2H and 31P NMR study of the interaction of general anesthetics with phosphatidylcholine membranes

The effect of the general anesthetics alpha-chloralose and chloral hydrate as well as the nonanesthetic beta-chloralose on the order of phosphatidylcholine and phosphatidylcholine-cholesterol liposomes has been examined by 2H nuclear magnetic resonance. Chloral hydrate interacts with the hydrophilic head-group region, causing a change in the torsion angle of the C alpha-C beta bond. The membrane interior is also disordered by the presence of this agent. alpha-Chloralose, on the other hand, disorders only the central position of the membrane. beta-Chloralose produces little significant change in bilayer order.     

Analysis of the Rem2 - voltage dependant calcium channel beta subunit interaction and Rem2 interaction with phosphorylated phosphatidylinositide lipids

Voltage dependant calcium channels (VDCC) play a critical role in coupling electrical excitability to important physiological events such as secretion by neuronal and endocrine cells. Rem2, a GTPase restricted to neuroendocrine cell types, regulates VDCC activity by a mechanism that involves interaction with the VDCC beta subunit (Ca(V)beta). Mapping studies reveal that Rem2 binds to the guanylate kinase domain (GK) of the Ca(V)beta subunit that also contains the high affinity binding site for the pore forming and voltage sensing VDCC alpha subunit (Ca(V)alpha) interaction domain (AID). Moreover, fine mapping indicates that Rem2 binds to the GK domain in a region distinct from the AID interaction site, and competitive inhibition studies reveal that Rem2 does not disrupt Ca(V)alpha - Ca(V)beta binding. Instead, the Ca(V)beta subunit appears to serve a scaffolding function, simultaneously binding both Rem2 and AID. Previous studies have found that in addition to Ca(V)beta binding, Rem2 must be localized to the plasma membrane to inhibit VDCC function. Plasma membrane localization requires the C-terminus of Rem2 and binding studies indicate that this domain directs phosphorylated phosphatidylinositide (PIP) lipids association. Plasma membrane localization may provide a unique point of regulation since the ability of Rem2 to bind PIP lipids is inhibited by the phosphoserine dependant binding of 14-3-3 proteins. Thus, in addition to Ca(V)beta binding, VDCC blockade by Rem2 is likely to be controlled by both the localized concentration of membrane PIP lipids and direct 14-3-3 binding to the Rem2 C-terminus.     

Studies on the interaction of anesthetic steroids with phosphatidylcholine using 2H and 13C solid state NMR

The effects of the anesthetic steroid alphaxalone and its inactive analog delta 16-alphaxalone on model phospholipid membranes were studied using 13C and 2H solid-state nuclear magnetic resonance spectroscopy. Aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) with specific 13C and 2H labels as endogenous probes at the carbonyl and the C-7 methylene groups, respectively, of the sn-2 chain were used to study the conformational and dynamical properties of the bilayer as a function of temperature. There were no significant changes between the 13C and 2H spectra of the DPPC preparation containing the inactive steroid and that of DPPC with no drug. However, the physiologically active steroid produces significant spectral 2H and 13C changes. These changes include a reduction of the main phase transition temperature and a broadening of that transition. Alphaxalone also increases the relative number of gauche conformers in the liquid-crystalline phase of DPPC and increases the rate of axial diffusion in both the gel and liquid-crystalline phase. The thermotropic properties of the above preparations, as monitored by differential scanning calorimetry, were congruent with the spectroscopic data.     

AFM study of interaction forces in supported planar DPPC bilayers in the presence of general anesthetic halothane

In spite of numerous investigations, the molecular mechanism of general anesthetics action is still not well understood. It has been shown that the anesthetic potency is related to the ability of an anesthetic to partition into the membrane. We have investigated changes in structure, dynamics and forces of interaction in supported dipalmitoylphosphatidylcholine (DPPC) bilayers in the presence of the general anesthetic halothane. In the present study, we measured the forces of interaction between the probe and the bilayer using an atomic force microscope. The changes in force curves as a function of anesthetic incorporation were analyzed. Force measurements were in good agreement with AFM imaging data, and provided valuable information on bilayer thickness, structural transitions, and halothane-induced changes in electrostatic and adhesive properties.     

AFM study of interaction forces in supported planar DPPC bilayers in the presence of general anesthetic halothane

In spite of numerous investigations, the molecular mechanism of general anesthetics action is still not well understood. It has been shown that the anesthetic potency is related to the ability of an anesthetic to partition into the membrane. We have investigated changes in structure, dynamics and forces of interaction in supported dipalmitoylphosphatidylcholine (DPPC) bilayers in the presence of the general anesthetic halothane. In the present study, we measured the forces of interaction between the probe and the bilayer using an atomic force microscope. The changes in force curves as a function of anesthetic incorporation were analyzed. Force measurements were in good agreement with AFM imaging data, and provided valuable information on bilayer thickness, structural transitions, and halothane-induced changes in electrostatic and adhesive properties.     

Oligomerization and maturation of Na,K-ATPase: functional interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha subunit

Subunit assembly plays an essential role in the maturation of oligomeric proteins. In this study, we have characterized the main structural and functional consequences of the assembly of alpha and beta subunits of Na,K-ATPase. Xenopus oocytes injected with alpha and/or beta cRNA were treated with brefeldin A, which permitted the accumulation of individual subunits or alpha-beta complexes in the ER. Only alpha subunits that are associated with beta subunits become resistant to trypsin digestion and cellular degradation. Similarly, assembly with beta subunits is necessary and probably sufficient for the catalytic alpha subunit to acquire its main functional properties at the level of the ER, namely the ability to adopt different ligand- dependent conformations and to hydrolyze ATP in an Na(+)- and K(+)- dependent, ouabain-inhibitable fashion. Not only the alpha but also the beta subunit undergoes a structural change after assembly, which results in a global increase in its protease resistance. Furthermore, extensive and controlled proteolysis assays on wild-type and NH2- terminally modified beta subunits revealed a K(+)-dependent interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha subunit, which is likely to be involved in the modulation of the K(+)- activation of the Na,K-pump transport activity. Thus, we conclude that the ER assembly process not only establishes the basic structural interactions between individual subunits, which are required for the maturation of oligomeric proteins, but also distinct, functional interactions, which are involved in the regulation of functional properties of mature proteins.     

NMR structure and dynamics of the second transmembrane domain of the neuronal acetylcholine receptor beta 2 subunit

Structure and backbone dynamics of a selectively [(15)N]Leu-labeled 28-residue segment of the extended second transmembrane domain (TM2e) of the human neuronal nicotinic acetylcholine receptor (nAChR) beta(2) subunit were studied by (1)H and (15)N solution-state NMR in dodecylphosphocholine micelles. The TM2e structure was determined on the basis of the nuclear Overhauser effects (NOEs) and the hydrogen bond restraints, which were inferred from the presence of H(alpha)(i)-H(N)(i+3), H(alpha)(i)-H(beta)(i+3), and H(alpha)(i)-H(N)(i+4) NOE connectivity and from the slow amide hydrogen exchange with D(2)O. The TM2e structure of the nAChR beta(2) subunit contains a helical region between T4 and K22. Backbone dynamics were calculated using the model-free approach based on the (15)N relaxation rate constants, R(1) and R(2), and on the (15)N-[(1)H] NOE. The data acquired at 9.4 and 14.1 T and calculations using different dynamic models demonstrated no conformational exchange and internal motions on the nanosecond time scale. The global tumbling time of TM2e in micelles was 14.4 +/- 0.2 ns; the NOE values were greater than 0.63 at 9.4 T, and the order parameter, S(2), was 0.83-0.96 for all (15)N-labeled leucine residues, suggesting a restricted internal motion. This is the first report of NMR structure and backbone dynamics of the second transmembrane domain of the human nAChR beta(2) subunit in a membrane-mimetic environment, providing the basis for subsequent studies of subunit interactions in the transmembrane domain complex of the neuronal nAChR.     

NMR study of volatile anesthetic binding to nicotinic acetylcholine receptors

New lines of evidence suggest that volatile anesthetics interact specifically with proteins. Direct binding analysis, however, has been largely limited to soluble proteins. In this study, specific interaction was investigated between isoflurane, a clinically important volatile anesthetic, and membrane-bound nicotinic acetylcholine receptors (nAChRs) from Torpedo electroplax, using (19)F nuclear magnetic resonance spectroscopy and gas chromatography. The receptors were reconstituted into 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles. After correcting for nonspecific partitioning into the lipid, the equilibrium dissociation constant, K(d), of isoflurane binding to nAChR at 15 degrees C was found to be 0.36 +/- 0.03 mM. This value is within the clinically relevant concentration range of the agent. Based on the receptor concentrations in the vesicle suspension assayed by the bicinchoninic acid method and the fraction of bound isoflurane, X(b), determined by gas chromatography, an estimate of an average of 9-10 specifically bound isoflurane molecules can be made for each receptor, or two for each subunit. Upon binding, the transverse relaxation time constant (T(2)) of (19)F resonance of isoflurane is decreased by nearly three orders of magnitude, indicating a dramatic reduction in the mobility of specifically bound isoflurane. Kinetic analysis reveals that the off rate of binding, k(-1), is 1.7 x 10(4) s(-1). The on rate, k(+1), can thus be calculated to be approximately 4.8 x 10(7) M(-1) s(-1), suggesting a nearly diffusion-limited association. This is in contrast to anesthetic binding to a soluble protein, bovine serum albumin (BSA), where k(+1) and k(-1) are at least an order of magnitude slower. It is concluded that the presence of lipids may be critical for the correct evaluation of binding kinetics between volatile anesthetics and neuronal receptors.     

Physical interaction between interleukin-12 receptor beta 2 subunit and Jak2 tyrosine kinase: Jak2 associates with cytoplasmic membrane-proximal region of interleukin-12 receptor beta 2 via amino-terminus

IL-12 is a heterodimeric cytokine, composed of p40 and p35 subunits, that exerts its biological effects by binding to specific cell surface receptors. Two human IL-12 receptor proteins, designated IL-12R beta 1 and IL-12R beta 2, have been previously identified. IL-12R beta 2 has box 1 motif, box 2 motif, and three tyrosine residues in its cytoplasmic domain. In response to IL-12, Jak2 and Tyk2, family members of Janus family protein tyrosine kinases, are phosphorylated in PHA-activated T lymphocytes. The present study demonstrates that Jak2 binds to the cytoplasmic membrane-proximal region of IL-12R beta 2, and box 2 motif and tyrosine residues in the cytoplasmic domain were not required for binding. The amino-terminus of Jak2 is necessary for association with IL-12R beta 2.     

Interactions of the local anesthetic tetracaine with membranes containing phosphatidylcholine and cholesterol: a 2H NMR study

The interactions of the local anesthetic tetracaine with multilamellar dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol have been investigated by deuterium nuclear magnetic resonance of specifically deuteriated tetracaines, DMPC and cholesterol. Experiments were performed at pH 5.5, when the anesthetic is primarily charged, and at pH 9.5, when it is primarily uncharged. The partition coefficients of the anesthetic in the membrane have been measured at both pH values for phosphatidylcholine bilayers with and without cholesterol. The higher partition coefficients obtained at pH 9.5 reflect the hydrophobic interactions between the uncharged form of the anesthetic and the hydrocarbon region of the bilayer. The lower partition coefficients for the DMPC/cholesterol system at both pH values suggest that cholesterol, which increases the order of the lipid chains, decreases the solubility of tetracaine into the bilayer. For phosphatidylcholine bilayers, it has been proposed [Boulanger, Y., Schreier, S., & Smith, I. C. P. (1981) Biochemistry 20, 6824-6830] that the charged tetracaine at low pH is located mostly at the phospholipid headgroup level while the uncharged tetracaine intercalates more deeply into the bilayer. The present study suggests that the location of tetracaine in the cholesterol-containing system is different from that in pure phosphatidylcholine bilayers: the anesthetic sits higher in the membrane. An increase in temperature results in a deeper penetration of the anesthetic into the bilayer. Moreover, the incorporation of the anesthetic into DMPC bilayers with or without cholesterol results in a reduction of the lipid order parameters both in the plateau and in the tail regions of the acyl chains, this effect being greater with the charged form of the anesthetic.     

Allelic variants interaction of CLOCK gene and G-protein beta3 subunit gene with diurnal preference

The 3111 C/T single nucleotide polymorphism (SNP) in the CLOCK gene and the 825C/T SNP in the G-protein β3 subunit gene (GNB3) have been reported to influence diurnal preference. This study has attempted to characterize the association between the CLOCK gene and GNB3 polymorphisms and diurnal preference in healthy Korean college students. All subjects completed the 13-item Composite Scale for Morningness (CSM). The interaction between the 3111 C/T SNP in the CLOCK gene and the 825 C/T SNP in the GNB3 gene significantly influenced diurnal preference, according to the CSM Performance subscore (F=10.94, p=0.001). However, when the different polymorphisms of the two genes were analyzed independently, no direct correlations with diurnal preference were detected. The CLOCK gene 3111 C/T SNP and GNB3 gene 825 C/T SNP were found to manifest a gene-gene interaction that affects diurnal preference.     

NMR study of the interaction of retinoids with phospholipid bilayers

The interaction of three vitamin A derivatives or retinoids: all-trans-retinoic acid, 13-cis-retinoic acid and retinol with multilamellar phospholipid bilayers was studied using a combination of 2H- and 31P-NMR measurements. The following model membrane systems were used: (1) dipalmitoylphosphatidylcholine (DPPC) bilayers; (2) bilayers composed of a mixture of DPPC and bovine heart phosphatidylcholine (PC); (3) mixed PC/phosphatidylethanolamine (PE) bilayers. Only a weak interaction was observed between 13-cis-retinoic acid and DPPC membranes. Addition of all-trans-retinoic acid at a molar ratio of 1:2 to the lipid causes a small decrease (5 C degrees) in the gel to liquid crystalline phase-transition temperature of DPPC, a small increase in the order parameters of the lipid side-chains of single component bilayers and no measurable effect in the other lipid systems studied. Considerably larger perturbation in the lipid bilayer structure is introduced by addition of retinol which, at a molar ratio of 1:2 to the lipid, lowered the gel to liquid crystalline phase-transition temperature of DPPC by 21 C degrees and caused a decrease of order parameters of the lipid side-chains in all three lipid bilayer systems. These effects are consistent with intercalation of retinol molecules into the bilayer interior. The results for the mixed PC/PE bilayers indicate that the presence of retinol caused lateral separation of PE- and retinol-enriched regions.     

Examination of subunit interaction in human ADH: carboxymethylation of the heterodimer beta 2 gamma 1.

Conflicting results have been obtained on whether the subunits of the human dimeric enzyme alcohol dehydrogenase interact kinetically. To examine this question, chemical modification by iodoacetate was used to selectively inactivate the beta 2 subunit of the heterodimeric isozyme beta 2 gamma 1. Subsequent studies of the modified beta 2 gamma 1 which, presumably, has only one functional active site per dimeric molecule, indicate that it is still active. Moreover, the properties of this hybrid are similar to those of the unmodified subunit. From these results, it is fair to conclude that the individual subunits of alcohol dehydrogenase contribute to the activity of the dimeric isozymes in an independent manner.     

CD98hc (SLC3A2) interaction with the integrin beta subunit cytoplasmic domain mediates adhesive signaling

In mammals, beta1 integrin adhesion receptors generate signals that mediate cell spreading, migration, proliferation, and survival. CD98, a heterodimeric transmembrane protein, physically associates with certain integrin beta subunit cytoplasmic domains (tails) via its heavy chain, CD98hc (SLC3A2), and loss of CD98hc impairs integrin signaling. Here we have used the lack of CD98hc interaction with the Drosophila integrin betaPS tail for a homology scanning analysis that implicated the C-terminal 8 residues of beta3 (Thr(755)-Thr(802)) in CD98hc binding. We then identified point mutations in the beta3 C terminus (T755K and T758M) that abolish CD98hc association and a double mutation in the corresponding residues in the betaPS tail (K839T,M842T), which resulted in gain of CD98hc interaction. Furthermore, the loss of function beta3(T755K) mutation or the gain of function beta3/betaPS(K839T,M842T) led to a loss or gain of integrin-mediated cell spreading, respectively. Thus, we have identified critical integrin residues required for CD98hc interaction and in doing so have shown that CD98c interaction with the integrin beta tail is required for its ability to mediate integrin signaling. These studies also provide new insights into how CD98hc may cooperate with other cytoplasmic domain binding proteins to modulate integrin functions and into the evolution of integrin signaling.     

An interaction between alpha-actinin and the beta 1 integrin subunit in vitro

A number of cytoskeletal-associated proteins that are concentrated in focal contacts, namely alpha-actinin, vinculin, talin, and integrin, have been shown to interact in vitro such that they suggest a potential link between actin filaments and the membrane. Because some of these interactions are of low affinity, we suspect the additional linkages also exist. Therefore, we have used a synthetic peptide corresponding to the cytoplasmic domain of beta 1 integrin and affinity chromatography to identify additional integrin-binding proteins. Here we report our finding of an interaction between the cytoplasmic domain of beta 1 integrin and the actin-binding protein alpha-actinin. Beta 1-integrin cytoplasmic domain peptide columns bound several proteins from Triton extracts of chicken embryo fibroblasts. One protein at approximately 100 kD was identified by immunoblot analysis as alpha-actinin. Solid phase binding assays indicated that alpha-actinin bound specifically and directly to the beta 1 peptide with relatively high affinity. Using purified heterodimeric chicken smooth muscle integrin (a beta 1 integrin) or the platelet integrin glycoprotein IIb/IIIa complex (a beta 3 integrin), binding of alpha-actinin was also observed in similar solid phase assays, albeit with a lower affinity than was seen using the beta 1 peptide. alpha-Actinin also bound specifically to phospholipid vesicles into which glycoprotein IIb/IIIa had been incorporated. These results lead us to suggest that this integrin-alpha-actinin linkage may contribute to the attachment of actin filaments to the membrane in certain locations.     

Assignment of proton resonances in the NMR spectrum of carbonmonoxy hemoglobin beta subunit tetramers

Isolated beta chains from human adult hemoglobin at millimolar concentration are mainly associated to form beta 4 tetramers. We were able to obtain relevant two-dimensional proton nuclear magnetic resonance (NMR) spectra of such supermolecular complexes (Mr approximately 66,000) in the carboxylated state. Analysis of the spectra enabled us to assign the major part of the proton resonances corresponding to the heme substituents. We also report assignments of proton resonances originating from 12 amino acid side chains mainly situated in the heme pocket. These results provide a basis for a comparative analysis of the tertiary heme structure in isolated beta(CO) chains in solution and in beta(CO) subunits of hemoglobin crystals. The two structures are generally similar. A significantly different position, closer to the heme center, is predicted by the NMR for Leu-141 (H19) in isolated beta chains. Comparison of the assigned resonances of conserved amino acids in alpha chains, beta chains and sperm whale myoglobin indicates a close similarity of the tertiary heme pocket structure in the three homologous proteins. Significant differences were noted on the distal heme side, at the position of Val-E11, and on Leu-H19 and Phe-G5 position on the proximal side.     

The beta 4 subunit cytoplasmic domain mediates the interaction of alpha 6 beta 4 integrin with the cytoskeleton of hemidesmosomes.

The alpha 6 beta 4 integrin is structurally distinct from all the other known integrins because the cytoplasmic domain of beta 4 is unusually large and contains four type III fibronectin-like modules toward its C-terminus. To examine the function of the beta 4 cytoplasmic tail, we have expressed full-length and truncated human beta 4 cDNAs in rat bladder epithelial 804G cells, which form hemidesmosome-like adhesions in vitro. The cDNA encoded wild-type beta 4 subunit associated with endogenous alpha 6 and was recruited at the cell surface within hemidesmosome-like adhesions. A recombinant form of beta 4, lacking almost the entire cytoplasmic domain associated with alpha 6, reached the cell surface but remained diffusely distributed. A beta 4 molecule lacking almost the entire extracellular portion did not associate with alpha 6 but was correctly targeted to the hemidesmosome-like adhesions. Thus, the cytoplasmic portion of beta 4 contains sequences that are required and may be sufficient for the assembly of the alpha 6 beta 4 integrin into hemidesmosomes. To localize these sequences we examined the properties of additional mutant forms of beta 4. A truncated beta 4 subunit, lacking the most C-terminal pair of type III fibronectin homology domains, was incorporated into hemidesmosome-like adhesions, but another recombinant beta 4 molecule, lacking both pairs of type III fibronectin repeats, was not. Finally a recombinant beta 4 molecule, which was created by adjoining the region of the cytoplasmic domain including all type III repeats to the transmembrane segment, was efficiently recruited in hemidesmosome-like adhesions. Taken together these results suggest that the assembly of the alpha 6 beta 4 integrin into hemidesmosomes is mediated by a 303-amino acid region of beta 4 tail that comprises the first pair of type III fibronectin repeats and the segment between the second and third repeats. These data imply a function of a specific segment of the beta 4 cytoplasmic domain in interaction with cytoskeletal components of hemidesmosomes.     

The G protein beta 2 complementary DNA encodes the beta 35 subunit

Antisera were generated against synthetic peptides that correspond to amino acid sequences deduced from a cDNA (designated beta 2) that encodes a second form of the beta subunit of guanine nucleotide-binding regulatory proteins (G proteins). The specificity of interactions of these antisera with purified G protein beta subunits indicates that the beta 2 cDNA encodes the beta 35 form of this polypeptide. This hypothesis is confirmed by the use of these antisera to detect expression of the beta 2 cDNA in COS-m6 cells.     

Weak interaction of spectrin with phosphatidylcholine-phosphatidylserine multilayers: a 2H and 31P NMR study

Spectrin from human erythrocytes binds to bilayer dispersions of both DMPC and DMPS:DMPC (1:1, w/w). However, no effect of bound spectrin on the conformation of the lipid head groups, as measured from the deuterium quadrupolar splittings of DMPC or DMPS specifically deuterated in the polar head groups, was detected in 1:1 mixtures of the two lipids containing either deuterated DMPC or DMPS. Neither the phase transition of the DMPS:DMPC mixtures, nor the spin-lattice relaxation time (T1) of the deuterated DMPS head group, was affected by spectrin. These results argue against any strong interaction of spectrin with phosphatidylserine and rule out the possibility that spectrin is responsible for the maintenance of PS in the inner monolayer of the erythrocyte membrane during the whole life-span of this cell.