A new class of scorpion toxin binding sites related to an A-type K+ channel: pharmacological characterization and localization in rat brain.

A new scorpion toxin (3751.8 Da) was isolated from the Buthus martensi venom, sequenced and chemically synthesized (sBmTX3). The A-type current of striatum neurons in culture completely disappeared when 1 microM sBmTX3 was applied (Kd=54 nM), whereas the sustained K+ current was unaffected. 125I-sBmTX3 specifically bound to rat brain synaptosomes (maximum binding=14 fmol x mg(-1) of protein, Kd=0.21 nM). A panel of toxins yet described as specific ligands for K+ channels were unable to compete with 125I-sBmTX3. A high density of 125I-sBmTX3 binding sites was found in the striatum, hippocampus, superior colliculus, and cerebellum in the adult rat brain.

Improved cholesterol management in coronary heart disease patients enrolled in an HMO.

The purpose of the study was to describe the effect of physician reminders on the measurement of low-density lipoprotein cholesterol (LDL-C) levels and treatment to achieve an LDL-C goal of < or = 100 mg/dL in coronary heart disease (CHD) patients. After reminders were initiated, the number of CHD patients without a documented LDL-C was reduced from 30% to 18%, between January 1997 and July 1998, and the percentage of CHD patients achieving the LDL-C goal improved from 10% to 27%. Thus, reminders can be an effective tool in improving cholesterol management of CHD patients. In contrast, a cholesterol-lowering clinic made available to some physicians, in addition to the reminders, was rarely used.

Syntheses of optically pure beta-hydroxyaspartate derivatives as glutamate transporter blockers.

DL-threo-beta-benzyloxyaspartate (DL-TBOA) is a non-transportable blocker of the glutamate transporters that serves as an indispensable tool for the investigation of the physiological roles of the transporters. To examine the precise interaction between a blocker and the transporters, we synthesized the optically pure isomers (L- and D-TBOA) and its erythro-isomers. L-TBOA is the most potent blocker for the human excitatory amino acid transporters (EAAT1-3), while D-TBOA revealed a difference in the pharmacophores between EAAT1 and EAAT3. We also synthesized the substituent variants (methyl or naphthylmethyl derivatives) of L-TBOA. The results obtained here suggest that bulky substituents are crucial for non-transportable blockers.

Structural and functional consequences of the presence of a fourth disulfide bridge in the scorpion short toxins: solution structure of the potassium channel inhibitor HsTX1.

We have determined the three-dimensional structure of the potassium channel inhibitor HsTX1, using nuclear magnetic resonance and molecular modeling. This protein belongs to the scorpion short toxin family, which essentially contains potassium channel blockers of 29 to 39 amino acids and three disulfide bridges. It is highly active on voltage-gated Kv1.3 potassium channels. Furthermore, it has the particularity to possess a fourth disulfide bridge. We show that HsTX1 has a fold similar to that of the three-disulfide-bridged toxins and conserves the hydrophobic core found in the scorpion short toxins. Thus, the fourth bridge has no influence on the global conformation of HsTX1. Most residues spatially analogous to those interacting with voltage-gated potassium channels in the three-disulfide-bridged toxins are conserved in HsTX1. Thus, we propose that Tyr21, Lys23, Met25, and Asn26 are involved in the biological activity of HsTX1. As an additional positively charged residue is always spatially close to the aromatic residue in toxins blocking the voltage-gated potassium channels, and as previous mutagenesis experiments have shown the critical role played by the C-terminus in HsTX1, we suggest that Arg33 is also important for the activity of the four disulfide-bridged toxin. Docking calculations confirm that, if Lys23 and Met25 interact with the GYGDMH motif of Kv1.3, Arg33 can contact Asp386 and, thus, play the role of the additional positively charged residue of the toxin functional site. This original configuration of the binding site of HsTX1 for Kv1.3, if confirmed experimentally, offers new structural possibilities for the construction of a molecule blocking the voltage-gated potassium channels.

DL-threo-beta-benzyloxyaspartate, a potent blocker of excitatory amino acid transporters.

DL-threo-beta-Benzyloxyaspartate (DL-TBOA), a novel derivative of DL-threo-beta-hydroxyaspartate, was synthesized and examined as an inhibitor of sodium-dependent glutamate/aspartate (excitatory amino acid) transporters. DL-TBOA inhibited the uptake of [14C]glutamate in COS-1 cells expressing the human excitatory amino acid transporter-1 (EAAT1) (Ki = 42 microM) with almost the same potency as DL-threo-beta-hydroxyaspartate (Ki = 58 microM). With regard to the human excitatory amino acid transporter-2 (EAAT2), the inhibitory effect of DL-TBOA (Ki = 5.7 microM) was much more potent than that of dihydrokainate (Ki = 79 microM), which is well known as a selective blocker of this subtype. Electrophysiologically, DL-TBOA induced no detectable inward currents in Xenopus laevis oocytes expressing human EAAT1 or EAAT2. However, it significantly reduced the glutamate-induced currents, indicating the prevention of transport. The dose-response curve of glutamate was shifted by adding DL-TBOA without a significant change in the maximum current. The Kb values for human EAAT1 and EAAT2 expressed in X. laevis oocytes were 9.0 microM and 116 nM, respectively. These results demonstrated that DL-TBOA is, so far, the most potent competitive blocker of glutamate transporters. DL-TBOA did not show any significant effects on either the ionotropic or metabotropic glutamate receptors. Moreover, DL-TBOA is chemically much more stable than its benzoyl analog, a previously reported blocker of excitatory amino acid transporters; therefore, DL-TBOA should be a useful tool for investigating the physiological roles of transporters.

Purification, characterization, and synthesis of three novel toxins from the Chinese scorpion Buthus martensi, which act on K+ channels.

Three novel toxins belonging to the scorpion K+ channel-inhibitor family were purified to homogeneity from the venom of the Chinese scorpion Buthus martensi. They have been identified according to their molecular mass (3800-4300 Da) and their neurotoxicity in mice and characterized as 37-amino acid peptides. One of them shows 81-87% sequence identity with members of the kaliotoxin group (named BmKTX), whereas the other two, named BmTX1 and BmTX2, show 65-70% identity with toxins of the charybdotoxin group. Their chemical synthesis by the Fmoc methodology allowed us to show that BmKTX, unlike BmTX1 and BmTX2, possesses an amidated C-terminal extremity. Toxicity assays in vivo established that they are lethal neurotoxic agents in mice (LD50s of 40-95 ng per mouse). Those toxins proved to be potent inhibitors of the voltage-gated K+ channels, as they were able to compete with [125I]kaliotoxin for its binding to rat brain synaptosomes (IC50s of 0.05-1 nM) and to block the cloned voltage-gated K+ channel Kv1.3 from rat brain, expressed in Xenopus oocytes (IC50s of 0.6-1.6 nM). BmTX1 and BmTX2 were also shown to compete with [125I]charybdotoxin for its binding to the high-conductance Ca2+-activated K+ channels present on bovine aorta sarcolemmal membranes (IC50s of 0.3-0.6 nM). These new sequences show multipoint mutations when compared to the other related scorpion K+ channel toxins and should prove to be useful probes for studying the diverse family of K+ channels.

New beta-hydroxyaspartate derivatives are competitive blockers for the bovine glutamate/aspartate transporter.

Four subtypes of excitatory amino acid transporters (EAAT1-4) have been identified in the mammalian brain. A number of pharmacological agents have been developed to study their intrinsic properties and function. Up to now, blockers were available only for EAAT2, whereas all the inhibitors of glutamate uptake active on the other subtypes were proved to be substrates of the transporters. We synthesized five new derivatives of DL-threo-beta-hydroxyaspartic acid, a well known general substrate of EAATs, and investigated their potential blocking activity on the cloned bovine EAAT1 expressed in the Xenopus oocyte system, by using radiotracer and voltage-clamp techniques. Two of our derivatives proved to be substrates for bovine EAAT1, with reduced electrogenicity compared with their parent compound, and an affinity of 40 and 64 microM. The last three derivatives displayed a blocking activity on bovine EAAT1. The affinity of DL-threo-beta-benzoyloxyaspartate and DL-threo-beta-(1-naphthoyl)oxyaspartate was determined by Schild analysis as 17.2 and 52.1 microM, respectively. These blockers should help in the better understanding of the key intrinsic properties of EAAT1. Moreover, they appear as good candidates for a general blocking activity on EAATs.

Characterization of four toxins from Buthus martensi scorpion venom, which act on apamin-sensitive Ca2+-activated K+ channels.

Four peptidyl inhibitors of the small-conductance Ca2+-activated K+ channels (SK(Ca)) have been isolated from the venom of the Chinese scorpion Buthus martensi. These peptides were identified by screening C18 HPLC fractions of the crude venom by means of mass analysis by matrix-assisted-laser-desorption/ionization time-of-flight mass spectrometry, and toxicological tests in mice. Edman degradation analysis of the purified peptides showed sequences of 28-31 amino acids including 6 cysteine residues. Three of the sequences were similar to the P01 peptides from Androctonus scorpions, showing 76% sequence similarity for the most closely related, named BmP01, and 46% for the other two, named BmP02 and BmP03. Like the P01 peptides, these molecules showed a low toxic activity in mice after intracerebroventricular injection, and competed (K0.5 > 1 microM) with iodinated apamin for binding to its receptor site from rat brain, which has been proved to be the SK(Ca) channels. The fourth toxin was structurally related to the P05/leiurotoxin I toxin family, with 90% similarity, and was named BmP05. This toxin exhibited a high toxic activity with lethal effects in mice. Due to its small representation in the venom [less than 0.01% (by mass)], its biological properties have been assessed on the synthetic analogue of BmP05, which was assembled on a solid phase by means of Fmoc methodology. The synthetic peptide was physicochemically identical to the natural peptide, as shown by comparison of their molecular masses and amino acid compositions, and by their coelution after coinjection on capillary electrophoresis. These results confirmed the primary structure of BmP05 including an amidated C-terminus. Similarly to natural BmP05, synthetic BmP05 produced toxic and lethal effects after intracerebroventricular injection in mice (LD50 = 37 ng), and was able to compete with iodinated apamin for binding to its receptor in rat brain (K0.5 = 20 pM).

Slow inhibition of Na+ current in crayfish axons by 2-(1non-8enyl)-5-(1non-8enyl)pyrrolidine (Pyr9), a synthetic derivative of an ant venom alkaloid

2,5-Dialkylpyrrolidines present in the venom of ants from the genus Monomorium are natural insecticides causing a flaccid paralysis. The mechanism of action of 2-(1non-8enyl)-5-(1non-8enyl)pyrrolidine (Pyr9), a synthetic derivative of 2,5-dialkylpyrrolidines, has been investigated in vitro on preparations of the ventral nerve cord of the crayfish Procambarus clarkii. Our results clearly indicate that Pyr9 blocks spike conduction without affecting the resting potential. Voltage-clamp experiments carried out on axons demonstrate that this blockade is due to a dual expression of Na+ current inhibition: a tonic inhibition developing slowly (90 % of inhibition within 20 min for a Pyr9 concentration of 50 µmol l-1) and independently of stimulation, and a phasic inhibition developing during repetitive stimulation (5 Hz), the accumulation kinetics of which is 0.072 pulse-1 at 5 Hz, according to the Courtney model. These findings suggest that tonic and phasic inhibition are due to different mechanisms. In addition, Pyr9 induces a shift of the Na+ inactivation curve towards more hyperpolarized potentials, which is in agreement with a higher affinity of Pyr9 for inactivated than for resting Na+ channels.

A four-disulphide-bridged toxin, with high affinity towards voltage-gated K+ channels, isolated from Heterometrus spinnifer (Scorpionidae) venom.

A new toxin, named HsTX1, has been identified in the venom of Heterometrus spinnifer (Scorpionidae), on the basis of its ability to block the rat Kv1.3 channels expressed in Xenopus oocytes. HsTX1 has been purified and characterized as a 34-residue peptide reticulated by four disulphide bridges. HsTX1 shares 53% and 59% sequence identity with Pandinus imperator toxin1 (Pi1) and maurotoxin, two recently isolated four-disulphide-bridged toxins, whereas it is only 32-47% identical with the other scorpion K+ channel toxins, reticulated by three disulphide bridges. The amidated and carboxylated forms of HsTX1 were synthesized chemically, and identity between the natural and the synthetic amidated peptides was proved by mass spectrometry, co-elution on C18 HPLC and blocking activity on the rat Kv1.3 channels. The disulphide bridge pattern was studied by (1) limited reduction-alkylation at acidic pH and (2) enzymic cleavage on an immobilized trypsin cartridge, both followed by mass and sequence analyses. Three of the disulphide bonds are connected as in the three-disulphide-bridged scorpion toxins, and the two extra half-cystine residues of HsTX1 are cross-linked, as in Pi1. These results, together with those of CD analysis, suggest that HsTX1 probably adopts the same general folding as all scorpion K+ channel toxins. HsTX1 is a potent inhibitor of the rat Kv1.3 channels (IC50 approx. 12 pM). HsTX1 does not compete with 125I-apamin for binding to its receptor site on rat brain synaptosomal membranes, but competes efficiently with 125I-kaliotoxin for binding to the voltage-gated K+ channels on the same preparation (IC50 approx. 1 pM).

A new configuration for voltage clamp of axons used to demonstrate nerve conduction blockade by a 2,5-disubstituted pyrrolidine.

An original voltage-clamp technique on axons from crayfish Procambarus clarkii is described in this paper. Its advantages are: a fast dissection leading to the availability of several fibers of different diameters (10-500 microns) that may contain different ion channels; and use of a double-electrode voltage clamp on a chosen fiber with good clamping characteristics (short time clamp and good space clamp, small leak conductance). Because of the absence of exogenous lipidic phase in the superfusion chamber, this technique appears particularly suited to studying how liposoluble neurotoxins affect nerve conduction. This method has been successfully applied to test the effect of a synthetic derivative (2-(1non-8enyl)-5(1non-8enyl)pyrrolidine (Pyr 9)) of ant venom alkaloids from Monomorium species on nerve conduction. We present here evidence of a strong blocking effect on inward current involved in spike conduction. The resting potential of the treated axons did not change and it appears that only the inward current was affected.