Tetracaine Binding Research Articles

Interactions of new lactoglobulin variants with tetracaine: crystallographic studies of ligand binding to lactoglobulin mutants possessing single substitution in the binding pocket.

β-Lactoglobulin (BLG) like other lipocalins can be modified by mutagenesis to re-direct its ligand binding properties. Local site-directed mutagenesis was used to change the geometry of the BLG ligand binding pocket and therefore change BLG ligand preferences. The presented studies are focused on previously described mutants L39Y, I56F, L58F, F105L, and M107L and two new BLG variants, L39K and F105A, and their interactions with local anesthetic drug tetracaine. Binding of tetracaine to BLG mutants was investigated by X-ray crystallography. Structural analysis revealed that for tetracaine binding, the shape of the binding pocket seems to be a more important factor than the substitutions influencing the number of interactions. Analyzed BLG mutants can be classified according to their binding properties to variants: capable of binding tetracaine in the β-barrel (L58F, M107L); capable of accommodating tetracaine on the protein surface (I56F) and unable to bind tetracaine (F105L). Variants L39K, L39Y, and F105A, had a binding pocket blocked by endogenous fatty acids. The new tetracaine binding site was found in the I56F variant. The site localized on the surface near Arg124 and Trp19 was previously predicted by in silico studies and was confirmed in the crystal structure.

High-Throughput Screening of Drug–Lipid Membrane Interactions via Counter-Propagating Second Harmonic Generation Imaging

Here we report the use of counter-propagating second harmonic generation (SHG) to image the interactions between the local anesthetic tetracaine and a multicomponent planar supported lipid bilayer array in a label-free manner. The lipid bilayer arrays, prepared using a 3D continuous flow microspotter, allow the effects of lipid phase and cholesterol content on tetracaine binding to be examined simultaneously. SHG images show that tetracaine has a higher binding affinity to liquid-crystalline phase lipids than to solid-gel phase lipids. The presence of 28 mol % cholesterol decreased the binding affinity of tetracaine to bilayers composed of the mixed chain lipid, 1-steroyl-2-oleoyl-sn-glycero-3-phophocholine (SOPC), and the saturated lipids 1,2-dimyristoyl-sn-glycero-3-phophocholine (DMPC) and 1,2-dipamitoyl-sn-glycero-3-phophocholine (DPPC) while having no effect on diunsaturated 1,2-dioleoyl-sn-glycero-3-phophocholine (DOPC). The maximum surface excess of tetracaine increases with the degree of unsaturation of the phospholipids and decreases with cholesterol in the lipid bilayers. The paper demonstrates that SHG imaging is a sensitive technique that can directly image and quantitatively measure the association of a drug to a multicomponent lipid bilayer array, providing a high-throughput means to assess drug-membrane interactions.

Structural Determinants of Drugs Acting on the Nav1.8 Channel

The aim of this work is to study the role of pore residues on drug binding in the Na(V)1.8 channel. Alanine mutations were made in the S6 segments, chosen on the basis of their roles in other Na(V) subtypes; whole cell patch clamp recordings were made from mammalian ND7/23 cells. Mutations of some residues caused shifts in voltage dependence of activation and inactivation, and gave faster time course of inactivation, indicating that the residues mutated play important roles in both activation and inactivation in the Na(V)1.8 channel. The resting and inactivated state affinities of tetracaine for the channel were reduced by mutations I381A, F1710A, and Y1717A (for the latter only inactivated state affinity was measured), and by mutation F1710A for the Na(V)1.8-selective compound A-803467, showing the involvement of these residues for each compound, respectively. For both compounds, mutation L1410A caused the unexpected appearance of a complete resting block even at extremely low concentrations. Resting block of native channels by compound A-803467 could be partially removed ("disinhibition") by repetitive stimulation or by a test pulse after recovery from inactivation; the magnitude of the latter effect was increased for all the mutants studied. Tetracaine did not show this effect for native channels, but disinhibition was seen particularly for mutants L1410A and F1710A. The data suggest differing, but partially overlapping, areas of binding of A-803467 and tetracaine. Docking of the ligands into a three-dimensional model of the Na(V)1.8 channel gave interesting insight as to how the ligands may interact with pore residues.

Identification of Binding Sites in the Nicotinic Acetylcholine Receptor for TDBzl-etomidate, a Photoreactive Positive Allosteric Effector

Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of gamma-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 microm. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (>10 microm) increased the submaximal response to acetylcholine (10 microm) with a 2.5-fold increase at 60 microm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [(3)H]tetracaine and [(3)H]phencyclidine to Torpedo nAChR-rich membranes (IC(50) values of 0. 8 mm). nAChR-rich membranes were photolabeled with [(3)H]TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (deltaLeu-265) and M2-13 (alphaVal-255 and deltaVal-269), whereas labeling of alphaM2-10 (alphaSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in gammaM3 (gammaMet-299), a residue that contributes to the same pocket in the nAChR structure as alphaM2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the alpha and gamma subunits (labeling of alphaM2-10 and gammaMet-299) likely to be a site for positive allosteric modulation.

Noncompetitive antagonist binding sites in the torpedo nicotinic acetylcholine receptor ion channel. Structure-activity relationship studies using adamantane derivatives.

We used a series of adamantane derivatives to probe the structure of the phencyclidine locus in either the resting or desensitized state of the nicotinic acetylcholine receptor (AChR). Competitive radioligand binding and photolabeling experiments using well-characterized noncompetitive antagonists such as the phencyclidine analogue [piperidyl-3,4-(3)H(N)]-N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([(3)H]TCP), [(3)H]ethidium, [(3)H]tetracaine, [(14)C]amobarbital, and 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) were performed. Thermodynamic and structure-function relationship analyses yielded the following results. (1) There is a good structure-function relationship for adamantane amino derivatives inhibiting [(3)H]TCP or [(3)H]tetracaine binding to the resting AChR. (2) Since the same derivatives inhibit neither [(14)C]amobarbital binding nor [(125)I]TID photoincorporation, we conclude that these positively charged molecules preferably bind to the TCP locus, perhaps interacting with alphaGlu(262) residues at position M2-20. (3) The opposite is true for the neutral molecule adamantane, which prefers the TID (or barbiturate) locus instead of the TCP site. (4) The TID site is smaller and more hydrophobic (it accommodates neutral molecules with a maximal volume of 333 +/- 45 A(3)) than the TCP locus, which has room for positively charged molecules with volumes as large as 461 A(3) (e.g., crystal violet). This supports the concept that the resting ion channel is tapering from the extracellular mouth to the middle portion. (5) Finally, although both the hydrophobic environment and the size of the TCP site are practically the same in both states, there is a more obvious cutoff in the desensitized state than in the resting state, suggesting that the desensitization process constrains the TCP locus. A plausible location of neutral and charged adamantane derivatives is shown in a model of the resting ion channel.

Allosterically linked noncompetitive antagonist binding sites in the resting nicotinic acetylcholine receptor ion channel

Previous studies have established the presence of overlapping binding sites for the noncompetitive antagonists (NCAs) amobarbital, tetracaine, and 3-trifluoromethyl-3-( m-[ 125 I ]iodophenyl) diazirine ([ 125 I ]TID) within the ion channel of the Torpedo nicotinic acetylcholine receptor (AChR) in the resting state. These well-characterized NCAs and competitive radioligand binding and photolabeling experiments were employed to better characterize the interaction of the dissociative anesthetics ketamine and thienylcycloexylpiperidine (TCP) with the resting AChR. Our experiments yielded what appear to be conflicting results: (i) both ketamine and TCP potentiated [ 125 I ]TID photoincorporation into AChR subunits; and (ii) ketamine and TCP had very little effect on [ 14 C ]amobarbital binding. Nevertheless, (iii) both ketamine and TCP completely displaced [ 3 H ]tetracaine binding ( K is∼20.9 and 2.0 μM, respectively) by a mutually exclusive mechanism. To reconcile these results we propose that, in the resting ion channel, TCP and ketamine bind to a site that is spatially distinct from the TID and barbiturate locus, while tetracaine bridges both binding sites.

Elucidation of Biphasic Alterations on Acetylcholinesterase (AChE) Activity and Membrane Fluidity in the Structure–Functional Effects of Tetracaine on AChE-Associated Membrane Vesicles

Tetracaine-induced biphasic structure–functional alterations were investigated in acetylcholinesterase (AChE)-associated membrane vesicles from the electric organ ofTorpedo californica.Enzyme assays showed that tetracaine exhibits a biphasic effect on the activity of membrane-bound AChE: increasing it at low concentrations (<12 mM) and decreasing it at high concentrations (>12 mM). Fluorescence-polarization experiments demonstrated that tetracaine affects the fluidity of lipid hydrocarbon chains of these membranes in a biphasic manner: increasing it at <20 mM and decreasing it at >20 mM. This small molecule also alters the fluidity of the negatively charged lipid head group: increasing it at <13 mM and remaining essentially at the same level at >13 mM. The positively charged lipid head group is unaffected. Contrasting effects on AChE activity with changes in membrane fluidity showed that [tetracaine] for AChE activity is comparable to that for the fluidity of the negatively charged lipid head group (12 mM versus 13 mM), but lower than that for a biphasic effect on the fluidity of lipid hydrocarbon chains (12 mM versus 20 mM). Differential scanning microcalorimetry showed that, due to membrane protein–lipid interaction, the lipid-phase transition temperature (tml) is higher for AChE-associated membrane vesicles than for isolated lipids from these membranes. An overall disordering of the membranes by tetracaine, as inferred from the lowering oftml, was also demonstrated. These findings suggested that binding of tetracaine to the lipid polar head group and membrane protein–lipid interaction may contribute to a higher [tetracaine] in inducing a comparable biphasic effect on membrane fluidity. At high [tetracaine], charge interactions between the tetracaine cation and the negatively charged lipid head group may result in a new lipid phase in the membranes, which could reverse the increase in membrane fluidity, resulting in the observed biphasic effect. Although both tetracaine and alcohol are amphiphilic species, they exhibit distinctive structure–functional effects on the membranes, as shown by comparing the results obtained on tetracaine with those previously reported for alcohol. The present observations may have significant physiological implications and may be of importance in understanding the biochemical effects of tetracaine in correlation with its physiological impact.

Effects of i.v. anaesthetic agents and halothane on [3H]tetracaine binding to rat cerebrocortical membranes

In an attempt to determine if there is any overlap in local and general anaesthetic binding sites, we have examined the effects of thiopental, phenobarbital, pentobarbital, propofol, ketamine (racemic and R(+)/S(-)), alfaxalone, etomidate and halothane on [3H]tetracaine binding to rat cerebrocortical membranes. Membranes were prepared in Tris HCI 50 mmol litre-1, pH 7.4, by homogenization and centrifugation. Binding assays were performed in 1-ml volumes of Tris HCI buffer at room temperature or 37 degrees C for halothane. Binding of [3H]tetracaine was displaced dose-dependently by unlabelled tetracaine with a mean pIC50 value of 6.91 (SEM 0.07) (123 nmol litre-1). With the exception of propofol (at high concentrations), all i.v. anaesthetic agents failed to displace the binding of [3H]tetracaine. In contrast, halothane produced a dose-dependent and statistically significant reduction in total [3H]tetracaine binding at clinically achievable concentrations (0.289, 0.885 and 1.484 mmol litre-1 equivalent to 1.0, 3.1 and 5.1 rat MAC) without markedly affecting the pIC50. Collectively these data may suggest some overlap in the binding sites for [3H]tetracaine and volatile but not i.v. general anaesthetic agents.

Effects of amiloride and its analogues on [ 3H]batrachotoxinin-A 20-α benzoate binding, [ 3H]tetracaine binding and 22Na influx

The ability of amiloride and its analogues to inhibit [ 3H]batrachotoxinin-A 20-α benzoate ([ 3H]BTX-B) and [ 3H]tetracaine binding to rat synaptosomes and to a rat heart membrane preparation was tested. Their ability to inhibit 22Na influx was determined with rat synaptosomes. 5-N-substituted analogues were generally more potent in inhibiting [ 3H]BTX-B and [ 3H]tetracaine binding than compounds substituted on the guanidine group. However, the inhibition was not competitive. Amiloride and some of its analogues were as active or more active in inhibiting [ 3H]tetracaine binding than they were in inhibiting [ 3H]BTX-B binding. 22Na influx was inhibited with the same relative potencies as [ 3H]BTX-B binding and a good correlation was found between the two inhibitions. These results show an effect of amiloride and its analogues on the voltage-sensitive Na + channels, which could partly explain the inotropic effects of these drugs.

Binding sites for [ 3H]tetracaine in synaptosomal sodium channel preparations from mouse brain

The present study was an attempt to answer the question whether the local anesthetic [ 3H]tetracaine labels sodium channels in mouse brain synaptosomes. Binding of [ 3H]tetracaine had a K d of 0.19 μM and a B max ranging from 3.7 to 5.2 pmol/mg of protein. Local anesthetics other than tetracaine and cocaine-related compounds inhibited [ 3H]tetracaine binding with Hill numbers between 0.3 and 0.6. Initiaton of the dissociation of [ 3H]tetracaine binding by an excess of unlabeled tetracaine resulted in non-linear curves. These results are consonant with site heterogeneity, negative cooperativity, or complexities arising from the use of synaptosomal preparations instead of broken membranes. There were important differences between the absolute values of the potency of various local anesthetic drugs and cocaine-related compounds in inhibiting binding of [ 3H]tetracaine and those in inhibiting binding of [ 3H]batrachotoxinin A 20-α-benzoate. Although there were some effects of manipulating the state of the channel with activating toxins on the apparent rate of association and dissociation of [ 3H]tetracaine binding, the equilibrium binding was not much affected by the toxins. The results indicate that further characterization is necessary before accepting [ 3H]tetracaine binding as a valid tool for studying sodium channels.

Anti‐anginal arylalkylamines and sodium channels: [3H]‐batrachotoxinin‐A 20‐α‐benzoate and [3H]‐tetracaine binding

[3H]-batrachotoxinin-A 20-alpha-benzoate ([3H]-BTX-B) and [3H]-tetracaine are useful ligands for the study of sodium channels. Inhibition of their binding by various anti-anginal drugs was tested on a rat synaptosomal preparation and on a heart membrane preparation. Diphenylalkylamines and structurally related drugs inhibited [3H]-BTX-B binding in both the synaptosomal preparation and heart membrane preparation. They were almost inactive on [3H]-tetracaine binding. These results suggest that activity of arylalkylamines could be mediated by an interaction on the sodium channel.

3H]‐tetracaine binding on rat synaptosomes and sodium channels

[3H]-tetracaine binding was studied in a rat synaptosomal preparation. [3H]-tetracaine bound to a single class of binding sites with a mean KD of 188 +/- 28 nM and a mean maximal binding capacity of 13 +/- 0.7 pmol mg-1 protein. [3H]-tetracaine binding was inhibited by tetracaine, procaine and by beta-adrenoceptor blocking agents which possess local anaesthetic properties. [3H]-tetracaine binding was not modified by neurotoxins interacting specifically with the sodium channels.

Vesicle membrane-water partition coefficients determined from Fourier transform pulsed-gradient spin-echo NMR based self-diffusion data. Application to anesthetic binding in tetracaine-phosphatidylcholine-water systems

Multicomponent self-diffusion data on dioleoyl(DOL)- and dipalmitoyllecithin (DPL) vesicle-water systems have been determined using a Fourier transform NMR technique. The self-diffusion of vesicles is characterized by diffusion coefficients two magnitudes lower than that of small molecules in solution. Consequently, the degree of binding of small molecules is strongly reflected in their time-averaged self-diffusion coefficient in vesicle-water systems. This provides a new basis for the determination of vesicle-water partition equilibria. The feasibility of the technique has been investigated in one anesthetic-lipid system and is found to be very good. The binding of the hydrochloride form of tetracaine to DOL vesicles at pH 3 and 7 ( K p = 30–50) is found to be very much lower than that of the neutral molecule at pH 9 ( K p = 800–900). No significant difference in the tetracaine binding characteristics was found between DOL, DOL-cholesterol and DPL systems.

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.