Block of high-threshold calcium channels by the synthetic polyamines sFTX-3.3 and FTX-3.3.

A polyamine component of Agelenopsis aperta spider venom designated FTX is reported to be a selective antagonist of P-type calcium channels in the mammalian brain. Consequently, this component has frequently been used as a pharmacological tool to determine the presence, distribution, and function of P-type channels in physiological systems. We describe antagonism of calcium channels by the synthesized polyamine FTX-3.3, which has the proposed structure of natural FTX. We also examined a corresponding polyamine amide, sFTX-3.3. These polyamines are critically evaluated for antagonism of three high-threshold calcium channel subtypes in rat neurons through the use of the whole-cell patch-clamp technique. FTX-3.3 (IC50 = approximately 0.13 mM) is approximately twice as potent as sFTX-3.3 (IC50 = approximately 0.24 mM) against P-type channels and approximately 3-fold more potent against N-type channels (FTX-3.3, IC50 = approximately 0.24 mM; sFTX-3.3, IC50 = approximately 0.70 mM). Both polyamines also block L-type calcium channels with similar potencies. sFTX-3.3 (1 mM) and FTX-3.3 (0.5 mM) typically block 50% and 65% of Bay K8644-enhanced L-type current, respectively. Antagonism of each calcium channel subtype is voltage dependent, with less inhibition of Ba2+ currents at more-positive potentials. These data show that both sFTX-3.3 and FTX-3.3 antagonize P-, N-, and L-type calcium channels in mammalian Purkinje and superior cervical ganglia neurons with similar IC50 values.

Functional expression and genetic alteration of an alpha scorpion neurotoxin.

The alpha neurotoxin Lqh alpha IT is toxic to both insects and mammals but exhibits a bioactivity ratio favoring insects (insect/mammal approximately 2). With the objective of increasing this ratio by genetic manipulation of the amino acid sequence, a cDNA clone encoding Lqh alpha IT was used to produce recombinant variants of the toxin in a high efficiency bacterial expression system. The unmodified recombinant toxin, isolated from inclusion bodies and renatured in vitro, exhibited chemical and biological properties indistinguishable from those of the authentic native toxin. Alteration of the toxin by site-directed mutagenesis led to a substantial reduction in anti-mammalian toxicity (mouse LD50 reduced 6.4-fold) but only a slight reduction (x 1.5) in the insect ED50 value for paralysis. The reduction in anti-mammalian toxicity was correlated with a approximately 2-fold reduction of its potency for slowing of sodium channel inactivation in mammalian neurons, while no change in mutant toxin binding affinity to insect neuronal receptors was registered. These results demonstrate for the first time expression of a recombinant sodium channel neurotoxin in Escherichia coli and the use of site-directed mutagenesis to improve phylogenetic selectivity. This recombinant approach provides a promising strategy for optimizing the selective toxicity of peptide neurotoxins.

Gating kinetics of the quisqualate-sensitive glutamate receptor of locust muscle studied using agonist concentration jumps and computer simulations.

Outside-out patches excised from extrajunctional membrane of locust muscle were subjected to "concentration jumps" of L-glutamate, using the liquid filament switch technique, to study channel opening and closing rates, desensitization onset, and recovery from desensitization of a quisqualate-sensitive glutamate receptor (qGluR). Based on data obtained from these experimental studies, computer modeling techniques have been used in an attempt to simulate the behavior of qGluR during a concentration jump of L-glutamate. A linear model with three closed states (one unliganded, one monoliganded, and one biliganded), one open state (binding two molecules of L-glutamate), and two desensitization states (the one monoliganded, the other biliganded) leading from the unliganded closed state simulated all of the experimentally observed behavior. The results are discussed in the context of previous equilibrium studies in which desensitization was inhibited with concanavalin A and for which a ten-state model was required to simulate the behavior of qGluR.