Modification of the philanthotoxin-343 polyamine moiety results in different structure-activity profiles at muscle nicotinic ACh, NMDA and AMPA receptors.

Voltage-dependent, non-competitive inhibition by philanthotoxin-343 (PhTX-343) analogues, with reduced charge or length, of nicotinic acetylcholine receptors (nAChR) of TE671 cells and ionotropic glutamate receptors (N-methyl-D-aspartate receptors (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR)) expressed in Xenopus oocytes from rat brain RNA was investigated. At nAChR, analogues with single amine-to-methylene or amine-to-ether substitutions had similar potencies to PhTX-343 (IC(50)=16.6 microM at -100 mV) whereas PhTX-(12), in which both secondary amino groups of PhTX-343 were replaced by methylenes, was more potent than PhTX-343 (IC(50)=0.93 microM at -100 mV). Truncated analogues of PhTX-343 were less potent. Inhibition by all analogues was voltage-dependent. PhTX-343 (IC(50)=2.01 microM at -80 mV) was the most potent inhibitor of NMDAR. At AMPAR, most analogues were equipotent with PhTX-343 (IC(50)=0.46 microM at -80 mV), apart from PhTX-83, which was more potent (IC(50)=0.032 microM at -80 mV), and PhTX-(12) and 4,9-dioxa-PhTX-(12), which were less potent (IC(50)s>300 microM at -80 mV). These studies show that PhTX-(12) is a selective nAChR inhibitor and PhTX-83 is a selective AMPAR antagonist.

A single amino acid change makes a rat neuronal sodium channel highly sensitive to pyrethroid insecticides.

Two amino acid substitutions in a housefly sodium channel, L1014F in domain IIS6 and M918T in the IIS4-S5 linker, have been identified in kdr and super-kdr pyrethroid-resistant phenotypes, respectively. Unlike their native insect counterparts, mammalian sodium channels are only weakly sensitive to pyrethroids. Do the sodium channels of mammal and pyrethroid-resistant housefly share similar structural characteristics that account for their low pyrethroid sensitivities? We report here that substitution of isoleucine for methionine at position 874 (equivalent to the super-kdr site 918 in the housefly) in the rat IIA alpha-subunit causes a 100-fold increase in sensitivity.

Solid phase synthesis and biological evaluation of enantiomerically pure wasp toxin analogues PhTX-343 and PhTX-12.

PhTX-343 and PhTX-12, analogues of the natural polyamine wasp toxin PhTX-433, were synthesised in 40-60% yields as pure enantiomers using solid phase synthesis techniques. Capillary electrophoresis procedures were developed for chiral separation and determination of enantiomeric purity (ee) of the enantiomers of PhTX-343 and PhTX-12. The methods were optimised with respect to chiral selector, buffer pH, and temperature around the capillary. Thus, rac-PhTX-343 was resolved using a separation buffer containing 30 mM heptakis-(2, 6-di-O-methyl)-beta-cyclodextrin in 50 mM 6-aminocarproic acid (pH 4. 0) at 15 degrees C. rac-PhTX-12 was not resolvable in this system, but could be resolved using a separation buffer containing 10% w/v of dextrin 10, a linear maltodextrin, in 50 mM 6-aminocaproic acid (pH 4.0) at 15 degrees C. Using these methods, the optical purity of the synthetic enantiomers was determined to be ee > 99%. The enantiomers were also characterised by chiroptical methods. The antagonist potency of the enantiomers was tested on nicotinic acetylcholine receptors (human muscle-type nAChR) expressed in TE671 cells, ionotropic glutamate receptors in Xenopus laevis oocytes (expressing recombinant GluR1flop receptors), and locust muscle ionotropic glutamate receptors sensitive to quisqualate (qGluR). The potencies of each pair of enantiomers were similar (eudismic ratio close to 1).

Functional analysis of a rat sodium channel carrying a mutation for insect knock-down resistance (kdr) to pyrethroids.

Pyrethroid insensitivity in resistant (kdr) insects has been correlated with a leucine to phenylalanine replacement in the S6 transmembrane segment of domain II of the axonal sodium channel alpha(para)-subunit. An alpha-subunit of rat brain type II sodium channel containing this mutation has been expressed and its sensitivity to permethrin compared with that of the wild-type channel. The steady-state activation curve of the mutant was shifted 14 mV in the depolarizing direction. We propose that an equivalent shift of the sodium current activation curve in kdr insects could account for their low sensitivity to permethrin toxicity.