusions. The interaction is attractive at large separations and is repulsive at short separations. This interaction stabilizes the emulsion and leads to the formation of inclusion dimers. The sizes of the dimers depend only on the type of the orientational order and the sizes of the inclusions; hence, our calculation of this size can be used to test for the presence of a hidden order parameter in liquid ordered phases. The behavior in the presence of strong thermal fluctuations will be discussed as well.

Coarse-grained modeling of interactions of lipid bilayers with supports

We characterize the differences between supported and unsupported lipid bilayer membranes using a mesoscopic simulation model and a simple particle-based realization for a flat support on to which the lipids are adsorbed. We show that the nanometer roughness of the support affects membrane binding strength very little. We then compare the lipid distributions and pressure profiles of free and supported membranes. The surface localization of the proximal leaflet breaks the symmetry seen in a free bilayer, and we quantify the entropic penalty for binding and the increased lateral compression modulus.

The Role of Lipids for the Functional Integrity of Porin: An FTIR Study Using Lipid and Protein Reporter Groups

We have investigated the temperature-dependent interaction of the porins OmpF from Paracoccus denitrificans and OmpG from Escherichia coli with lipid molecules after reconstitution in lecithin. Effects of incubation at increased temperatures on activity were tested by functional experiments for OmpG and compared with previously published results of OmpF in order to understand the activity loss of OmpF with monomerization. Protein-lipid interaction was monitored by different reporter groups both from lipid molecules and from protein. OmpF loses its activity by approximately 90% at 50 degrees C while OmpG does not show a temperature-dependent change in activity between room temperature and 50 degrees C. The interaction between OmpF and lipid molecules is severely altered in a two-step mechanism at 55 and approximately 75 degrees C for OmpF. The first step is attributed to changes in the degree of interaction between the aromatic girdle of OmpF and the interfacial region of the lipid bilayer, leading to monomerization of this trimeric porin. The second step at 75 degrees C is attributed to the changes in lipid-porin monomer interaction. Around 90 degrees C, reconstituted porin aggregates. For OmpG, changes in lipid-protein interaction were observed starting from approximately 80 degrees C because of temperature-induced breakdown of its folding. This study provides deeper understanding of porin-lipid bilayer interaction as a function of temperature and can explain the functional breakdown by monomerization while porin secondary structure is still preserved.

Interactions of Lipid Bilayers with Supports: A Coarse-Grained Molecular Simulation Study

The study of lipid structure and phase behavior at the nanoscale is of utmost importance due to implications in understanding the role of the lipids in biochemical membrane processes. Supported lipid bilayers play a key role in understanding real biological systems, but they are vastly underrepresented in computational studies. In this paper, we discuss molecular dynamics simulations of supported lipid bilayers using a coarse-grained model. We first focus on the technical implications of modeling solid supports for biomembrane simulations. We then describe noticeable influences of the support on the systems. We are able to demonstrate that the bilayer system behavior changes when supported by a hydrophilic surface. We find that the thickness of the water layer between the support and the bilayer (the inner-water region in the latter part of this paper) adapts through water permeation on the microsecond time scale. Additionally, we discuss how different surface topologies affect the bilayer. Finally, we point out the differences between the two leaflets induced by the support.

The RGS2 Gene Product from a Candidate Hypertension Allele Shows Decreased Plasma Membrane Association and Inhibition of Gq

Hypertension is a leading risk factor for the development of cardiovascular disease. Data from human and animal studies suggest that RGS2, a potent inhibitor of G(q) signaling, is important for blood pressure regulation. Several RGS2 mutations in the Japanese population have been found to be associated with hypertension. The product of one of these alleles, R44H, is mutated within the amino terminal amphipathic alpha-helix domain, the region responsible for plasma membrane-targeting. The functional consequence of this mutation and its potential link to the development of hypertension, however, are not known. In this study, we showed that R44H was a weaker inhibitor of receptor-mediated G(q) signaling than wild-type RGS2. Confocal microscopy revealed that YFP-tagged R44H bound to the plasma membrane less efficiently than wild-type RGS2. R44 is one of the basic residues positioned to stabilize lipid bilayer interaction of the RGS2 amphipathic helix domain. Tryptophan fluorescence and circular dichroism studies of this domain showed that the R44H mutation prevented proper entrenchment of hydrophobic residues into the lipid bilayer without disrupting helix-forming capacity. Together, these data suggest that decreasing the side-chain length and flexibility at R44 prevented proper lipid bilayer association and function of RGS2. Finally, the R44H protein did not behave as a dominant-negative interfering mutant. Thus, our data are consistent with the notion that a R44H missense mutation in human RGS2 produces a hypomorphic allele that may lead to altered receptor-mediated G(q) inhibition and contribute to the development of hypertension in affected subjects.

Glycine residues G338 and G342 are important determinants for serotonin transporter dimerisation and cell surface expression

Compelling evidence has been provided that Na + and Cl −-dependent neurotransmitter transporter proteins form oligomeric complexes. Specific helix–helix interactions in lipid bilayers are thought to promote the assembly of integral membrane proteins to oligomeric structures. These interactions are determined by selective transmembrane helix packing motifs one of which is the Glycophorin A motif (GxxxG). This motif is present in the sixth transmembrane domain of most transporter proteins. In order to investigate, whether this motif is important for proper expression and function of the serotonin transporter (SERT), we have analysed the effect of mutating the respective glycine residues Gly338 and Gly342 to valine upon transient expression of the respective cDNAs in HEK293 cells. As revealed by western blotting, wildtype SERT is found in monomeric and dimeric forms while both mutants are expressed as monomers solely. Confocal microscopy revealed that the wildtype SERT is expressed at the cell surface, whereas both mutant proteins are localised in intracellular compartments. Failure of integration into the cell membrane is responsible for a total loss of [ 3H]5HT uptake capability by the mutants. These findings show that in the SERT protein the integrity of the GxxxG motif is essential for dimerisation and proper targeting of the transporter complex to the cell surface.

Is Lipid Bilayer Binding a Common Property of Inhibitor Cysteine Knot Ion-Channel Blockers?

Recent studies of several ICK ion-channel blockers suggest that lipid bilayer interactions play a prominent role in their actions. Structural similarities led to the hypothesis that bilayer interactions are important for the entire ICK family. We have tested this hypothesis by performing direct measurements of the free energy of bilayer partitioning (Δ G) of several peptide blockers using our novel quenching-enhanced fluorescence titration protocol. We show that various ICK peptides demonstrate markedly different modes of interaction with large unilamellar lipid vesicles. The mechanosensitive channel blocker, GsMTx4, and its active diastereomeric analog, D-GsMTx4, bind strongly to both anionic and zwitterionic membranes. One potassium channel gating modifier, rHpTx2gs, interacts negligibly with both types of vesicles at physiological pH, whereas another, SGTx1, interacts only with anionic lipids. The slope of Δ G dependence on surface potential is very shallow for both GsMTx4 and D-GsMTx4, indicating complex interplay of their hydrophobic and electrostatic interactions with lipid. In contrast, a cell-volume regulator, GsMTx1, and SGTx1 exhibit a very steep Δ G dependence on surface potential, resulting in a strong binding only for membranes rich in anionic lipids. The high variability of 5 kcal/mole in observed Δ G shows that bilayer partitioning is not a universal property of the ICK peptides interacting with ion channels.