Sodium channel polypeptides in central nervous systems of various insects identified with site directed antibodies (original) (raw)
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Solubilization and characterization of the insect neuronal sodium channel
Neuroscience Letters, 1991
Locust neuronal sodium channels were solubilized by 1% cholate and 0.2% Triton X-100, and their functionality was monitored by [~H]saxitoxin (STX) binding assays. About 40% of STX binding activity was recovered in the solubilized fraction without affecting affinity (Kd = 0.5 nM) and the time and temperature dependent STX binding activity was significantly stabilized in the presence of 20 nM STX. Partial purification by an anion exchange resin yielded a 20% recovery and a 3.5 times increase in the specific STX binding activity. Identification of the locust solubilized sodium channels by immunoprecipitation and radiophosphorylation revealed a Mr of 245,000 on SDS-PAGE. The present solubilized preparation will enable the study of the unique pharmacology of insect sodium channels.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2002
The ability of the excitatory anti-insect-selective scorpion toxin AahIT (Androctonus australis hector) to exclusively bind to and modify the insect voltage-gated sodium channel (NaCh) makes it a unique tool to unravel the structural differences between mammalian and insect channels, a prerequisite in the design of selective pesticides. To localize the insect NaCh domain that binds AahIT, we constructed a chimeric channel composed of rat brain NaCh alpha-subunit (rBIIA) in which domain-2 (D2) was replaced by that of Drosophila Para (paralytic temperature-sensitive). The choice of D2 was dictated by the similarity between AahIT and scorpion beta-toxins pertaining to both their binding and action and the essential role of D2 in the beta-toxins binding site on mammalian channels. Expression of the chimera rBIIA-ParaD2 in Xenopus oocytes gave rise to voltage-gated and TTX-sensitive NaChs that, like rBIIA, were sensitive to scorpion alpha-toxins and regulated by the auxiliary subunit bet...
Density of sodium channels in insect synaptic nerve endings
Neurochemistry International, 1990
Specific binding of [ll-3H]saxitoxin (STX) and activity of ouabain sensitive adenosinetriphosphatase (Na ÷, K+-ATPase) were determined in neuronal membrane fractions using a subcellular preparation from the central nervous system of the cockroach Periplaneta americana. The nerve ending fractions (synaptosomes) contained 90-95% of the total specific activity of ouabain sensitive Na +, K +-ATPase, and 60-70% of specific STX binding of the crude nerve homogenate. Sodium influx induced by veratridine in synaptosomes was inhibited by saxitoxin at a half-maximal concentration of 4 nM, and kinetics were consistent with reversible binding of one molecule of saxitoxin to each sodium channel receptor site, with an equilibrium dissociation constant (Ko) of approx. 3 nM. The density of saturable binding sites was 2 pmol/mg protein which was estimated to correspond to about 95 binding sites per/tm 2 of synaptic membrane. The results of transport and binding data show that insect synaptosomes possess the capability to conduct inward sodium currents at least comparable to those found in other neuronal membranes, and thus provide a physiologically viable preparation to assess the effect of cation fluxes on the synaptic transmitter release process.
Variability among insect sodium channels revealed by selective neurotoxins
Insect Biochemistry and Molecular Biology, 1994
Binding assays with the radioiodinated depressant (LqhlTz) and excitatory (AalT) insect selective neurotoxins derived from scorpion venoms to neuronal membrane preparations derived from cockroach, fly and lepidopterous larvae coupled with the employment of sodium channel site-directed antibodies resulted in the following information. (1) The two toxins were shown to bind with high affinity to the insect neuronal preparations and revealed similar binding constants in each of the various preparations.
The Journal of Neuroscience, 2002
The ability of the excitatory anti-insect-selective scorpion toxin AahIT (Androctonus australis hector) to exclusively bind to and modify the insect voltage-gated sodium channel (NaCh) makes it a unique tool to unravel the structural differences between mammalian and insect channels, a prerequisite in the design of selective pesticides. To localize the insect NaCh domain that binds AahIT, we constructed a chimeric channel composed of rat brain NaCh ␣-subunit (rBIIA) in which domain-2 (D2) was replaced by that of Drosophila Para (paralytic temperaturesensitive). The choice of D2 was dictated by the similarity between AahIT and scorpion -toxins pertaining to both their binding and action and the essential role of D2 in the -toxins binding site on mammalian channels. Expression of the chimera rBIIA-ParaD2 in Xenopus oocytes gave rise to voltage-gated and TTX-sensitive NaChs that, like rBIIA, were sensitive to scorpion ␣-toxins and regulated by the auxiliary subunit  1 but not by the insect TipE. Notably, like Drosophila Para/TipE, but unlike rBIIA/ 1 , the chimera gained sensitivity to AahIT, indicating that the phyletic selectivity of AahIT is conferred by the insect NaCh D2. Furthermore, the chimera acquired additional insect channel properties; its activation was shifted to more positive potentials, and the effect of ␣-toxins was potentiated. Our results highlight the key role of D2 in the selective recognition of anti-insect excitatory toxins and in the modulation of NaCh gating. We also provide a methodological approach to the study of ion channels that are difficult to express in model expression systems.
Biochemistry, 1992
Site-directed antibodies corresponding to conserved putative extracellular segments of sodium channels, coupled with binding studies of radiolabeled insect-selective scorpion neurotoxins, were employed to clarify the relationship between the toxins' receptor sites and the insect sodium channel. (1) The depressant insect toxin LqhIT2 was shown to possess two noninteracting binding sites in locust neuronal membranes: a high-affinity (KD, = 0.9 f 0.6 nM) and low-capacity (Bmax, = 0.1 f 0.07 pmol/mg) binding site as well as a low-affinity (KD, = 185 f 13 nM) and high-capacity (Bmaxz = 10.0 f 0.6 pmol/mg) binding site. The high-affinity site serves as a target for binding competition by the excitatory insect toxin AaIT. The binding of LqhIT2 was significantly inhibited in a dose-dependent manner by each of four site-directed antibodies. The binding inhibition resulted from reduction in the number of binding sites. (4) The antibodymediated inhibition of [1251]AaIT binding differs from that of LqhIT2: three out of the four antibodies which inhibited LqhIT2 binding only partially affected AaIT binding. Two antibodies, one corresponding to extracellular and one to intracellular segments of the channel, did not affect the binding of either toxin. These data suggest that the receptors to the depressant and excitatory insect toxins (a) comprise an integral part of the insect sodium channel, (b) are formed by segments of external loops in domains I, 111, and IV of the sodium channel, and (c) are localized in close proximity but are not identical in spite of the competitive interaction between these toxins.
Insect Biochemistry and Molecular Biology, 2006
In a previous study, we showed that two alternative exons (G1 and G2 encoding IIIS3-S4) were involved in the differential sensitivity of two cockroach sodium channel splice variants, BgNa v 1-1 and BgNa v 2-1 (previously called KD1 and KD2), to deltamethrin, a pyrethroid insecticide . Alternative splicing of an insect sodium channel gene generates pharmacologically distinct sodium channels. J. Neurosci. 22, 5300-5309.). Here, we report the identification of an amino acid residue in exon G2 that contributes to the low deltamethrin sensitivity of BgNa v 2-1. Replacement of A1356 in BgNa v 2-1 with the corresponding V1356 in BgNa v 1-1 enhanced the sensitivity of the BgNa v 2-1 channel to deltamethrin by six-fold. Conversely, substitution of V1356 with A1356 in BgNa v 1-1 produced a recombinant BgNa v 1-1 channel that was 5-fold more resistant to deltamethrin. These results demonstrate that A1356 contributes to the low sensitivity of BgNa v 2-1 to deltamethrin. A1356V substitution also shifted the voltage-dependence of activation by 10 mV in the hyperpolarizing direction. Possible mechanisms by which this amino acid change affects the action of pyrethroids on the sodium channel are discussed.
Sodium Channel Auxiliary Subunits
Microbial physiology, 2007
Voltage-gated ion channels are well known for their functional roles in excitable tissues. Excitable tissues rely on voltage-gated ion channels and their auxiliary subunits to achieve concerted electrical activity in living cells. Auxiliary subunits are also known to provide functional diversity towards the transport and biogenesis properties of the principal subunits. Recent interests in pharmacological properties of these auxiliary subunits have prompted significant amounts of efforts in understanding their physiological roles. Some auxiliary subunits can potentially serve as drug targets for novel analgesics. Three families of sodium channel auxiliary subunits are described here:  1 and  3,  2 and  4, and temperature-induced paralytic E (TipE). While sodium channel -subunits are encoded in many animal genomes, TipE has only been found exclusively in insects. In this review, we present phylogenetic analyses, discuss potential evolutionary origins and functional data available for each of these subunits. For each family, we also correlate the functional specificity with the history of evolution for the individual auxiliary subunits.
Invertebrate Neuroscience, 1999
The amino acid sequence BCNG-1 (brain cyclic nucleotide gated 1, of the mouse), the ®rst member of mamalian I h channels, was used to construct a set of polymerase chain reaction (PCR) primers from possibly conserved regions. Reverse transcription-PCR with Drosophila melanogaster mRNA yielded in a PCR product, which exhibited a high homology to BCNG-1. Using these PCR products to screen a D. melanogaster head cDNA library we isolated a cDNA encoding a member of a new class of putative voltage-and cyclic nucleotide-gated potassium channels from D. melanogaster. The most important features of the amino acid sequence predicted from the cDNA were a C-terminal cyclic nucleotide-binding region, an S4-voltage sensor and a putative potassium-selective pore-forming motif. The high homology of 51% to the sea urchin I h channel, which belongs to the same class of ion channels as BCNG-1, leads us to suggest that the Drosophila cDNA is the ®rst insect member of a new class of hyperpolarization-activated and cyclic nucleotide-gated channels. As shown by in situ hybridization, a pronounced mRNA expression was detected in neuronal tissue, including sensory tissue like the compound eyes, and the olfactory and the auditory organs. Key words Voltage-activated channel á Cyclic nucleotide-gated channel á Drosophila melanogaster á Olfaction á Signal transduction