Scanning mutagenesis of the I-II loop of the Cav2.2 calcium channel identifies residues Arginine 376 and Valine 416 as molecular determinants of voltage dependent G protein inhibition.

Direct interaction with the beta subunit of the heterotrimeric G protein complex causes voltage-dependent inhibition of N-type calcium channels. To further characterize the molecular determinants of this interaction, we performed scanning mutagenesis of residues 372-387 and 410-428 of the N-type channel alpha1 subunit, in which individual residues were replaced by either alanine or cysteine. We coexpressed wild type Gbeta1gamma2 subunits with either wild type or point mutant N-type calcium channels, and voltage-dependent, G protein-mediated inhibition of the channels (VDI) was assessed using patch clamp recordings. The resulting data indicate that Arg376 and Val416 of the alpha1 subunit, residues which are surface-exposed in the presence of the calcium channel beta subunit, contribute significantly to the functional inhibition by Gbeta1. To further characterize the roles of Arg376 and Val416 in this interaction, we performed secondary mutagenesis of these residues, coexpressing the resulting mutants with wild type Gbeta1gamma2 subunits and with several isoforms of the auxiliary beta subunit of the N-type channel, again assessing VDI using patch clamp recordings. The results confirm the importance of Arg376 for G protein-mediated inhibition and show that a single amino acid substitution to phenylalanine drastically alters the abilities of auxiliary calcium channel subunits to regulate G protein inhibition of the channel.

A model genetic system for testing the in vivo function of peptide toxins.

We have developed a model genetic system for analyzing the function of peptide toxins from animal venoms. We engineered and propagated strains of Drosophila melanogaster expressing heat-inducible transgenes encoding either kappa-ACTX-Hv1c or omega-ACTX-Hv1a, two insect-specific neurotoxic peptides found in the venom of the Australian funnel-web spider Hadronyche versuta. Heat induction of transgene expression for 20 min was sufficient to kill all transgenic flies, indicating that the ion channels targeted by these toxins are viable insecticide targets. The unusual phenotype of flies induced to express omega-ACTX-Hv1a recapitulates that of a hypomorphic allele of the high-voltage-activated calcium channel Dmca1D, suggesting that this is likely to be the target of omega-ACTX-Hv1a.

Scanning mutagenesis of omega-atracotoxin-Hv1a reveals a spatially restricted epitope that confers selective activity against insect calcium channels.

We constructed a complete panel of alanine mutants of the insect-specific calcium channel blocker omega-atracotoxin-Hv1a. Lethality assays using these mutant toxins identified three spatially contiguous residues, Pro10, Asn27, and Arg35, that are critical for insecticidal activity against flies (Musca domestica) and crickets (Acheta domestica). Competitive binding assays using radiolabeled omega-atracotoxin-Hv1a and neuronal membranes prepared from the heads of American cockroaches (Periplaneta americana) confirmed the importance of these three residues for binding of the toxin to target calcium channels presumably expressed in the insect membranes. At concentrations up to 10 microm, omega-atracotoxin-Hv1a had no effect on heterologously expressed rat Cav2.1, Cav2.2, and Cav1.2 calcium channels, consistent with the previously reported insect selectivity of the toxin. 30 microm omega-atracotoxin-Hv1a inhibited rat Cav currents by 10-34%, depending on the channel subtype, and this low level of inhibition was essentially unchanged when Asn27 and Arg35, which appears to be critical for interaction of the toxin with insect Cav channels, were both mutated to alanine. We propose that the spatially contiguous epitope formed by Pro10, Asn27, and Arg35 confers specific binding to insect Cav channels and is largely responsible for the remarkable phyletic selectivity of omega-atracotoxin-Hv1a. This epitope provides a structural template for rational design of chemical insecticides that selectively target insect Cav channels.

Australian funnel-web spiders: master insecticide chemists.

Arthropods are the most diverse animal group on the planet. Their ability to inhabit a vast array of ecological niches has inevitably brought them into conflict with humans. Although only a small minority are classified as pest species, they nevertheless destroy about a quarter of the world's annual crop production and transmit an impressive array of pathogens of human and veterinary public health importance. Arthropod pests have been controlled almost exclusively with chemical insecticides since the introduction of DDT in the 1940s. However, the evolution of resistance to many insecticides, coupled with increased awareness of the potential environmental and human and animal health impacts of these chemicals, has stimulated the search for new insecticidal compounds, novel molecular targets, and alternative control methods. Spider venoms are complex chemical cocktails that have evolved to kill or paralyze arthropod prey, and they represent a largely untapped reservoir of insecticidal compounds. This review focuses on several families of invertebrate-specific peptide neurotoxins that were isolated from the venom of Australian funnel-web spiders. These peptides are promising insecticide leads because of their selectivity for invertebrates and activity on previously unvalidated targets. These toxins should facilitate the development of novel target-based screens for new insecticide leads, while their mapped pharmacophores will provide templates for rational design of mimetics that act at these target sites. Furthermore, genes encoding these toxins can be used to improve the efficacy of insect-specific viruses.

Redox-based control of the gamma heavy chain ATPase from Chlamydomonas outer arm dynein.

The outer dynein arm from Chlamydomonas flagella contains two redox-active thioredoxin-related light chains associated with the alpha and beta heavy chains; these proteins belong to a distinct subgroup within the thioredoxin family. This observation suggested that some aspect of dynein activity might be modulated through redox poise. To test this, we have examined the effect of sulfhydryl oxidation on the ATPase activity of isolated dynein and axonemes from wildtype and mutant strains lacking various heavy chain combinations. The outer, but not inner, dynein arm ATPase was stimulated significantly following treatment with low concentrations of dithionitrobenzoic acid; this effect was readily reversible by dithiol, and to a lesser extent, monothiol reductants. Mutational and biochemical dissection of the outer arm revealed that ATPase activation in response to DTNB was an exclusive property of the gamma heavy chain, and that enzymatic enhancement was modulated by the presence of other dynein components. Furthermore, we demonstrate that the LC5 thioredoxin-like light chain binds to the N-terminal stem domain of the alpha heavy chain and that the beta heavy chain-associated LC3 protein also interacts with the gamma heavy chain. These data suggest the possibility of a dynein-associated redox cascade and further support the idea that the gamma heavy chain plays a key regulatory role within the outer arm.