Rat Brain Synaptosomal Membranes Research Articles

Delineation of the Functional Site of α-Dendrotoxin: THE FUNCTIONAL TOPOGRAPHIES OF DENDROTOXINS ARE DIFFERENT BUT SHARE A CONSERVED CORE WITH THOSE OF OTHER Kv1 POTASSIUM CHANNEL-BLOCKING TOXINS

We identified the residues that are important for the binding of alpha-dendrotoxin (alphaDTX) to Kv1 potassium channels on rat brain synaptosomal membranes, using a mutational approach based on site-directed mutagenesis and chemical synthesis. Twenty-six of its 59 residues were individually substituted by alanine. Substitutions of Lys5 and Leu9 decreased affinity more than 1000-fold, and substitutions of Arg3, Arg4, Leu6, and Ile8 by 5-30-fold. Substitution of Lys5 by norleucine or ornithine also greatly altered the binding properties of alphaDTX. All of these analogs displayed similar circular dichroism spectra as compared with the wild-type alphaDTX, indicating that none of these substitutions affect the overall conformation of the toxin. Substitutions of Ser38 and Arg46 also reduced the affinity of the toxin but, in addition, modified its dichroic properties, suggesting that these two residues play a structural role. The other residues were excluded from the recognition site because their substitutions caused no significant affinity change. Thus, the functional site of alphaDTX includes six major binding residues, all located in its N-terminal region, with Lys5 and Leu9 being the most important. Comparison of the functional site of alphaDTX with that of DTX-K, another dendrotoxin (Smith, L. A., Reid, P. F., Wang, F. C., Parcej, D. N., Schmidt, J. J., Olson, M. A., and Dolly, J. O. (1997) Biochemistry 36, 7690-7696), reveals that they only share the predominant lysine and probably a leucine residue; the additional functional residues differ from one toxin to the other. Comparison of the functional site of alphaDTX with those of structurally unrelated potassium channel-blocking toxins from venomous invertebrates revealed the common presence of a protruding key lysine with a close important hydrophobic residue (Leu, Tyr, or Phe) and few additional residues. Therefore, irrespective of their phylogenetic origin, all of these toxins may have undergone a functional convergence. The functional site of alphaDTX is topographically unrelated to the "antiprotease site" of the structurally analogous bovine pancreatic trypsin inhibitor.

Cobatoxins 1 and 2 from Centruroides noxius Hoffmann constitute a subfamily of potassium-channel-blocking scorpion toxins.

Potassium-channel-blocking scorpion toxins (alpha-K-toxins) have been shown to be valuable tools for the study of potassium channels. Here we report two toxins, cobatoxin 1 and 2, of 32 amino acids, containing three disulphide bridges, that were isolated from the venom of the Mexican scorpion Centruroides noxius. Their primary sequences show less than 40% identity to other alpha-K-toxins. It is therefore proposed that they belong to subfamily 9. The cDNA of cobatoxin 1 encodes a putative signal peptide, a putative short propeptide, the mature peptide and two amino acids that are processed to leave cobatoxin 1 amidated at the C-terminus. In rat brain synaptosomal membranes cobatoxin 1 and cobatoxin 2 bind to a common binding site of alpha-K-toxins with Ki values of 109 pM and 87 pM, respectively. Moreover, they block the Shaker and Kv1.1 K+ channels with moderate affinities, with Kd values of around 0.7 microM and 4.1 microM (Shaker) and 0.5 microM and 1.0 microM (Kv1.1), respectively. A three-dimensional model of cobatoxin 1 was generated and used to interpret the obtained functional data on a structural basis.

Modulation of rat brain synaptosomal plasma membrane achieved by atracurium and its metabolite laudanosine.

Atracurium besylate and laudanosine cause excitement and seizures when introduced into the central nervous system of laboratory animals. We examined the modulation of lipid-protein interaction in the lipid environment of rat brain synaptosomal plasma membrane (SPM)-bound enzymes as a possible mechanism leading to these effects. The effect of various concentrations of atracurium besylate and laudanosine, or of varying duration of SPM, on the activity of Na+/K+-stimulated ATPase, Mg2+-stimulated ATPase and 5'-nucleotidase were assessed. The modulation of lipid protein interaction by laudanosine was estimated on the basis of the temperature dependence and cooperative behaviour of Na+/K+-stimulated ATPase. The effect of atracurium besylate or laudanosine on Na+/K+-stimulated ATPase activity was biphasic. Maximal enzyme stimulation appeared at 10(-4) M atracurium besylate or 10(-8) M laudanosine, and at 30 min of pre-incubation with both drugs. Arrhenius plots of Na+/K+-stimulated ATPase showed a transition temperature of 23.0 +/- 1.2 degrees C in control SPM and shifted to 16.5 +/- 0.9 degrees C (p < 0.01) in SPM treated with 10(-8) M laudanosine. The Hill coefficients for the allosteric inhibition of Na+/K+-stimulated ATPase by fluoride decreased from 1.99 +/- 0.22 in controls to 1.06 +/- 0.11 (p < 0.001) in the presence of 10(-8) M laudanosine. Our results suggest that laudanosine, one of the principal metabolites of atracurium besylate, affects nerve cell function in rats through the perturbation of the membrane lipid structure accompanied by SPM-bound enzyme dysfunction.

Two similar peptides from the venom of the scorpion Pandinus imperator, one highly effective blocker and the other inactive on K + channels

Two novel peptides, named Pi4 and Pi7, were purified from the venom of the scorpion Pandinus imperator, and their primary structures were determined. These peptides have 38 amino acids residues, compacted by four disulfide bridges, instead of the normal three found in most K +-channel specific toxins. Both peptides contain 25 identical amino acid residues in equivalent positions (about 66% identity), including all eight half-cystines. Despite the fact that their C-terminal sequence comprising amino acid residues 27 to 37 are highly conserved (10 out of 11 amino acids are identical), Pi4 blocks completely and reversibly Shaker B K +-channels (a Kv1.1 sub-family type of channel) at 100 nM concentration, whereas Pi7 is absolutely inactive at this concentration. Similar effects were observed in binding and displacement experiments to rat brain synaptosomal membranes using 125I-Noxiustoxin, a well known K +-channel specific toxin. In this preparation Pi4 displaces the binding of radiolabeled Noxiustoxin with ic 50 in the order of 10 nM, whereas Pi7 is ineffective at same concentration. Comparative analysis of Pi4 and Pi7 sequences with those obtained by site directed mutagenesis of Charybdotoxin, another very well studied K +-channel blocking toxin, shows that the substitution of lysine (in Pi4) for arginine (in Pi7) at position 26, might be one of the important `point mutations' responsible for such impressive variation in blocking properties of both toxins, here described.

Effect of desmopressin on rat brain synaptosomal membranes: a pilot study

Desmopressin (DDAVP) is a synthetic analogue of arginine vasopressin (AVP) that is indicated for the treatment of diabetes insipidus, nocturnal enuresis, and some hemostatic disorders. Some activities of DDAVP are attributed to the interaction with the AVP cell surface receptor; other activities are uncertain. In the present study we investigated the effect of DDAVP on rat brain synaptosomal membranes by means of a fluorescence technique. The membrane fluidity of synaptosomal membranes was studied by measuring the steady-state fluorescence anisotropy of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene incorporated into the membrane. The results show that the addition of DDAVP (10 −6 mol/L) induced a significant and time-limited increase in the lipid order in the exterior part of the membrane. These data demonstrate that DDAVP interacts with synaptosomal membranes. It is reasonable to propose that this interaction induces changes in cell response or function. Further studies are needed to elucidate the molecular basis of this effect.

Modulation of synaptosomal plasma membrane-bound enzyme activity through the perturbation of plasma membrane lipid structure by bupivacaine.

We investigated modulations of lipid dynamics and lipid-protein interactions of rat brain synaptosomal plasma membrane (SPM) as one of the possible mechanisms by which the local anesthetic bupivacaine (BPV) has an adverse effect on nerve cell function, with SPM-bound enzyme activity used as a functional probe. The kinetics of BPV impact on the activity of the endoenzymes Ca2+/Mg2+-stimulated ATPase and Na+/K+-stimulated ATPase and the active concentrations of the drug were relevant to those that produce biphasic systemic toxicity. Arrhenius plots of these enzymes showed a transition temperature of 26.6 +/- 1.8 degrees C and 24.5 +/- 1.2 degrees C (mean +/- SD), respectively, in control SPM, which shifted to 17.1 +/- 0.95 degrees C (P < 0.01) and 18.2 +/- 0.85 degrees C (P < 0.05) in SPM treated with 10(-5) M BPV. The Hill coefficients for the allosteric inhibition of Ca2+/Mg2+-stimulated ATPase by Na+ and Na+/K+-stimulated ATPase by fluoride decreased from 1.73 +/- 0.20 and 1.95 +/- 0.25, respectively, in controls to 0.92 +/- 0.09 (P < 0.001) and 1.09 +/- 0.11 (P < 0.001) in the presence of 10(-5) M BPV. The fluidity perturbation in the microenvironment of the ectoenzyme acetylcholinesterase was observed only at 5 x 10(-3) M BPV, as confirmed by the disparity in transition temperature between the controls (22.3 +/- 1.2 degrees C) and the BPV-treated SPM (17.5 +/- 0.8 degrees C, P < 0.01) and that in the Hill coefficient in the two groups: 2.15 +/- 0.24 and 0.97 +/- 0.12 (P < 0.001), respectively. We propose that under physiological conditions, the neutral and protonated forms of local anesthetics can affect nerve cell function through the asymmetric perturbation of the membrane lipid structure, accompanied by synaptosomal plasma membrane-bound enzyme dysfunction.

Modulation of Synaptosomal Plasma Membrane-Bound Enzyme Activity Through the Perturbation of Plasma Membrane Lipid Structure by Bupivacaine

We investigated modulations of lipid dynamics and lipid-protein interactions of rat brain synaptosomal plasma membrane (SPM) as one of the possible mechanisms by which the local anesthetic bupivacaine (BPV) has an adverse effect on nerve cell function, with SPM-bound enzyme activity used as a functional probe. The kinetics of BPV impact on the activity of the endoenzymes Ca2+/Mg2+-stimulated ATPase and Na+/K+-stimulated ATPase and the active concentrations of the drug were relevant to those that produce biphasic systemic toxicity. Arrhenius plots of these enzymes showed a transition temperature of 26.6 +/- 1.8[degree sign]C and 24.5 +/- 1.2[degree sign]C (mean +/- SD), respectively, in control SPM, which shifted to 17.1 +/- 0.95[degree sign]C (P < 0.01) and 18.2 +/- 0.85[degree sign]C (P < 0.05) in SPM treated with 10-5 M BPV. The Hill coefficients for the allosteric inhibition of Ca2+/Mg2+-stimulated ATPase by Na+ and Na+/K+-stimulated ATPase by fluoride decreased from 1.73 +/- 0.20 and 1.95 +/- 0.25, respectively, in controls to 0.92 +/- 0.09 (P < 0.001) and 1.09 +/- 0.11 (P < 0.001) in the presence of 10-5 M BPV. The fluidity perturbation in the microenvironment of the ectoenzyme acetylcholinesterase was observed only at 5 x 10-3 M BPV, as confirmed by the disparity in transition temperature between the controls (22.3 +/- 1.2[degree sign]C) and the BPV-treated SPM (17.5 +/- 0.8[degree sign]C, P < 0.01) and that in the Hill coefficient in the two groups: 2.15 +/- 0.24 and 0.97 +/- 0.12 (P < 0.001), respectively. Implications: We propose that under physiological conditions, the neutral and protonated forms of local anesthetics can affect nerve cell function through the asymmetric perturbation of the membrane lipid structure, accompanied by synaptosomal plasma membrane-bound enzyme dysfunction. (Anesth Analg 1997;85:1337-43)

Lipid Composition and Fatty Acid Profiles of Myelin and Synaptosomal Membranes of Rat Brain in Response to the Consumption of Different Fats

Experiments were performed to ascertain the effect of dietary fats like safflower, mustard, peanut, and coconut oil, which differ widely in the essential fatty acid components on the distribution patterns of specific lipids such as cholesterol, phospholipids, and cerebrosides and the fatty acid profile of the latter two lipid fractions obtained from myelin and synaptosomal membranes of three anatomical regions of the rat brain (cerebrum, cerebellum, and brain stem). Weanling male CFY rats received diets adequate in all essential nutrients but varied with respect to the nature of dietary fat. The fat was fed at the 20% level in the diet for 16 weeks. Myelin and synaptosomal membranes from cerebrum, cerebellum, and brain stem of these rat brains were prepared using discontinuous sucrose density gradient ultracentrifugation. Cholesterol to phospholipid molar ratio remained constant in the synaptosomal membranes in cerebrum and brainstem regions irrespective of the dietary fat treatment, except in the cerebellar region of coconut and mustard oil-fed animals, which showed significantly higher ratio. The cerebroside content of myelin membranes obtained from cerebra and cerebella of these animals were significantly lower compared with those of groundnut and safflower oil-fed rats. Fatty acid compositions of myelin and cerebroside fractions of these membranes were determined. Mustard oil feeding resulted in the enrichment of synaptosomal phospholipid fraction with docosahexaenoic (22:6 n-3) acid (DHA) in the cerebral and cerebellar regions. On the contrary, myelin phospholipid fractions of these regions of mustard oil-fed group were characterized by a lower degree of unsaturation. Lignoceric (24:0) and nervonic (24:1 n-9) acids, the marker fatty acids of myelin, were significantly lower in the brain regions of coconut and mustard oil-fed animals. Results indicate that the brain can adapt to essential fatty acid deficiency in response to coconut oil feeding by retaining high concentrations of polyunsaturated fatty acid in its membranes. On the other hand, dietary fats rich in linoleic (18:2 n-6) acid (safflower) or linolenic acid (18:3 n-3) (mustard oil) significantly elevated the polyunsaturated fatty acid content. In addition, they also altered the long chain fatty acids (like lignoceric and nervonic acids) of a stable membrane, like myelin.

Maurotoxin, a four disulfide bridge toxin from Scorpio maurus venom: purification, structure and action on potassium channels

A new toxin acting on K + channels, maurotoxin (MTX), has been purified to homogeneity from the venom of the chactoid scorpion Scorpio maurus. MTX is a basic single chain 34 amino acid residue polypeptide, amidated at its C terminal, and crosslinked by four disulfide bridges. It shows 29–68% sequence identity with other K + channel toxins, and presents an original disulfide pattern, the last two half-cystine residues (31–34) being connected. Although the first three disulfide bonds have not been defined experimentally, modelling based on the structure of charybdotoxin favored two combinations out of six, one of which has two bridges (3–24 and 9–29) in common with the general motif of scorpion toxins. The last bridge would connect residues 13 and 19. MTX inhibits the binding to rat brain synaptosomal membranes of both [ 125I]apamin, a SK Ca channel blocker (IC 50 5 nM), and [ 125I]kaliotoxin, a Kv channel blocker (IC 50 30 pM). MTX blocks the Kv1.1, Kv1.2 and Kv1.3 currents expressed in Xenopus oocytes with IC 50 of 45, 0.8 and 180 nM, respectively. MTX represents a member of a new class of short toxins with 4 disulfide bridges, active on voltage-dependent K + channel and also competing with apamin for binding to its receptor.

A potassium-channel toxin from the sea anemone Bunodosoma granulifera, an inhibitor for Kv1 channels. Revision of the amino acid sequence, disulfide-bridge assignment, chemical synthesis, and biological activity.

The potassium channel toxin secreted by the sea anemone Bunodosoma granulifera (BgK) is a 37-amino-acid peptide containing three disulfide bridges. Because a synthetic peptide corresponding to the reported sequence of BgK was found not to fold properly, the sequence was determined again. The new sequence differed from the previous one in the C-terminal tetrapeptide, which contains two cysteines involved in disulfide bridging. The revised sequence is: V C R D W F K E T A C R H A K S L G N C R T S Q K Y R A N C A K T C E L C. The toxin BgK was synthesized according to the new sequence and folded successfully. Disulfide bridges were assigned by peptide mapping on both natural and synthetic forms to be between Cys2-Cys37, Cys11-Cys30 and Cys20-Cys34. The toxin contains a C-terminal free carboxylate as shown by comparing the native toxin with two synthetic peptides containing the C-terminus in either the carboxylate or carboxamido form. Synthetic BgK inhibits binding of 125I-alpha-dendrotoxin to rat brain synaptosomal membranes, similarly to natural BgK (nanomolar range). No activity was observed on maxi-K+ channels incorporated into planar lipid bilayers. The ability of BgK to block voltage-dependent K+ channels was determined from recordings of whole cell currents in Xenopus oocytes injected with cRNA encoding three cloned Kv1 channels (Kv1.1, Kv1.2, Kv1.3) and one Kv3 (Kv3.1) channel. The Shaker-related Kv1 channels are equally affected by BgK, while the Shaw-related channel Kv3.1 is insensitive up to 0.125 microM toxin. Indeed, half blockage of the current through the three Kv1 channels tested occurred in the same concentration range (Kd = 6 nM for Kv1.1, 15 nM for Kv1.2, 10 nM for Kv1.3). The specificity of BgK for the Shaker-related K+ channels indicates that BgK is able to discriminate a large group of neuronal Kv1 channels in situ. The sequence, the disulfide bridge pattern, the secondary structure and the biological activity of BgK demonstrated that the sea anemone toxins, i.e. BgK, ShK and Kaliseptine, constitute novel molecular probes useful for investigating K+ channel properties.

ATP-Sensitive Anion Channel from Rat Brain Synaptosomal Membranes Incorporated into Planar Lipid Bilayers

An anion channel was incorporated from rat brain synaptic plasma membrane fractions into planar lipid bilayers. The single-channel conductance was found to be 48.5 pS in choline-Cl solution (300 microM cis/100 microM trans). The anion selectivity of the channel was rather low (PCl/Pcholine = 1.7). The gating rate of the channel did not change with membrane potential over the range of -50 mV to 50 mV. Several drugs, which are known as inhibitors of anion channels, were found to be efficient inhibitors for the synaptosomal anion channel. 4-Acetoamino-4'-isothiocyanostilbene-2,2'-disulfonic acid, ethacrynic acid, indanyloxyacetic acid, and 5-nitro-2-(3-phenylpropylamino) benzoic acid inhibited the channel from the cis side of the membrane, corresponding to the cytoplasmic side of the plasma membrane. We found that the channel is regulated by intracellular ATP at millimolar concentrations. Other nucleotides, ADP and GTP, inhibited the channel as well. Glibenclamide, which is known as an inhibitor of an ATP-regulated potassium channel, inhibited the channel at micromolar concentrations from the trans side of the membrane. It is likely that the synaptosomal anion channel is a member of the ATP-binding cassette superfamily.

Synthesis and expression of the gene coding for noxiustoxin, a K + channel-blocking peptide from the venom of the scorpion Centruroides noxius

A set of six synthetic overlapping oligonucleotides coding for noxiustoxin were coupled into a continuous DNA fragment by means of recursive polymerase chain reaction. The polymerase chain reaction product was digested with SalI and HindIII, ligated into the E. coli vector pCSP105 and expressed as a fusion protein. The fusion protein was purified and digested with trypsin and the hydrolysis products were separated by high-performance liquid chromatography. Approximately 1.3 mg of recombinant noxiustoxin per liter of culture was obtained. Amino acid analysis and N-terminal amino acid sequence of the recombinant noxiustoxin confirmed the nucleotide sequence of the cloned DNA. Binding experiments using rat brain synaptosomal membranes revealed that recombinant noxiustoxin displaced bound radioactive native NTX with a similar efficiency to cold native noxiustoxin.

Okadaic acid as a probe for regulation In Vitro of Mg 2+,Ca 2+-ATPase activity in rat cortical and cerebellar synaptosomal membranes

The in vitro effect of okadaic acid on basal phorbol 12-myristate 13-acetate (PMA)-, and cyclic adenosine monophosphate (cAMP)-stimulated Mg 2+-dependent Ca 2+-adenosine triphosphatase (ATPase) activity in synaptosomal membranes isolated from rat brain cortex and cerebellum was investigated. The basal activity was enhanced by okadaic acid in both examined regions. This inhibitor differed in the regulation of Mg 2+,Ca 2+-ATPase activity in PMA- and cAMP-incubated membranes. Stimulation by calmodulin (CaM) of basal Mg 2+,Ca 2+-ATPase activity declined in cortex and cerebellum after treatment with okadaic acid. The presence of PMA or cAMP decreases the stimulatory effect of CaM. These results suggest that Mg 2+,Ca 2+-ATPase activity in the rat-brain synaptosomal membrane may be regulated in vitro by dephosphorylation processes.

A novel structural class of K+-channel blocking toxin from the scorpion Pandinus imperator.

A novel peptide was purified and characterized from the venom of the scorpion Pandinus imperator. Analysis of its primary structure reveals that it belongs to a new structural class of K+-channel blocking peptide, composed of only 35 amino acids, but cross-linked by four disulphide bridges. It is 40, 43 and 46% identical to noxiustoxin, margatoxin and toxin 1 of Centruroides limpidus respectively. However, it is less similar (26 to 37% identity) to toxins from scorpions of the geni Leiurus, Androctonus and Buthus. The disulphide pairing was determined by sequencing heterodimers produced by mild enzymic hydrolysis. They are formed between Cys-4-Cys-25, Cys-10-Cys-30, Cys-14-Cys-32 and Cys-20-Cys-35. Three-dimensional modelling, using the parameters determined for charybdotoxin, showed that is it possible to accommodate the four disulphide bridges in the same general structure of the other K+-channel blocking peptides. The new peptide (Pil) blocks Shaker B K+ channels reversibly. It also displaces the binding of a known K+-channel blocker, [125I]noxiustoxin, from rat brain synaptosomal membranes with an IC50 of about 10 nM.

Primary alcohols and phosphatidylcholine metabolism in rat brain synaptosomal membranes via phospholipase D.

Phospholipase D of rat brain synaptosomal membranes was tested with phosphatidylcholine as the substrate for its specificity in the use of primary alcohols as transphosphatidylation co-substrates. The efficiency of the reaction was related to the hydrophobicity and the membrane penetrating capacity of the alcohol molecule. Phosphatidylalcohol formation could be detected up to 1-octanol but not for alcohols with longer hydrocarbon chains (C(9), C(10)). With increasing alcohol concentration the transphosphatidylation activity of the phospholipase D reached an optimum and then declined abruptly. Alcohol concentrations required for maximal transphosphatidylation reaction generally decreased with increasing hydrophobicities of the alcohols. Nevertheless 1-butanol and 4-chloro-1-butanol were the most efficient cosubstrates, sharing identical optimal conditions. Transphosphatidylation works at the cost of phosphatidic acid formation. Phosphatidic acid itself was transformed to diacylglycerol, probably by a contaminating phosphatidic acid phosphohydrolase.

Comparative study of the effect of amiridine on biological membranes

The effects of amiridine and of the comparable drugs tacrine and piracetam on synaptosomes and membranes of sarcoplasmic reticulum were studied by electron paramagnetic resonance; in addition, the effects of these drugs on the activity of Ca2+, Mg2+-dependent ATPase regulating calcium transport in neurons were investigated. In concentrations of 10−7 to 10−5 M the drugs did not affect the structure of synaptosomal membranes of rat brain. Amiridine and tacrine in a concentration of 0.1 mM reduced the rate of calcium ion transport across the sarcoplasmic reticulum membrane by inhibiting the function of Ca2+, Mg2+-dependent ATPase and induced marked changes of the structural rigidity of the protein part of the membrane.

SNX-325, a novel calcium antagonist from the spider Segestria florentina

A novel selective calcium channel antagonist peptide, SNX-325, has been isolated from the venom of the spider Segestria florentina. The peptide was isolated using as bioassays the displacement of radioiodinated omega-conopeptide SNX-230 (MVIIC) from rat brain synaptosomal membranes, as well as the inhibition of the barium current through cloned expressed calcium channels in oocytes. The primary sequence of SNX-325 is GSCIESGKSCTHSRSMKNGLCCPKSRCNCRQIQHRHDYLGKRKYSCRCS, which is a novel amino acid sequence. Solid-phase synthesis resulted in a peptide that is chromatographically identical with the native peptide and which has the same configuration of cysteine residues as the spider venom peptide omega-Aga-IVa [Mintz, I. M., et al., (1992) Nature 355, 827-829]. At micromolar concentrations, SNX-325 is an inhibitor of most calcium, but not sodium or potassium, currents. At nanomolar concentrations, SNX-325 is a selective blocker of the cloned expressed class B (N-type), but not class C (cardiac L), A, or E, calcium channels. SNX-325 is approximately equipotent with the N-channel selective omega-conopeptides (GVIA and MVIIA as well as closely related synthetic derivatives) in blocking the potassium induced release of tritiated norepinephrine from hippocampal slices (IC50s, 0.1-0.5 nM) and in blocking the barium current through cloned expressed N-channels in oocytes (IC50s 3-30 nM). By contrast, SNX-325 is 4-5 orders of magnitude less potent than is SNX-111 (synthetic MVIIA) at displacing radioiodinated SNX-111 from rat brain synaptosomal membranes. SNX-325 will be a useful comparative tool in further defining the function and pharmacology of the N- and possibly other types of high-voltage activated calcium channels.

Monoclonal antibodies against noxiustoxin.

Noxiustoxin, a 39-amino acid residue peptide isolated from the venom of the Mexican scorpion Centruroides noxius, has previously been shown to affect voltage-dependent K+ channels. Here we describe the isolation and characterization of monoclonal antibodies (MAbs) against this toxin and their use in structure-function relationship studies. Six hybridoma clones (BNTX4, -12, -14, -16, -18, and -21) producing MAbs against noxiustoxin were isolated. The epitopes defined by the MAbs are overlapping or in close proximity because no MAb pair could bind simultaneously to the toxin. All the MAbs inhibited to various degrees the binding of the toxin to its receptor sites on rat brain synaptosomal membranes. The venom from other Centruroides species was shown to contain components cross-reacting with the MAbs, suggesting the existence of other NTX-like toxins.

Characterization of a potassium channel toxin from the Caribbean sea anemone Stichodactyla helianthus

A peptide toxin, ShK, that blocks voltage-dependent potassium channels was isolated from the whole body extract of the Caribbean sea anemone Stichodactyla helianthus. It competes with dendrotoxin I and α-dendrotoxin for binding to synaptosomal membranes of rat brain, facilitates acetylcholine release at an avian neuromuscular junction and suppresses K + currents in rat dorsal root ganglion neurones in culture. Its amino acid sequence is R 1SCIDTIPKS 10RCTAFQCKHS 20MKYRLSFCRK 30-TCGTC 35. There is no homology with other K + channel-blocking peptides, except for BgK from the sea anemone Bunodosoma granulifera. ShK and BgK appear to be in a different structural class from other toxins affecting K + channels.

Nitrous oxide and xenon enhance phospholipid-N-methylation in rat brain synaptic plasma membranes

Halothane and isoflurane increase the rate of phospholipid methylation (PLM) in rat brain synaptosomal membranes, a process linked to the coupling of neuronal excitation to neurotransmitter release. In contrast, synaptic plasma membrane (SPM) Ca 2+ ATPase (PMCA) pumping is reduced by exposure to halothane, isoflurane, xenon and nitrous oxide (N 2O). To examine further the relationship between PLM, PMCA and anesthetic action, we investigated the effect of clinically relevant concentrations of two less potent anesthetic gases, N 2O and xenon, on PLM in SPM. Biochemical assays were performed on SPM exposed to 1.3 MAC of N 2O (2 atm), 1.3 MAC of xenon (1.23 atm) or an equivalent pressure of helium for control. N 2O or xenon exposure increased PLM to 115% or 113%, respectively, of helium control (p < 0.02). Similar exposures to N 2O or xenon depressed PMCA activity to 78% and 85% of control (p < 0.05). Observations that PLM and PMCA are both altered by a wide variety of inhalation anesthetic agents at clinically relevant partial pressures lend support to a possible involvement and interaction of these processes in anesthetic action.