Vincent L. Salgado

Thiamethoxam is a neonicotinoid precursor converted to clothianidin in insects and plants

Neonicotinoid insecticides act agonistically on insect nicotinic acetylcholine receptors (nAChRs). Like imidacloprid (IMI), all neonicotinoids bind with high affinity (I 50 -values ∼ 1 nM) to [ 3 H]IMI binding sites on insect nAChRs. One notable omission is thiamethoxam (THIAM), showing binding affinities up to 10,000-fold less potent than other neonicotinoids, using housefly head membrane preparations. Clothianidin (CLOTHI) exhibits high activity as an agonist on isolated neurons at concentrations as low as 30 nM. Pharmacokinetic studies in different insect species revealed that THIAM was rapidly metabolized to CLOTHI, which shows high affinity to nAChRs in both binding assays and whole cell voltage clamp studies. When applied to cotton plants, THIAM was also quickly metabolized, with CLOTHI being the predominant neonicotinoid in planta briefly after application, as indicated by LC-MS/MS analyses. Our studies show that THIAM is likely to be a neonicotinoid precursor for CLOTHI and not active by itself.

Use‐ and Voltage‐Dependent Block of the Sodium Channel by Saxitoxin

Saxitoxin and tetrodotoxin have long fascinated electrophysiologists because of their remarkable specificity and potency of action on sodium channels in various excitable membranes.’.’ Much effort has been made to elucidate the mechanism of sodium channel blocking action by these toxins. Kao and Nishiyama’ first proposed that the tetrodotoxin and saxitoxin molecules, which are too large to pass through the sodium channel, physically blocked the passage of other ions by binding in the channel, acting on the channel like a cork in a bottle, with the guanidinium group of the toxin being the cork. This seemed to be a reasonable hypothesis then since free guanidinium ions can pass through the channel, and since the TTX and STX molecules are thought to be too bulky to pass through the Na channel. This notion was further elaborated by Hille! By comparing the structure of TTX with STX, and by incorporating the theory of ion permeation through the channel, Hille proposed a hypothetical structural basis to account for the Na channel blocking action of these two toxins. Hille proposed that the progress of an ion through the open sodium channel can be simulated by the passage of the ion over a series of energy barriers according to Eyring rate theory.’ The highest energy barrier in the channel is viewed as being the basis for the channel’s selectivity among different permeating ions. This barrier is viewed mechanistically as being the narrowest part of the channel, and would require ions to shed at least some of their water of hydration. Hille termed this constriction the “selectivity filter,” proposing that it consists of a 3 x 5A oxygen-lined constriction that allows close interaction of the partially dehydrated ion with a highly negative field strength site, presumably an ionized carboxylic acid. This selectivity filter is also the binding site for both H+ and CaZ+ ions6. Since the site is located within the membrane field, the blocking action is expected to vary with voltage according to a Boltzmann distribution. This is indeed the case for the block in the presence of H+ and Ca2+ ions.6.’ In Hille’s model for toxin block, the guanidinium group was pictured as being bound to a carboxylic acid group in the selectivity filter, with five hydrogen bonds forming between the toxin molecule and the lining of the sodium channel. This TTX-carboxylic acid binding hypothesis was supported by experiments with trimethyloxonium tetrafluoroborate (TMO), a carboxyl group alkylating reagent. After the treatment of nerve or muscle membranes with TMO, TTX could no longer bind to the sodium channel’ nor block the sodium currents? However, the TTX-resistant channels had normal selectivity and were still sensitive to hydrogen ion block, suggesting that the carboxyl group necessary for TTX binding is distinct from that in the selectivity filter. Two other lines of evidence further argue against STX and TTX binding to the

Kinetic properties of single sodium channels modified by fenvalerate in mouse neuroblastoma cells

Abstract(1) The kinetic properties of single sodium channels modified by the pyrethroid fenvalerate have been analyzed by patch clamp techniques using the cultured mouse neuroblastoma cells. (2) Fenvalerate drastically prolonged the open time of single sodium channels from the normal value of 5 ms to several hundred milliseconds during a depolarizing pulse. The channels remained open after termination of a depolarizing pulse for as long as several seconds. (3) The channel lifetime varied with the membrane potential, attained a maximum at −70 mV, and decreased with hyperpolarization and depolarization from −70 mV. (4) Prolonged openings of the modified channels allowed a current-voltage curve for a single channel to be plotted by sweeping a ramp pulse. The single channel conductance had a value of 11 pS and was linear over potentials ranging from 0 to −100 mV. (5) Power density spectral analysis of the open channel current noise indicated a single Lorentzian curve with a cut-off frequency at 90 Hz, indicating that the increase in noise during channel opening resulted from a relatively slow kinetic process. (6) The probability of the channel being modified by fenvalerate was independent of the length of time during which the channel was opened. This observation suggests that channel modification had taken place before the channel opened. This study of the prolonged opening at the single channel level provides a new insight into open channel properties and the kinetics of channel modification.