Linda M. Hall

Courtship-stimulating volatile compounds from normal and mutant Drosophila

Abstract Volatile compounds from Drosophila melanogaster males and females dramatically affect male courtship behaviour. These substances, which have been extracted from flies of different ages and genotypes, have been analysed by gas chromatography (GC) and in behavioural assays. Extracts from virgin females and males have different gas chromatographic profiles, which may reflect the fact that extract from virgin females stimulates high levels of courtship between males over short distances, while extract from mature wild-type males does not affect sexual behaviour. However, volatile compounds from very young males or males expressing the fruitless ( fru ) mutation do stimulate courtship between males, and chromatographic profiles of young male and fru male extracts differ from the GC profile of extracts from mature wild-type males.

Cloning, sequence analysis and chromosome localization of a Drosophila muscarinic acetylcholine receptor

Two cDNA clones (3.7 kb and 4.8 kb) encoding a Drosophila muscarinic acetylcholine receptor were isolated from a Drosophila head cDNA library and characterized by automated DNA sequence analysis. The Drosophila muscarinic receptor contains 788 amino acids with a calculated M r of 84 807 and displays greater than 60% homology with mammalian muscarinic receptors. The muscarinic receptor maps to the tip of the right arm of the second chromosome of the Drosophila genome.

On the mechanism by which saxitoxin binds to and blocks sodium channels.

Saxitoxin is a highly specific inhibitor of sodium channels in excitable membranes, usually with high affinity, and showing rapid and complete reversibility. Inasmuch as saxitoxin (STX) and another agent, tetrodotoxin (TTX), bind with high affinity to most sodium channels, studies of their binding site and of their mechanism of action may reveal interesting information about the physiology of the channel itself. In this paper we report the results of four approaches to examine the action of STX: (1) studies of the binding of natural derivatives of saxitoxin, (2) studies of the potency of synthetic analogues of STX, (3) studies of the interference of STX binding by divalent metal and monovalent organic cations, and (4) studies of the interaction between saxitoxin and drugs that modify the gating of Na channels.

Evolution of striatal opiate receptors

Abstract The phylogenetic trend in the relative proportions of two opiate receptor subtypes from invertebrates to humans was identified. The binding of tritiated leu-enkephalin and dihydromorphine was compared in cryostat-cut striatal sections from 11 vertebrates and the anterior ganglion from 2 arthropods. As an independent measure of species relatedness, we used the deviations from the human sequence of amino acids in the molecule cytochrome-C. We show that the percentage of leu-enkephalin binding decreases as the relatedness to human increases.

The tip-E Mutation of Drosophila Decreases Saxitoxin Binding and Interacts with Other Mutations Affecting Nerve Membrane Excitability

A recessive temperature-sensitive paralytic mutation, tip-E, is associated with reduced binding of [3H]saxitoxin to voltage-sensitive sodium channels in membranes from adult Drosophila heads. There is a decrease of 30-40% in the number of [3H]saxitoxin-binding sites per mg protein (Bmax), but the dissociation constant (Kd) for [3H]saxitoxin binding is normal in the remaining population of binding sites. This decrease is not due to a general hypotrophy of neural tissue since the number of alpha-bungarotoxin binding sites is normal in tip-E mutants. Although saxitoxin binding is reduced in vitro, pharmacological experiments suggest that tip-E mutants have close to the wild-type number of sodium channels in vivo. This suggestion is supported by the observation that at permissive temperatures tip-E only marginally suppresses a mutation which causes enhanced membrane excitability. However, even at permissive temperatures tip-E interacts synergistically with mutations that decrease membrane excitability. In this case, the double mutants exhibit reduced viability and/or longevity. We postulate that either the structure of sodium channels or their microenvironment is altered in tip-E mutants resulting in an increased liability of binding sites in vitro.

Saxitoxin binding to sodium channels in head extracts from wild-type and tetrodotoxin-sensitive strains of Drosophila melanogaster

Extracts prepared from heads of Drosophila melanogaster show high-affinity binding (KD = 1.9 nM) of [3H]saxitonin, a compound known to bind to and block voltage-sensitive sodium channels in other organisms. The interaction between saxitoxin and the Drosophila saxitoxin receptor is non-cooperative and reversible with a half-life of 18.3 s for binding at 4 degrees C. The saturable binding is specifically inhibited by tetrodotoxin with a K1 = 0.30 nM. The number of saturable binding sites in the extract is 97 fmol/mg protein. Since approx. 50% of the binding activity is recovered in the extract, the number of binding sites in the head is estimated to be 6.4 fmol/mg head. Nerve conduction in Drosophila larvae is completely blocked after 20 min in a bathing solution containing 200 nM tetrodotoxin. A comparison between the binding and the electrophysiological studies in Drosophila and other organisms suggests that the Drosophila saxitoxin receptor is part of the voltage-sensitive sodium channel involved in the propagation of action potentials. A mutant (ttxs), which is abnormally sensitive to dietary tetrodotoxin, is shown to be indistinguishable from wild type with respect to [3H]saxitonin-binding properties and physiological sensitivity to tetrodotoxin. These studies provide techniques which can be used to identify mutants with defects in the saxitoxin-binding component of the sodium channel.

A Drosophila mutation that reduces sodium channel number confers resistance to pyrethroid insecticides

Abstract The no action potential, temperature-sensitive (nap ts ) mutation in Drosophila melanogaster confers resistance to the pyrethroids fenvalerate and permethrin. Additionally we have found that females are more resistant to pyrethroids than males. Piperonyl butoxide can suppress this resistance in a long pyrethroid exposure (>45 min) but has less of an effect during shorter exposures. Since the pyrethroid resistance of nap ts can be suppressed by piperonyl butoxide, the nap mutant appears to define a phenotypically different locus than the kdr mutant in house flies. Previous work has demonstrated that the nap ts mutation decreases the number of saxitoxin-binding sodium channels. We propose that the presence of fewer sodium channels in mutant flies delays knockdown by pyrethroids, which act as channel agonists. This delay allows other mechanisms to come into play which, in turn, allow survivial. This model is supported by the finding that the nap resistance can be mimicked by feeding wild-type flies a sublethal dose of tetrodotoxin to reduce the number of functional channels. Although the nap ts mutation is resistant to sodium channel agonists, the same single gene mutation causes enhanced sensitivity to the sodium channel antagonist, tetrodotoxin. These observations suggest that an effective way to deal with this form of hereditary insecticide resistance might be to simultaneously administer a sodium channel antagonist, along with the pyrethroid. However, our results illustrate a difficulty with such an approach. We demonstrate that sublethal doses of a channel antagonist cause “pharmacological” resistance to pyrethroids even in wild-type strains.

Genetic variants in an acetylcholine receptor from drosophila melanogaster

The central nervous system of the fruit fly Drosophila mclanogastcr has been shown [l--6] to be a rich source of an a-bungarotoxin-binding component with the properties expected of a nicotinic acetylcholine receptor. In view of the present confusion over the relation of a-bungarotoxin-binding activity to the nicotinic acetylcholine receptor in the vertebrate central nervous system [7,8], it is important to note that oc-bungarotoxin blocks synaptic transmission in the insect central nervous system and thus appears to be binding to a functional acetylcholine receptor. This blockade has been demonstrated in the cereal nerve, giant fiber synapses in the terminal abdominal ganglion of the cockroach Periplatwta americana when the desheathed ganglion is bathperfused with 1 Ow6 M toxin. Resting potentials and action potentials recorded within the ganglion are unchanged after complete blockade of synaptic transmission (D. B. Sattelle, B. Hue, I. D. Harrow, J. I. Gepner and L. M. H., unpublished observations). The primary motivation for extending a-bungarotoxin binding studies to Drosophila is to open the possibility of isolating mutants with altered receptors that can be used in the analysis of receptor structure, function and role in behavior and development. We describe here an experimental strategy for the detection of genetic variants that affect acetylcholine receptor structure. As a first step we have screened for nicotine-resistant flies in order to enrich for genetic variants affecting receptor structure. Nicotine, one of the oldest insecticides [9], is very effective for killing Drosophila, presumably because of its inter-

Native and detergent-solubilized membrane extracts from Drosophila heads contain binding sites for phenylalkylamine calcium channel blockers

Abstract Membrane extracts from Drosophila melanogaster heads contain binding sites for [ 3 H]D-600, a verapamil-like calcium channel blocker. Scatchard analysis suggested a two binding site model with a dissociation constant ( K d ) of 3.3 ± 0.28 nM and a B max of 1.3 ± 0.14 pmol/mg protein for the high affinity site and a K d of 150 ± 28 nM and a B max of 29 ± 10 pmol/mg protein for the low affinity site. The rank order of affinity of phenylalkylamine binding to the high affinity site was (−)-D-888 > (±)-verapamil > (±)-D-600. Displacement of (−)-[ 3 H])D-888 binding by phenylalkylamines is stereoselective. The diphenylpiperazine cinnarizine is an effective inhibitor of [ 3 H]D-600 binding. However, [ 3 H]D-600 binding is only partially inhibited by diltiazem and bepridil at 10 −4 M concentrations, and is not affected by dihydropyridine calcium channel drugs at concentrations −5 M. Moreover, only extremely low specific [ 3 H]nitrendipine, (+)-[ 3 H]PN200-110, or d-(cis)- [ 3 H]diltiazem binding was detectable. The phenylalkylamine photoaffinity probe, [N-methyl- 3 H]LU 49888, labeled two Proteinase-K sensitive proteins of 136,000 and 27,000 Da. The high molecular weight, trypsin-sensitive band behaves like the high affinity phenylalkylamine receptor, while the low molecular weight, trypsin-insensitive band has properties of the low affinity receptor. Digitonin-solubilized binding activity has a sedimentation coefficient of 12 S on a sucrose gradient.

USE OF NEUROTOXINS FOR BIOCHEMICAL AND GENETIC ANALYSIS OF MEMBRANE PROTEINS INVOLVED IN CELL EXCITABILITY

In recent years the fruit fly Drosophila melanogaster has become increasingly popular for use in genetic approaches to problems in neurobiology. As a result, a variety of single gene mutations which affect nervous system function have become available to the investigator. (See other chapters in this volume and the following recent reviews for descriptions of the types of mutants which are available: Pak and Pinto, 1976; Ward, 1977; Kankel and Ferrus, 1979; Pak, 1979). Although electrophysiological studies have been used to provide clues about possible sites of action of these mutations (Ikeda et al., 1976; Siddiqi and Benzer, 1976; Jan et al., 1977; Wu et al., 1978), biochemical experiments will be necessary to identify the molecular site of the mutant defect. Mutations which affect enzymes important to nervous system function such as those involved in neurotransmitter synthesis and degradation are relatively straightforward to identify and analyze since most enzyme activities can be readily monitored in extracts. Thus, mutations affecting the enzymes choline acetyltransferase and acetylcholinesterase have been identified in Drosophila. (See chapter by J.C. Hall et al. in this volume).