The Bacillus thuringiensis CryIAa toxin binds a cadherin-like protein (BtR175) on the brush-border membranes of the Bombyx mori midgut columnar cells, which are the targets. By introducing the BtR175 gene with a baculovirus, Spodoptera frugiperda Sf9 cells expressed BtR175 protein on the cell membrane and became susceptible to the CryIAa toxin. The toxin bound the cadherin repeat adjacent to the membrane and made a pore that passed inorganic ions, causing the cell to swell and burst. This was not observed with a BtR175 variant lacking the toxin-binding site. This in vitro experiment mimicked the specific insecticidal action of the toxin in vivo well.z 1999 Federation of European Biochemical Societies.
Neuropeptides are a ubiquitous class of signaling molecules. In our attempt to understand the generation of feeding behavior in Aplysia, we have sought to identify and fully characterize the neuropeptides operating in this system. Preliminary evidence indicated that Mytilus inhibitory peptide (MIP)-like peptides are present and operating in the circuitry that generates feeding in Aplysia. MIPs were originally isolated from the bivalve mollusc Mytilus edulis, and related peptides have been identified in other invertebrate species, but no precursor has been identified. In this study, we describe the isolation and characterization of novel Aplysia MIP-related peptides (AMRPs) and their precursor. Several AMRPs appear to have some structural and functional features similar to vertebrate opioid peptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to confirm that all 14 AMRPs predicted by the precursor are processed in isolated neurons. Northern analysis, whole-mount in situ hybridization, and immunohistochemistry are used to map the abundant expression of these peptides in the CNS and peripheral tissues such as the digestive tract, vasculature, and the reproductive organs. Physiological studies demonstrate that the rank order of the inhibitory actions of these peptides is different for three target muscles. These results underscore the importance of using a multidisciplinary approach to identifying and characterizing the actions of neuropeptides in an effort to gain understanding of their role in systems of interest. The widespread distribution of the AMRPs indicates that they may be operating in many different systems of Aplysia.
Although potassium channels play a variety of roles in shaping the electrical properties of neurons, little is known about how these channels are constituted in neurons. To examine the assembly and physiological function of A-type K+ channels in mature differentiated neurons, we have developed a higly efficient gene transfer method forAplysia neurons that has allowed us to express about 107 copies of the cloned Aplysia Shaker (Sh) K+ channel (AKOla) in single identified cells. We find that expression of AKOla phenocopies one of the native transient K+ currents (IAepa)J suggesIng that the native channel carrying IAd&pI is assembled as a homooligomer of AKOla. Overexpression of AKOla has substantial effect on the action potential, shortening its duration, enhancing its hyperpolarizing afterpotential, and depressing by more than half the amount of transmitter release by the action potential from the terminals. Thus, the AKOla channel not only contributes to the firing properties within a given neuron but also can regulate the signaling between interconnected cells.Potassium channels are important regulators of the resting potential, the action potential, and the excitability of neurons (1). In addition, they have a role in certain forms of procedural learning. For example, in the marine molluscs Aplysia (2-5) and Hermissenda (6-9) modulation of different species of K+ currents in sensory neurons contributes to several learned modifications of simple reflex behaviors. The cloning of the Shaker (Sh) locus in Drosophila (10-13) has now made it possible to study the functions of various K+ channels on the molecular level. However, K+ channels have so far not been successfully transfected into mature, differentiated nerve cells in the intact nervous system. As a result, it has not been possible to study cloned K+ channels in their native cellular environment and to determine, by detailed comparison of cloned and endogenous channels, the subunit composition of any endogenous K+ channel. G. R. MacGregor, Houston, TX) as a BamHI fragment (3.7 kb) and inserted into the BamHI site of pNEX, and the orientation was determined. To make pNEX-AKO1a, the full-length cDNA fragment (1.8 kb) of Aplysia Shaker (AKOla) having Cla I and Xmn I sites at 5' and 3' ends, respectively, was inserted into Acc I-and Sma I-digested pNEX.Microinjection of Plasmids into Aplysia Neurons. From Aplysia californica weighing 10-100 g (furnished by the Howard Hughes Medical Institute, Miami Facility) the abdominal ganglion was dissected and treated with protease IX (Sigma) at 10 mg/ml in L15 medium (15) for 1 hr at 34.50C.The ganglion was then pinned on a Sylgard plate and desheathed carefully to expose the neurons to the L15 medium containing an equal volume of Aplysia hemolymph or artificial seawater (ASW) that had the following composition: 460 mM NaCl/10 mM KCl/11 mM CaCl2/55 mM MgCl2/10 mM Hepes, pH 7.6. In a few experiments, neurons in primary cell culture were used. Primary cell culture techniques and media have been described (15). Withi...
We describe here the cloning of the Aplysia K+ channel AK01a.AK01a codes for a protein of 515 amino acids, shows considerable homology to other cloned potassium channels, and can be classified as a member of the ShakerK+ channel family. Expression of the AK01a channel in Xenopus oocytes produces a rapidly inactivating outward potassium current (IAK01a) resembling the A-type currents of Drosophila Shaker. Gating for this current is shifted to potentials considerably more positive than the traditional A-currents of Aplysia; we have, however, identified a novel transient potassium current (IAdepoI) in a subset of Aplysia neurons that has similar gating and pharmacological properties to IAK01a.
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