Characterization of the binding of the local anesthetics procaine and tetracaine to model membranes of phosphatidylethanolamine: a deuterium nuclear magnetic resonance study

The interaction of the local anesthetics tetracaine and procaine with multilamellar dispersions of phosphatidylethanolamine has been investigated by using 2H NMR of specifically deuterated anesthetics. Tetracaine was found to partition more strongly than procaine into the lipid. The 2H NMR spectra showed a quadrupole doublet and a narrow line, with the former corresponding to membrane-bound anesthetic and the latter to anesthetic free in solution. The integrated areas of the narrow line and of the doublet correspond to the concentrations of free and bound anesthetic predicted from the Kp values. There is no strong pH dependence for the quadrupole splittings of tetracaine, suggesting a similar depth of penetration into the lipid bilayer over the entire pH range. The data are consistent with a model in which tetracaine acts as a wedge to stabilize the phosphatidylethanolamine bilayer against transition to a hexagonal structure. Procaine is proposed to sit higher in the phosphatidylethanolamine bilayer than does tetracaine. The T1 values were generally shorter in the membrane than in solution, suggesting slower motions, particularly for the aromatic ring of tetracaine.     

Multiple binding sites for local anesthetics on reconstituted acetylcholine receptor membranes

Using electron spin resonance spectroscopy and a spin-labeled analog of a tertiary amine local anesthetic, we have identified several populations of the local anesthetic within reconstituted lipid membranes containing purified acetylcholine receptors. These populations represent the local anesthetic interacting with membrane lipid and with the acetylcholine receptor. The data also suggest the existence of at least two classes of binding sites for the local anesthetic on the acetylcholine receptor.     

Multiple sites of inhibition of mitochondrial electron transport by local anesthetics

Local anesthetics and alcohols were found to inhibit mitochondrial electron transport at several points along the chain. The anesthetics employed were the tertiary amines procaine, tetracaine, dibucaine, and chlorpromazine, and the alcohols were n-butanol, n-pentanol, n-hexanol, and benzyl alcohol. Uncoupled sonic submitochondrial particles from beef heart and rat liver were studied. We report the following: (1) All of the anesthetics were found to inhibit each of the segments of the electron transport chain assayed; these included cytochrome c oxidase, durohydroquinone oxidase, succinate oxidase, NADH oxidase, succinate dehydrogenase, succinate-cytochrome c oxidoreductase, and NADH-cytochrome c oxidoreductase. (2) NADH oxidase and NADH-cytochrome c oxidoreductase required the lowest concentrations of anesthetic for inhibition, and cytochrome c oxidase required the highest concentrations. (3) We conclude that there are several points along the chain at which inhibition occurs, the most sensitive being in the region of Complex I (NADH dehydrogenase). (4) Beef heart submitochondrial particles are less sensitive to inhibition than are rat liver particles. (5) Low concentrations of several of the anesthetics gave enhancement of electron transport activity, whereas higher concentrations of the same agents caused inhibition. (6) The concentrations of anesthetics (alcohol and tertiary amine) which gave 50% inhibition of NADH oxidase were lower than the reported concentrations required for blockage of frog sciatic nerve.     

Studies of chromophores in model membranes by polarized light-absorption spectroscopy. The orientation and binding of tetracaine and procaine.

A method for studying the orientation and binding of chromophores in macroscopically aligned membranes by polarized light absorption spectroscopy is described. Here tetracaine and procaine solubilized in a lamellar phase of octanoyl-1-glyceride (monooctanoin) and water have been investigated. Tetracaine is found to be located in the lipid region with a preferential orientation of the molecular long axis parallel to the hydrocarbon chains. The orientation of procaine, mainly residing in the water region, is very small.     

Metal binding sites in proteins: identification and characterization by paramagnetic NMR relaxation

A method is presented that allows the identification and quantitative characterization of metal binding sites in proteins using paramagnetic nuclear magnetic resonance spectroscopy. The method relies on the nonselective longitudinal relaxation rates of the amide protons and their dependence on the paramagnetic metal ion concentration and the pH, and on the three-dimensional structure of the protein. The method is demonstrated using Escherichia coli thioredoxin as a model protein and Ni(2+) as the paramagnetic metal ion. Through a least-squares analysis of the relaxation rates, it is found that Ni(2+) binds to a series of specific sites on the surface of thioredoxin. The strongest binding site is found near the N-terminus of the protein, where the metal ion is coordinated to the free NH(2) group of the N-terminal serine residue and the side chain carboxylate group of the aspartic acid residue in position 2. In addition, Ni(2+) binds specifically but more weakly to the surface-exposed side chain carboxylate groups of residues D10, D20, D47, and E85.     

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.     

Interaction of the local anesthetics dibucaine and tetracaine with sarcoplasmic reticulum membranes. Differential scanning calorimetry and fluorescence studies

The local anesthetics dibucaine and tetracaine inhibit the (Ca2+ + Mg2+)-ATPase from skeletal muscle sarcoplasmic reticulum [DeBoland, A. R., Jilka, R. L., & Martonosi, A. N. (1975) J. Biol. Chem. 250, 7501-7510; Suko, J., Winkler, F., Scharinger, B., & Hellmann, G. (1976) Biochim. Biophys. Acta 443, 571-586]. We have carried out differential scanning calorimetry and fluorescence measurements to study the interaction of these drugs with sarcoplasmic reticulum membranes and with purified (Ca2+ + Mg2+)-ATPase. The temperature range of denaturation of the (Ca2+ + Mg2+)-ATPase in the sarcoplasmic reticulum membrane, determined from our scanning calorimetry experiments, is ca. 45-55 degrees C and for the purified enzyme ca. 40-50 degrees C. Millimolar concentrations of dibucaine and tetracaine, and ethanol at concentrations higher than 1% v/v, lower a few degrees (degrees C) the denaturation temperature of the (Ca2+ + Mg2+)-ATPase. Other local anesthetics reported to have no effect on the ATPase activity, such as lidocaine and procaine, did not significantly alter the differential scanning calorimetry pattern of these membranes up to a concentration of 10 mM. The order parameter of the sarcoplasmic reticulum membranes, calculated from measurements of the polarization of the fluorescence of diphenylhexatriene, is not significantly altered at the local anesthetic concentrations that shift the denaturation temperature of the (Ca2+ + Mg2+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chain dynamics of selectively deuterated fatty acids in high-density lipoproteins studied by deuterium NMR

Deuterium order parameters have been determined for approximately 5 mol% selectively deuterated palmitic acid incorporated into the outer monolayer of high-density lipoproteins (HDL3). The values are SCD = 0.38 for [2,2-2H2]palmitic acid, 0.38 for [4,4-2H2]palmitic acid, 0.37 for [5,5,6,6-2H4]palmitic acid, 0.23 for [11,11,12,12-2H4]palmitic acid, and 0.05 for [16,16,16-2H3]palmitic acid. Comparison of the acyl chain order parameters in HDL3 with acyl chain order parameters determined recently [Parmar, Y.I., Wassall, S.R., & Cushley, R.J. (1984) J. Am. Chem. Soc. 106, 2434-2435] for approximately 5 mol% deuterated palmitic acid in sonicated unilamellar vesicles, composed of the same ratio of phosphatidylcholine/sphingomyelin (85/15 w/w) found in HDL3, shows that acyl chain order in the HDL3 monolayer is approximately 3-5 times higher than in the vesicle bilayer. The acyl chain order in the lipoprotein monolayer is approximately 1.5-2 times higher than in the bilayer of phosphatidylcholine multilamellar dispersions. Deuterium longitudinal relaxation times have been measured for deuterated palmitic acid in HDL3, and the values T1 approximately 16 ms for C2H2 and 170 ms for C2H3 groups are a factor of more than 2 times smaller than found in phospholipid bilayers.     

Direct observation of the properties of cholesterol in membranes by deuterium NMR

The properties of cholesterol in bilayers of egg phosphatidylcholine (PC) were investigated directly by means of ²H-NMR of specifically-deuterated species (C3, C7, C26, C27). Quadrupole splittings were a measure of molecular ordering, and relaxation times T₁ and T_2e were indicators of rates of motion. The importance of the use of echoes for spectral acquisition is emphasised, particularly to obtain accurate values of the quadrupole splitting. In the case of overlapping powder patterns from two labelled positions, the use of the absolute value mode of spectral presentation is shown to yield reasonable estimates of the individual quadrupole splittings. Spectral properties were monitored as a function of cholesterol concentration and temperature. Increasing cholesterol concentration led to a high degree of ordering for the rigid ring system of cholesterol, approaching a molecular order parameter of 0.8 at 50 mol% cholesterol. The isopropyl methyl groups were in all cases less ordered anmore mobile than the ring system, but responded in a similar fashion to variable cholesterol concentration and temperature. The observation of a minimum in the temperature dependence of T₁ for cholesterol-7,7-d₂ led to a direct estimate of its correlation time for molecular motion, 3.5 × 10^?9 s rad^?1. This indicates that the overall rate of motion of cholesterol is considerably slower than that of the lipids in which it is located. The short T_2e values suggest that the motional spectrum of cholesterol is rich in low frequencies. The parallel temperature and cholesterol dependences of quadrupole splittings for different positions on the rigid ring system of cholesterol indicate that the position of the axis of motional averaging of the molecule is not changing, and is the same as that determined in an earlier study. It is emphasised that the steep temperature dependence and small quadrupole splittings for the chain isopropyl methyl groups of cholesterol do not necessarily indicate a high degree of disorder, but may be due to their axes of motional averaging lying at angles close to 54° with respect to the director of the ordered lipids.     

Multiple dopamine binding sites: subcellular localization and biochemical characterization.

Abstract— The biochemical and pharmacological characteristics of dopamine agonist and antagonist binding to rat striatal subcellular fractions were studied and compared to the localization of dopamine–sensitive adenylate cyclase activity. The highest specific activity of adenylate cyclase sensitive to dopamine was associated almost exclusively with the crude synaptic membrane fraction (P₂). Using [³H]-haloperidol, [³H]apomorphine and [³H]spiroperidol as markers for the dopamine receptor, high affinity and stereoselective specific binding was observed for the crude synaptic fraction and the microsomal fraction (P₃). Analysis of the binding of [³H]haloperidol to the striatal microsomal preparation revealed a homogeneous receptor site with a K_d value of 3.0 nm . The data for [³H]haloperidol binding to the crude synaptosomal fraction showed two saturable binding sites with K_d values of 2.5 nm and 12.5 nm . A similar heterogeneous binding profile was observed in the P₂ fraction using [³H]apomorphine. The K_d values for [³H]apomorphine in this fraction were determined to be 1.2 nm and 7.2 nm . The effects of various biochemical parameters including ionic strength, salt concentration and pH on the binding of [³H]haloperidol to the P₂ fraction were also studied. Overall, these data show that the subcellular localization of multiple binding sites in the crude synaptosomal fraction and the identification of specific binding to purified synaptosomes correlate with the subcellular distribution of striatal dopamine-sensitive adenylate cyclase activity.     

A study of mobility and order in model membranes using 2H NMR relaxation rates and quadrupole splittings of specifically deuterated lipids

²H NMR spectra have been observed for several selectively deuterated phospholipid and fatty acid probes intercalated in the liquid crystalline phase of egg phosphatidylcholine in aqueous dispersion. For unsonicated lamellar dispersions and planar multibilayers, quadrupole splittings may be observed which lead directly to a value for the order parameter for the carbon-deuterium bond. Sonicated dispersions yield high-resolution spectra, from which spin-lattice relaxation rates and correlation times for rotational diffusion can be obtained. The presence of cholesterol in the dispersion has no effect on the quadrupole splittings and relaxation rates for ²H in the choline methyl groups, in contrast to its profound effect on the spectra for ²H in the hydrocarbon chains.     

Identification of trapped and boundary lipid binding sites in M13 coat protein/lipid complexes by deuterium NMR spectroscopy

The major coat protein of M13 bacteriophage has been incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, deuterated in the trimethyl segments of the choline headgroup (DMPC-d9). Two-component deuterium and phosphorus-31 NMR spectra have been observed from bilayer complexes containing the coat protein, indicating slow exchange (on the deuterium quadrupole anisotropy and phosphorus-31 chemical shift averaging time scales) of lipid molecules of less than 10(3) Hz between two motionally distinct environments in the complexes. The fraction of the isotropic spectral component increases with increasing M13 protein concentration, and this component is attributed to lipid headgroups, which are disordered relative to their order in protein-free bilayers. The activation energy of the fast local motions of the trimethyl groups of the choline residue in the headgroup decreases from 23 kJ mol-1 in the pure lipid bilayers to 20 kJ mol-1 for the protein-associated lipid headgroups. The chemical exchange rate of lipid molecules between the two motionally distinct environments has been estimated to be 20-50 Hz by steady-state line-shape simulations of the deuterium spectra of DMPC-d9/M13 coat protein complexes using exchange-coupled modified Bloch equations. The off-rate was, as expected from one-to-one exchange, independent of the L/P ratio; tau off -1 = 0.23 kHz. It is suggested that the protein-associated lipid may be trapped between closely packed parallel aggregates of M13 coat protein and that the high local concentration of protein in a one-dimensional arrangement in lipid bilayers may be required for the fast reassembly of phage particles before release from an infected cell.     

Partitioning of local anesthetics into membranes: surface charge effects monitored by the phospholipid head-group

The binding of the charged form of two local anesthetics, dibucaine and etidocaine, to bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was measured simultaneously with ultraviolet spectroscopy and deuterium magnetic resonance. Because of their amphiphilic molecular structure, both drugs intercalate between the lipid molecules, increasing the surface area and imparting a positive electric charge onto the membrane. The ultraviolet (UV) binding isotherms were therefore analyzed in terms of a model which specifically took into account the bilayer expansion as well as the charge-induced concentration variations near the membrane surface. By formulating a quantitative expression for the change in surface area upon drug intercalation and combining it with the Gouy-Chapman theory, the binding of charged dibucaine and etidocaine to the lipid membrane was best described by a partition equilibrium, with surface partition coefficients of 660 +/- 80 M-1 and 11 +/- 2 M-1 for dibucaine and etidocaine, respectively (pH 5.5, 0.1 M NaCl/50 mM buffer). Deuterium magnetic resonance demonstrated further that the binding of drug changed the head-group conformation of the lipid molecules. Invoking the intercalation model, a linear variation of the deuterium quadrupole splittings of the choline segments with the surface charge density was observed, suggesting that the phosphocholine head-group may act as a 'molecular electrometer' with respect to surface charges.     

Identification and characterization of endothelin binding sites in rat renal papillary and glomerular membranes

The present study was designed to identify and characterize specific endothelin binding sites in membranes of rat renal papillae and glomeruli which appear to be target tissues for this new peptide hormone. Saturation binding studies indicate that the sites have a high and uniform affinity. The dissociation constants averaged 662 +/- 151 and 1309 +/- 123 pM and the receptor densities 7666 +/- 920 and 5831 +/- 348 fmol/mg protein for papillary and glomerular membranes, respectively. Endothelin 1, endothelin 3 and sarafotoxin all inhibited [125I]-endothelin binding with IC50's in the 100-300 pM range, whereas unrelated peptides, namely angiotensin II, atrial natriuretic peptide, and platelet-derived growth factor failed to compete for [125I]-endothelin binding. Deletion of the carboxyterminal tryptophan in endothelin 1 reduced its affinity for glomerular binding sites by 2 orders of magnitude. Specific endothelin binding to these membranes was maximal at pH 4 and was markedly inhibited as the pH was raised above 8. When [125I]-endothelin is covalently linked to glomerular membrane binding sites, SDS-PAGE of these solubilized membranes followed by autoradiography reveals a predominant specifically labeled band of 45 kDa. Whether this band represents a subunit of the endothelin receptor(s), the receptor proper, or an intracellular endothelin binding protein remains to be determined.     

Mechanisms of use-dependent block of sodium channels in excitable membranes by local anesthetics

Many local anesthetics promote reduction in sodium current during repetitive stimulation of excitable membranes. Use-, frequency-, and voltage-dependent responses describe patterns of peak INa when pulse width, pulse frequency, and pulse amplitude are varied. Such responses can be viewed as reflecting voltage-sensitive shifts in equilibrium between conducting, unblocked channels and nonconducting, blocked channels. The modulated-receptor hypothesis postulates shifts in equilibrium as the result of a variable-affinity receptor and modified inactivation gate kinetics in drug-complexed channels. An alternative view considers drug blocking in the absence of these two features. We propose that drug binds to a constant-affinity channel receptor where receptor access is regulated by the channel gates. Specifically, we view channel binding sites as guarded by the channel gate conformation, so that unlike receptors where ligands have continuous access, blocking agent access is variable during the course of an action potential. During the course of an action potential, the m and h gates change conformation in response to transmembrane potential. Conducting channels with both gates open leave the binding site unguarded and thus accessible to drug, whereas nonconducting channels, with gates in the closed conformation, act to restrict drug access to unbound receptors and possibly to trap drug in drug-complexed channels. We develop analytical expressions characterizing guarded receptors as "apparently" variable-affinity binding sites and predicting shifts in "apparent" channel inactivation in the hyperpolarizing direction. These results were confirmed with computer simulations. Furthermore, these results are in quantitative agreement with recent investigations of lidocaine binding in cardiac sodium channels.     

Interactions of lectins with CHO cell surface membranes. II. Differential effects of local anesthetics on endocytosis of CON A and WGA binding sites

Using fibroblastic CHO cells, we have examined (1) the internalization and redistribution of surface binding sites for the lectins Concanavalin A and wheat germ agglutinin and (2) the sensitivity of these processes to putative inhibitors of cytoskeletal activity. Following initial exposure to fluorescein conjugated Con A (CAF) or WGA (WGAF) at 4° C, kinetic analysis of internalization and intracellular aggregation of lectin at 37°C indicated more rapid aggregate formation in the case of WGA than in the case of Con A. Treatment with tertiary amine local anesthetics (tetracaine, dibucaine, and xylocaine) or with a lysosomatrophic amine, m-dansyl cadaverine, blocked internalization of Con A but not of WGA. Similar differential effects on redistribution of Con A and WGA were not however observed with the antimicrotubule agents colchicine and nocodazole. Simultaneous treatment of cells with WGAF and with rhodamine labeled Con A (CAR) resulted in the accumulation of both labels in a central perinuclear aggregate; a similar experiment in the presence of local anesthetic resulted in a diffuse peripheral distribution of CAR and a central aggregate of WGAF. These results suggest (1) CHO cells possess at least two distinct pathways for lectin internalization and redistribution, which can be discriminated in terms of drug sensitivity; (2) CHO cells can sort out and independently internalize different populations and lectin binding sites; and (3) different pathways may be a manifestation of biochemically distinct linkages between cytoskeletal elements and various groups of surface glycoproteins. Present findings concur with our previous results concerning the mutual independence of the surface binding sites for Con A and WGA (Emerson and Juliano, 1982).