Membrane Binding Assays Research Articles

Crystal Structure of Lactadherin C2 Domain at 1.7Å Resolution with Mutational and Computational Analyses of Its Membrane-binding Motif

Lactadherin is a phosphatidyl-L-serine (Ptd-L-Ser)-binding protein that decorates membranes of milk fat globules. The major Ptd-l-Ser binding function of lactadherin has been localized to its C2 domain, which shares homology with the C2 domains of blood coagulation factor VIII and factor V. Correlating with this homology, purified lactadherin competes efficiently with factors VIII and V for Ptd-L-Ser binding sites, functioning as a potent anticoagulant. We have determined the crystal structure of the lactadherin C2 domain (Lact-C2) at 1.7A resolution. The bovine Lact-C2 structure has a beta-barrel core that is homologous with the factor VIII C2 (fVIII-C2) and factor V C2 (fV-C2) domains. Two loops at the end of the beta-barrel, designated spikes 1 and 3, display four water-exposed hydrophobic amino acids, reminiscent of the membrane-interactive residues of fVIII-C2 and fV-C2. In contrast to the corresponding loops in fVIII-C2 and fV-C2, spike 1 of Lact-C2 adopts a hairpin turn in which the 7-residue loop is stabilized by internal hydrogen bonds. Further, central glycine residues in two membrane-interactive loops may enhance conformability of Lact-C2 to membrane binding sites. Mutagenesis studies confirmed a membrane-interactive role for the hydrophobic and/or Gly residues of both spike 1 and spike 3. Substitution of spike 1 of fVIII-C2 into Lact-C2 also diminished binding. Computational ligand docking studies identified two prospective Ptd-l-Ser interaction sites. These results identify two membrane-interactive loops of Lact-C2 and provide a structural basis for the more efficient phospholipid binding of lactadherin as compared with factor VIII and factor V.

Derlin-1 Facilitates the Retro-Translocation of Cholera Toxin

Cholera toxin (CT) intoxicates cells by using its receptor-binding B subunit (CTB) to traffic from the plasma membrane to the endoplasmic reticulum (ER). In this compartment, the catalytic A1 subunit (CTA1) is unfolded by protein disulfide isomerase (PDI) and retro-translocated to the cytosol where it triggers a signaling cascade, leading to secretory diarrhea. How CT is targeted to the site of retro-translocation in the ER membrane to initiate translocation is unclear. Using a semipermeabilized-cell retro-translocation assay, we demonstrate that a dominant-negative Derlin-1-YFP fusion protein attenuates the ER-to-cytosol transport of CTA1. Derlin-1 interacts with CTB and the ER chaperone PDI as assessed by coimmunoprecipitation experiments. An in vitro membrane-binding assay showed that CTB stimulated the unfolded CTA1 chain to bind to the ER membrane. Moreover, intoxication of intact cells with CTB stabilized the degradation of a Derlin-1-dependent substrate, suggesting that CT uses the Derlin-1 pathway. These findings indicate that Derlin-1 facilitates the retro-translocation of CT. CTB may play a role in this process by targeting the holotoxin to Derlin-1, enabling the Derlin-1-bound PDI to unfold the A1 subunit and prepare it for transport.

3,4-Diamino-2,5-thiadiazole-1-oxides as potent CXCR2/CXCR1 antagonists

A series of novel and potent 3,4-diamino-2,5-thiadiazole-1-oxides were prepared and found to show excellent binding affinities for CXCR2 and CXCR1 receptors and excellent inhibitory activity of Gro-alpha and IL-8 mediated in vitro hPMN MPO release of CXCR2 and CXCR1 expressing cell lines. On the other hand, a closely related 3,4-diamino-2,5-thiadiazole-dioxide did not show functional activity despite its excellent binding affinities for CXCR2 and CXCR1 in membrane binding assays. A detailed SAR has been discussed in these two closely related structures.

Clustering and synaptic targeting of PICK1 requires direct interaction between the PDZ domain and lipid membranes

Protein interacting with c kinase 1 (PICK1) regulates the trafficking of receptors and ion-channels such as AMPA receptors. Traditionally, the PICK1 PDZ domain is regarded as an adaptor capable of binding to receptors trafficked by PICK1, and the lipid-binding BAR domain functions to tether PICK1 directly to membranes. Here, we show that the PICK1 PDZ domain can directly interact with lipid membranes. The PDZ domain and lipid membrane interaction is mediated by both a polybasic amino-acid cluster and a conserved 'Cys-Pro-Cys' motif located away from the peptide ligand-binding groove. Disruption of the PDZ and lipid membrane interaction totally abolished synaptic targeting of PICK1. Although mutation of the CPC motif did not affect the interaction between PICK1 and AMPA receptors, the mutant PICK1 was unable to cluster the GluR2 subunit of the receptor. In neurons, PICK1 containing the same mutation displayed dramatically compromised capacity in the trafficking of AMPA receptors. Taken together, our findings not only uncovered the novel lipid membrane-binding property of the PICK1 PDZ domain, but also provided direct evidence supporting the functional relevance of the PDZ-lipid interaction.

Live Cell Monitoring of μ-Opioid Receptor-mediated G-protein Activation Reveals Strong Biological Activity of Close Morphine Biosynthetic Precursors

G-protein activation by receptors is generally measured using (35)S-GTPgammaS binding assays in cell membranes and cannot be well assessed in intact cells. We have recently developed a fluorescence resonance energy transfer (FRET)-based approach to monitor G(i)-protein activation in living cells. Here we report that this technique can be used to determine structure-activity relationships of receptor agonists in intact cells. We have recently shown that morphine is biosynthesized de novo by mammals via a multistep pathway different from that in plants. However, the pharmacological properties of morphine precursors are poorly understood. Here, we directly monitored mu-opioid receptor (MOR)-mediated G(i)-protein activation in living cells by FRET and validated this method with classical GTPgammaS binding assays. Receptor binding studies and FRET measurements demonstrated that several (R)-configurated morphine precursors such as (R)-reticuline, salutaridine, salutaridinol, thebaine, and codeine were partial MOR agonists. Some closer precursors such as oripavine, codeinone, and morphinone activated G(i)-proteins as strongly as morphine, but with slightly lower potencies. The more distant the precursors were positioned in the pathway with respect to morphine, the less efficient and potent they were at MOR. Comparison of pharmacological properties of close morphine precursors and concentrations in which they occur in animal tissues suggests that they might activate MOR signaling under physiological conditions. Taken together, our data indicate that FRET-based assays of G-protein activation can serve to determine the abilities of compounds to activate G-protein signaling directly and in living cells.

Angiotensin II and Its Receptor Subtypes in the Human Retina

To quantify and evaluate the distribution of angiotensin II (Ang II) and its receptors in the human retina. Donor eyes were obtained within 12 hours postmortem and classified as hypertensive or normotensive and diabetic or nondiabetic, based on the donors' medical histories. Ang II in retina and vitreous was quantified by RIA. Ang II receptors were characterized and quantified by competitive membrane-binding assays. Ang II, its heptapeptide metabolite Ang-(1-7), and AT1 and AT2 receptors were localized by immunohistochemistry and confocal imaging. Levels of Ang II in the retina were significantly higher than in vitreous (P < 0.05). Ang II in the diabetic retina had a higher median compared with that in the nondiabetic retina. Ang II and Ang-(1-7) colocalized in retinal Müller cells. The retina had the highest levels of Ang II receptors that were significantly higher than the optic nerve, retinal pigment epithelium-choroid complex, and ciliary body-iris complex (P < 0.05). AT1 receptors were more abundant than AT2 receptors in the retina. Immunoreactivity for AT1 was detected in Müller cells and on blood vessels. AT2 receptors were localized throughout the Müller cells and nuclei of ganglion cells and neurons in the inner nuclear layer. In the human retina, identification of Ang II and its bioactive metabolite Ang-(1-7) in Müller cells suggests that these glial cells are able to produce and process Ang II. Ang receptors were localized in the blood vessels and neural cells. Local Ang II signaling may thus allow for autoregulation of neurovascular activity. Such an autonomous system could modulate the onset and severity of retinovascular disease.

Discovery, Synthesis, and Structure Activity of a Highly Selective α7 Nicotinic Acetylcholine Receptor Antagonist

Nicotinic acetylcholine receptors (nAChRs) that contain an alpha7 subunit are widely distributed in neuronal and nonneuronal tissue. These receptors are implicated in the release of neurotransmitters such as glutamate and in functions ranging from thought processing to inflammation. Currently available ligands for alpha7 nAChRs have substantial affinity for one or more other nAChR subtypes, including those with an alpha1, alpha3, alpha6, and/or alpha9 subunit. An alpha-conotoxin gene was cloned from Conus arenatus. Predicted peptides were synthesized and found to potently block alpha3-, alpha6-, and alpha7-containing nAChRs. Structure-activity information regarding conotoxins from distantly related Conus species was employed to modify the C. arenatus derived toxin into a novel, highly selective alpha7 nAChR antagonist. This ligand, alpha-CtxArIB[V11L,V16D], has low nanomolar affinity for rat alpha7 homomers expressed in Xenopus laevis oocytes, and antagonism is slowly reversible. Kinetic analysis provided insight into the mechanism of antagonism. alpha-CtxArIB interacts with five ligand binding sites per alpha7 receptor, and occupation of a single site is sufficient to block function. The peptide was also shown to be highly selective in competition binding assays in rat brain membranes. alpha-CtxArIB[V11L,V16D] is the most selective ligand yet reported for alpha7 nAChRs.

A GAPDH Mutant Defective in Src‐Dependent Tyrosine Phosphorylation Impedes Rab2‐Mediated Events

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple intracellular activities in addition to its role in gluconeogenesis. Indeed, we have reported that GAPDH is required for Rab2-mediated retrograde transport from vesicular tubular clusters (VTCs). These diverse GAPDH activities are the result of posttranslational modifications that confer a new function to the enzyme. In that regard, GAPDH is tyrosine phosphorylated by Src. To establish the functional significance of this modification for GAPDH activity in Rab2-dependent events, an amino acid substitution was made at tyrosine 41 (GAPDH Y41F). The inability of Src to phosphorylate purified recombinant GAPDH Y41F was confirmed in an in vitro kinase assay. The mutant was then employed in a quantitative membrane-binding assay that measures Rab2 recruitment of soluble components to VTCs. As we observed with GAPDH wild type, Rab2 promoted GAPDH Y41F binding to membranes in a dose-dependent manner, indicating that GAPDH tyrosine phosphorylation is not required for VTC association. However, GAPDH was tyrosine phosphorylated on VTCs. Importantly, GAPDH Y41F blocked vesicular stomatitis virus-G transport in an assay that reconstitutes endoplasmic reticulum to Golgi trafficking, indicating that phosphorylation of tyrosine 41 is essential for GAPDH activity in the early secretory pathway. The block in transport is because of the decreased binding of atypical protein kinase C iota/lambda to GAPDH Y41F, which reduces beta-coat protein association with the VTC and subsequent formation of Rab2-mediated retrograde vesicles. Our results suggest that Src plays a pivotal role in regulating the interaction of Rab2 effectors on the VTC.

Synthesis and In Vitro Evaluation of Novel 2‐oxo‐1,2‐dihydroquinoline CB2 Receptor Inverse Agonists

Aliphatic amides of 7-methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carboxylic acid 6 were synthesized and evaluated for their CB2 and CB1 receptor activities by a [35S]GTPgammaS membrane-binding assay. Compounds 7-18 produced CB2 inverse agonist activities with a variety of potencies (IC50: 1 nm-1 microm). By contrast, no considerable antagonism of CB1 receptor was observed by 1-10 microm concentrations indicating that synthesized compounds were CB2 receptor selective.

Antidepressant Principles of the Roots of Polygala tenuifolia

[(125)I]RTI-55-membrane binding assay-guided fractionation and separation of a water-soluble extract of the roots of Polygala tenuifolia gave five new oligosaccharide derivatives, polygalatenosides A-E (1-5). The structures of these new oligosaccharides were established on the basis of spectroscopic evidence. Polygalatenosides A and B (1 and 2) showed significant inhibitory activity, with IC(50) values of 30.0 and 6.04 microM, respectively, in this membrane binding assay and acted as norepinephrine reuptake inhibitors through blocking norepinephrine transport.

The Actin-depolymerizing Factor Homology and Charged/Helical Domains of Drebrin and mAbp1 Direct Membrane Binding and Localization via Distinct Interactions with Actin

Cytoskeletal dynamics are important for efficient function of the secretory pathway. ADP-ribosylation factor, ARF1, triggers vesicle coat assembly and, in concert with Cdc42, regulates actin polymerization and molecular motor-based motility. Drebrin and mammalian Abp1 (mAbp1) are actin-binding proteins found previously to bind to Golgi membranes in an ARF1-dependent manner in vitro. Despite sharing homology through two shared actin binding domains, drebrin and mAbp1 have different subcellular localization and bind to distinct actin structures on the Golgi apparatus. We find that the actin-depolymerizing factor homology (ADFH) and charged/helical actin binding domains of drebrin and mAbp1 are sufficient for regulated binding to Golgi membranes and subcellular localization. We have used mutant proteins and chimeras between mAbp1 and drebrin to identify motifs that direct targeting. We find that a linker region between the ADFH and charged/helical domains confers Golgi binding properties to mAbp1. mAbp1 binds to a specific actin pool through its ADFH/linker domain that is not bound by drebrin. Drebrin localization to the cell surface was found to involve motifs within the charged/helical domain. Our results indicate that targeting of these proteins is directed through multiple distinct interactions with the actin cytoskeleton. The mechanisms for selective recruitment of mAbp1 and drebrin to Golgi membranes indicate how actin-based structures are able to select specific actin-binding proteins and, thus, carry out multiple different functions within cells.

Src-dependent aProtein Kinase C ι/λ (aPKCι/λ) Tyrosine Phosphorylation Is Required for aPKCι/λ Association with Rab2 and Glyceraldehyde-3-phosphate Dehydrogenase on Pre-Golgi Intermediates

The small GTPase Rab2 is required for membrane transport between the endoplasmic reticulum (ER) and the Golgi complex. Rab2 associates with pre-Golgi intermediates (also termed vesicular tubular clusters; VTCs) that sort cargo to the anterograde pathway from recycling proteins retrieved to the ER. Our previous studies have shown that Rab2 stimulates atypical protein kinase C iota/lambda (aPKCiota/lambda) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) recruitment to VTCs. Both aPKCiota/lambda and GAPDH bind directly to Rab2 and aPKCiota/lambda and GAPDH interact. Based on the reports demonstrating aPKCiota-Src interaction and Src activity in the retrograde pathway (Golgi-ER), studies were initiated to learn whether Rab2 also promoted Src recruitment to VTCs. Using a quantitative membrane binding assay, we found that Rab2-stimulated Src membrane association in a dose-dependent manner. The recruited Src binds to aPKCiota/lambda and GAPDH on the membrane; however, Src does not interact with Rab2. The membrane-associated Src tyrosine phosphorylates aPKCiota/lambda on the VTC. To determine the consequence of aPKCiota/lambda tyrosine phosphorylation, the membrane binding assay was supplemented with the Src-specific tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP2). Although Rab2, Src, and GAPDH recruitment was not affected, the Rab2-PP2-treated membranes contained a negligible amount of aPKCiota/lambda. Since Rab2 requires aPKCiota/lambda for the downstream recruitment of beta-coat protein (beta-COP) to VTCs, the Rab2-PP2-treated membranes were evaluated for the presence of beta-COP. Like aPKCiota/lambda, the membranes contained a negligible amount of beta-COP that was reflected by the drastic reduction in Rab2-dependent vesicle formation. These data suggest that Src-mediated tyrosine phosphorylation of aPKCiota/lambda facilitates aPKCiota/lambda association with Rab2-Src-GAPDH on VTCs, which is ultimately necessary for the downstream recruitment of beta-COP and release of Rab2-mediated retrograde-directed vesicles.

Smoothened Regulates Activator and Repressor Functions of Hedgehog Signaling via Two Distinct Mechanisms

The secreted protein Hedgehog (Hh) plays an important role in metazoan development and as a survival factor for many human tumors. In both cases, Hh signaling proceeds through the activation of the seven-transmembrane protein Smoothened (Smo), which is thought to convert the Gli family of transcription factors from transcriptional repressors to transcriptional activators. Here, we provide evidence that Smo signals to the Hh signaling complex, which consists of the kinesin-related protein Costal2 (Cos2), the protein kinase Fused (Fu), and the Drosophila Gli homolog cubitus interruptus (Ci), in two distinct manners. We show that many of the commonly observed molecular events following Hh signaling are not transmitted in a linear fashion but instead are activated through two signals that bifurcate at Smo to independently affect activator and repressor pools of Ci.

Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+and PIP2Recognition by Its C2 Domain

The C2 domain of protein kinase Calpha (PKCalpha) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca2+ signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca2+-activated PKCalpha and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), dramatically enhances the Ca2+-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKCalpha Ca2+-binding loops (CBLs) and PIP2 binding site (beta-strands 3-4) into a different C2 domain exhibits native Ca2+-triggered targeting to plasma membrane and recognizes PIP2. Conversely, a hybrid containing the CBLs but lacking the PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKCalpha C2 domains possessing mutations in the PIP2 site target primarily to TGN and fail to recognize PIP2. Overall, these findings demonstrate that the CBLs are essential for Ca2+-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on beta-strands 3-4, which bind to plasma membrane PIP2.

ERp29 Triggers a Conformational Change in Polyomavirus to Stimulate Membrane Binding

Membrane penetration of nonenveloped viruses is a poorly understood process. We have investigated early stages of this process by studying the conformational change experienced by polyomavirus (Py) in the lumen of the endoplasmic reticulum (ER), a step that precedes its transport into the cytosol. We show that a PDI-like protein, ERp29, exposes the C-terminal arm of Py's VP1 protein, leading to formation of a hydrophobic particle that binds to a lipid bilayer; this reaction likely mimics initiation of Py penetration across the ER membrane. Expression of a dominant-negative ERp29 decreases Py infection, indicating ERp29 facilitates viral infection. Interestingly, cholera toxin, another toxic agent that crosses the ER membrane into the cytosol, is unfolded by PDI in the ER. Our data thus identify an ER factor that mediates membrane penetration of a nonenveloped virus and suggest that PDI family members are generally involved in ER remodeling reactions.

Cysteine-scanning Mutagenesis of Serotonin Transporter Intracellular Loop 2 Suggests an α-Helical Conformation

Like other proteins involved in neurotransmitter transport, serotonin transporter (SERT) activity is regulated by multiple intracellular signal transduction pathways. The second intracellular loop (IL2) of SERT contains consensus sequences for cGMP-dependent protein kinase and protein kinase C. A 24-residue region of SERT including IL2, from Ile-270 through Ser-293, was analyzed by cysteine-scanning mutagenesis and chemical modification. 2-(Aminoethyl)methanethiosulfonate hydrobromide (MTSEA) failed to inhibit serotonin transport or binding of the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane (beta-CIT) in intact cells expressing these mutants, but it inactivated beta-CIT binding in membrane preparations. From the pattern of sensitivity, IL2 appears to extend from Trp-271 through Ile-290, a significantly longer region than that initially predicted by hydropathy analysis. Six mutants reacted with MTSEA much faster than the others, and the pattern of the more reactive mutations suggested that IL2 is in an alpha-helical conformation. Some of the mutants had significantly elevated transport rates, suggesting a possible mechanism for the regulation of SERT activity.

Tetraamine‐modified octreotide and octreotate: labeling with 99mTc and preclinical comparison in AR4‐2J cells and AR4‐2J tumor‐bearing mice

Two somatostatin analogues, [(99m)Tc]Demotide and [(99m)Tc]Demotate 4, were compared with [(99m)Tc]Demotate 1, a previously reported somatostatin receptor subtype 2 (sst(2)) targeting tracer. Conjugates were prepared by coupling an open-chain tetraamine chelator to D-Phe(1) of [Tyr(3)]-octreotide or [Tyr(3)]-octreotate, respectively, via a p-benzylaminodiglycolic acid spacer adopting solid-phase peptide synthesis techniques. Peptide conjugates were collected in a highly pure form after chromatographic purification. Eventually, [(99m)Tc]Demotide and [(99m)Tc]Demotate 4 were obtained in approximately 1 Ci/micromol specific activity and >96% purity after labeling under alkaline conditions. Demotide and Demotate 4 exhibited similar high binding affinities for the sst(2) expressed in AR4-2J cells with IC(50) values 0.16 and 0.10 nM, respectively. The (radio)metallated analogues [(99m)Tc]Demotide and [(99m)Tc]Demotate 4 showed equally high affinities to the sst(2) during saturation binding assays in AR4-2J cell membranes (K(d)s 0.08 and 0.07 nM, respectively). During incubation at 37 degrees C with AR4-2J cells, the radiopeptides internalized effectively via a receptor-mediated process, with [(99m)Tc]Demotate 4 exhibiting a faster internalization rate than [(99m)Tc]Demotide. After injection in athymic mice bearing sst(2)-expressing AR4-2J tumors, the radiotracers showed high and specific uptake in the tumor (>25%ID/g at 1 h) and in the sst(2)-positive organs. However, both [(99m)Tc]Demotide and [(99m)Tc]Demotate 4 showed unfavorably higher background activity, especially in the abdomen, in comparison to [(99m)Tc]Demotate 1 and are, therefore, less suited than [(99m)Tc]Demotate 1 for sst(2)-targeted tumor imaging in man.

Long‐term Effect of Convulsive Behavior on the Density of Adenosine A1 and A2A Receptors in the Rat Cerebral Cortex

Adenosine is a neuromodulator that has been proposed to act as an anticonvulsant mainly via inhibitory A1 receptors, but recent data show that genetic deletion of facilitatory A 2A receptors might also attenuate convulsions. Since both A1 and A 2A receptors are prone to down- and upregulation in different stressful situations, we investigated if convulsive behavior leads to a long-term change in A1 and A 2A receptor density in the rat cerebral cortex. Stage 4-5 convulsions (Racine's scale) were induced in adult Wistar rats either through amygdala stimulation (kindling) or by intraperitoneal injection of kainate (10 mg/ml). Rats were killed after 4 weeks to evaluate adenosine A1 and A 2A receptor density in the cerebral cortex using both Western blot and membrane binding assays. The binding density of the A1 antagonist, 3H-DPCPX, decreased by 40. +/- 4.4% and by 20.7 +/- 0.5% after kindling or kainate injection. Likewise, A1 receptor immunoreactivity in cortical membranes from kindled or kainate-injected rats decreased by 19.1 +/- 3.3% and 12.7 +/- 5.7%, respectively. In contrast, the binding density of the A 2A receptor antagonist 3H-SCH 58261 increased by 293 +/- 34% and by 159 +/- 32% in cortical membranes from kindled or kainate-injected rats, and A 2A receptor immunoreactivity also increased by 151 +/- 12% and 79.6 +/- 7.0%. This indicates that after convulsive behavior there is a long-term decrease of A1 receptors accompanied by an increased density of A 2A receptors, suggesting that A 2A antagonists rather than A1 agonists may be more promising anticonvulsive drugs.

Giant liposomes in physiological buffer using electroformation in a flow chamber

We describe a method to obtain giant liposomes (diameter 10–100 μm) in solutions of high ionic strength to perform a membrane-binding assay under physiological conditions. Using electroformation on ITO electrodes, we formed surface-attached giant liposomes in solutions of glycerol in a flow chamber and then introduced solutions of high ionic strength (up to 2 M KCl) into this chamber. The ionic solution exchanged with the isoosmolar glycerol solution inside and outside the liposomes. An initial mismatch in index of refraction between the inside and outside of liposomes allowed for the observation of solution replacement. Ions and small polar molecules exchanged into and out of surface-attached liposomes within minutes. In contrast, liposomes formed in solutions of macromolecules retained molecules larger than 4 kDa, allowing for encapsulation of these molecules for hours or days even if the solution outside the liposomes was exchanged. We propose that solutes entered liposomes through lipid tubules that attach liposomes to the film of lipids on the surface of the ITO electrode. The method presented here makes it straightforward to perform flow-through binding assays on giant liposomes under conditions of physiological ionic strength. We performed a membrane-binding assay for annexin V, a calcium-dependent protein that binds to phosphatidylserine (PS). The binding of annexin V depended on the concentration of PS and decreased as ionic strength increased to physiological levels.

The subunit composition and pharmacology of α-Conotoxin MII-binding nicotinic acetylcholine receptors studied by a novel membrane-binding assay

The subunit composition and pharmacology of α-Conotoxin MII-binding (α-CtxMII) nicotinic acetylcholine receptors (nAChR) was studied by an improved [ 125I]-α-CtxMII membrane binding method. This binding method facilitates pharmacological studies that have been difficult to accomplish with [ 125I]-α-CtxMII autoradiography or α-CtxMII inhibition of [ 125I]-epibatidine binding. Binding densities and K d-values obtained by this [ 125I]-α-CtxMII membrane binding were similar to the values obtained by autoradiography or α-CtxMII inhibition of [ 125I]-epibatidine binding, verifying that each of these approaches measures the same nAChR population. Binding results with nAChR subunit-null mutant mice confirm and extend observations from earlier studies: [ 125I]-α-CtxMII binding measures two sets of α6β2* nAChR (α4α6β2β3 or α6β2β3). Most nicotinic agonists and antagonists show monophasic inhibition of [ 125I]-α-CtxMII binding, indicating that α4α6β2β3 and α6β2β3 have similar binding properties. Comparison of the binding and activation profiles of α6β2* nAChR to those of other nAChR subtypes (α4β2* and β4*) indicates that these receptors have distinctly different pharmacology indicating that it may be possible to target α6β2* nAChR selectively to develop compounds that might be therapeutically useful.