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Query: UNIPROT:P06889 (Mol)
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We report the isolation of cDNA clones containing the full 6.3-kb coding sequence of the para-type sodium channel gene of the housefly, Musca domestica. This gene has been implicated as the site of knockdown resistance (kdr), an important resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. The cDNAs predict a polypeptide of 2108 amino acids with close sequence homology (92% identity) to the Drosophila para sodium channel, and around 50% homology to vertebrate sodium channels, Only one major splice form of the housefly sodium channel was detected, in contrast to the Drosophila para transcript which has been reported to undergo extensive alternative splicing. Comparative sequence analysis of housefly strains carrying kdr or the more potent super-kdr factor revealed two amino acid mutations that correlate with these resistance phenotypes. Both mutations are located in domain II of the sodium channel. A leucine to phenylalanine replacement in the hydro-phobic IIS6 transmembrane segment was found in two independent kdr strains and six super-kdr strains of diverse geographic origin, while an additional methionine to threonine replacement within the intracellular IIS4-S5 loop was found only in the super-kdr strains. Neither mutation was present in five pyrethroid-sensitive strains. The mutations suggest a binding site for pyrethroids at the intracellular mouth of the channel pore in a region known to be important for channel inactivation.
Mol Gen Genet 1996 Aug 27
PMID:Identification of mutations in the housefly para-type sodium channel gene associated with knockdown resistance (kdr) to pyrethroid insecticides. 880 3

Using reverse transcription polymerase chain reactions (RT-PCR), the DNA sequence for the main membrane-spanning region (IS3 through IVS6) of the gene encoding the alpha-subunit of the para sodium channel of the German cockroach, Blattella germanica, has been determined. The overall structure of the open reading frame region of this B. germanica gene is very similar to that of the para gene of Drosophila melanogaster, and that of the partially sequenced para gene of Musca domestica. On the other hand, it is distinctly different from that of the DSC gene (Drosophila sodium channel). As a result of a side-by-side comparison of the para gene sequences of the susceptible CSMA strain and the kdr resistant VT strain of B. germanica, one mutation (TTG to TTC) at the approximate center of the IIS6 membrane-spanning segment was found to result in an amino acid change from L to F. While the functional meaning of this mutation for the operation of the para sodium channel remains to be studied, this region is very highly conserved among all sodium channels identified so far, and is one of the most hydrophobic areas of the entire alpha-subunit. For comparison, we have studied the same region of the para sodium channel of both kdr and susceptible SBO strain of the housefly, Musca domestica. We found the homologous type of mutation, CTT to TTT, resulting in the same amino acid alteration (L to F) at this site. However, in the case of houseflies both kdr and susceptible strains contained both L and F versions of the protein. The ratio of TTT to CTT was significantly higher in the kdr strain of M. domestica than in the three susceptible strains examined.
Mol Gen Genet 1996 Aug 27
PMID:Cloning and sequencing of the para-type sodium channel gene from susceptible and kdr-resistant German cockroaches (Blattella germanica) and house fly (Musca domestica). 880 4

The kdr insecticide resistance trait of the house fly (Musca domestica .L.), which confers reduced neuronal sensitivity to DDT and pyrethroid insecticides, was previously shown to exhibit tight genetic linkage to restriction fragment length polymorphism markers lying within a voltage-sensitive sodium channel gene that is homologous to the para gene of Drosophila melanogaster. In the present study, the 6315 nucleotide coding sequences of this voltage-sensitive sodium channel gene from insecticide-susceptible (NAIDM strain) and kdr (538ge strain) house flies were determined by automated direct DNA sequencing of PCR fragments obtained by amplification on first strand cDNA from adult heads. The deduced 2105-residue amino acid sequence from each strain exhibited overall structure and organization typical of sodium channel alpha subunit genes and was 90.0% identical to that of the D. melanogaster para gene product. We did not detect any splice variants among voltage-sensitive sodium channel cDNAs obtained from adult house fly head preparations. Comparison of the coding sequence of the voltage-sensitive sodium channel gene of the kdr house fly strain to that of the NAIDM strain revealed 12 amino acid differences in the 538ge strain. The significance of these polymorphisms as candidate resistance-conferring mutations is discussed.
Insect Biochem Mol Biol 1996 Apr
PMID:Characterization of voltage-sensitive sodium channel gene coding sequences from insecticide-susceptible and knockdown-resistant house fly strains. 881 80

Pseudohypoaldosteronism type 1 (PHA1, OMIM 264350) is a rare Mendelian disorder characterised by end-organ unresponsiveness to mineralocorticoids. Most steroid hormone insensitivity syndromes arise from mutations in the corresponding receptor, but available genetic evidence is against involvement of the mineralocorticoid receptor gene, MLR, in PHA1. A complete genome scan for PHA1 genes was undertaken using homozygosity mapping in 11 consanguineous families. Conclusive evidence of linkage with heterogeneity was obtained with a maximum two-locus admixture lod score of 9.9. The disease locus mapped to chromosome 16p12.2-13.11 in six families and to 12p13.1-pter in the other five families. The two chromosomal regions harbour genes for subunits of the amiloride-sensitive epithelial sodium channel: SCNN1B and SCNN1G on 16p and SCNN1A on 12p. Liddle's syndrome of hypertension and pseudoaldosteronism has been shown to arise from mutations in SCNN1B and SCNN1G. These results strongly suggest that PHA1 and Liddle's syndrome are allelic variants caused by mutations in genes encoding subunits of this sodium channel. These genes are of broad biological interest both in relation to sodium and water homeostasis in mammals and by virtue of their homology to the mec genes of Caenorhabditis elegans involved in mechanosensitivity and neuronal degeneration.
Hum Mol Genet 1996 Feb
PMID:Localisation of pseudohypoaldosteronism genes to chromosome 16p12.2-13.11 and 12p13.1-pter by homozygosity mapping. 882 86

The Pinceau is a paintbrush-like network of cerebellar basket cell axon branchlets embracing the initial segment of the Purkinje cell axon. Its electrical activity contributes to the control of the cerebellar cortical output through the Purkinje cell axon by generating an inhibitory field effect. In addition to the structural features of the Pinceau, its repertoire of voltage-gated ion channels is likely to be an important aspect of this function. Therefore, we investigated the fine structural distribution of voltage-activated potassium (Kv1.1, Kv1.2, Kv3.4) and sodium channel proteins in the Pinceau. The ultrastructural localization of potassium channel subunits was compared to the distribution of synapse-associated protein 90 (SAP90), a protein capable to induce in vitro clustering of Kv1 proteins. With an improved preembedding technique including ultrasmall gold particles, silver enhancement and gold toning, we could show that antibodies recognizing Kv1.1, Kv1.2 and SAP90 are predominantly localized to septate-like junctions, which connect the basket cell axonal branchlets. Kv3.4 immunoreactivity is not concentrated in junctional regions but uniformly distributed over the Pinceau and the pericellular basket surrounding the Purkinje cell soma. In contrast, voltage-activated sodium channels were not detected in the Pinceau, but localized to the Purkinje cell axon initial segment. The results suggest that Kv1.1 and Kv1.2 form heterooligomeric delayed rectifier type Kv channels, being colocalized to septate-like junctions by interaction with SAP90.
Brain Res Mol Brain Res 1996 Nov
PMID:Ultrastructural localization of Shaker-related potassium channel subunits and synapse-associated protein 90 to septate-like junctions in rat cerebellar Pinceaux. 891 80

The polar flagella of Vibrio alginolyticus are driven by sodium motive force and those motors are specifically and strongly inhibited by phenamil, an amiloride analog that is thought to interact with a sodium channel of the flagellar motor. To study the sodium ion coupling site, we isolated motility mutants resistant to phenamil and named the phenotype Mpa(r) for motility resistant to phenamil. The motility of the wild-type (Mpa(s)) was inhibited by 50 microM phenamil, whereas Mpa(r) strains were still motile in the presence of 200 microM phenamil. The Ki value for phenamil in the Mpa(r) strain was estimated to be five times larger than that in the Mpa(s) strain. However, the sensitivities to amiloride or benzamil, another amiloride analog, were not distinctly changed in the Mpa(r) strain. The rotation rate of the wild-type Na+-driven motor fluctuates greatly in the presence of phenamil, which can be explained in terms of a relatively slow dissociation rate of phenamil from the motor. We therefore studied the stability of the rotation of the Mpa(r) and Mpa(s) motors by phenamil. The speed fluctuations of the Mpa(r) motors were distinctly reduced relative to the Mpas motors. The steadier rotation of the Mpa(r) motors can be explained by an increase in the phenamil dissociation rate from a sodium channel of the motor, which suggests that a phenamil-specific binding site of the motor is mutated in the Mpa(r) strain.
J Mol Biol 1997 Jan 24
PMID:Vibrio alginolyticus mutants resistant to phenamil, a specific inhibitor of the sodium-driven flagellar motor. 901 45

The expression of sodium channel alpha-, beta 1- and beta 2-subunit mRNAs was examined in adult rat DRG neurons in dissociated culture at 1 day in vitro and within sections of intact ganglia by in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR). The results demonstrate that sodium channel alpha-subunit mRNAs are differentially expressed in small (< 25 microns diam), medium (25-45 microns diam.) and large (> 45 microns diam.) cultured DRG neurons at 1 day in vitro (div). Sodium channel mRNA I is expressed at higher levels in large neurons than small DRG neurons, while sodium channel mRNA II is variably expressed, with most cells lacking or exhibiting low levels of detectable signal of these mRNAs and limited numbers of neurons with moderate expression levels. DRG neurons generally exhibit negligible or low levels of hybridization signal for sodium channel mRNA III. Sodium channel mRNAs Na6 and NaG show similar patterns of expression, with most large and many medium DRG neurons exhibiting high levels of expression. The mRNA for the rat cognate of human sodium channel hNE-Na is detected in virtually every DRG neuron; most cells in all size classes exhibit moderate or high levels of hNE-Na expression. Sodium channel SNS mRNA is expressed in all size classes of DRG neurons, but shows greater expression in small and medium DRG neurons than in large neurons. The mRNA for the rat cognate of mouse sodium channel mNa 2.3 is not detected, or is detected at low levels, in most DRG neurons, regardless of size, although moderate expression is detected in some neurons. Sodium channel beta 1- and beta 2-subunit mRNAs exhibit similar expression patterns; they are detected in most DRG neurons, although the level of expression tends to be greater in large neurons than in small neurons. RT-PCR and in situ hybridization of intact adult DRG showed a similar pattern of expression of sodium channel mRNAs to that observed in DRG neurons in vitro. These results demonstrate that adult DRG neurons express multiple sodium channel mRNAs in vitro and in situ and suggest a molecular basis for the biophysical heterogeneity of sodium currents observed in these cells.
Brain Res Mol Brain Res 1996 Dec 31
PMID:Spinal sensory neurons express multiple sodium channel alpha-subunit mRNAs. 903 25

Sequencing of the sodium channel gene hscp (para-homologous) revealed that a T to A mutation encoding a Leu to His change (L1029H) is associated with pyrethroid resistance in populations of Heliothis virescens F. This mutation occurs at a position homologous to that of the mutation Leu to Phe (L1014F) reportedly associated with knock-down resistance (kdr) to pyrethroids in a house fly strain. Another mutation reportedly associated with super-kdr in house fly was not found in H. virescens samples. In previous work, we found that one haplotype Hpy3 of a marker locus Hpy that lies approximately 5 kb away from the L1029H polymorphism in the gene, increased in frequency among survivors of selection with pyrethroids, presumably through "hitch-hiking" with a resistance-conferring mutation. Not all resistant individuals examined in this study carried the L1029H mutation, but only those that also carried Hpy3 haplotypes. Resistance has been genetically linked to hscp markers for the strain PEG87, but this strain was found not to carry the L1029H mutation. Assuming that the L1029H mutation does in fact confer resistance, this suggests that more than one sodium channel mutation may be contributing to pyrethroid resistance in field populations of H. virescens.
Insect Biochem Mol Biol 1997 Jan
PMID:A novel mutation L1029H in sodium channel gene hscp associated with pyrethroid resistance for Heliothis virescens (Lepidoptera:Noctuidae). 906 24

Previous genetic and pharmacological studies suggest that knockdown-resistance (kdr) to pyrethroid insecticides likely results from a mutation(s) in the para sodium channel gene. In this study, para sodium channel genes from an insecticide-susceptible German cockroach strain, CSMA, and a kdr-type German cockroach strain, Ectiban-R, were cloned and sequenced. Comparison of the nucleotide sequences of paraCSMA and paraEctiban-R revealed two nucleotide differences at nt 1491 and nt 2979, respectively. Only the difference at nt 2979 (G in paraCSMA and C in paraEctiban-R) resulted in an amino acid change (Leu993 in CSMA and Phe993 in Ectiban-R). Leu993/Phe993 is located in the highly conserved membrane-spanning segment 6 of domain II (IIS6). Furthermore, reverse transcription-polymerase chain reaction (RT-PCR) sequence analysis using another two insecticide-susceptible and one kdr German cockroach strain, revealed that C2979 was specifically associated with kdr resistant strains, whereas only G2979 was present in all susceptible strains. These results suggest that a single amino acid change from Leu993 to Phe993 is responsible for the kdr-type resistance in German cockroach.
Insect Biochem Mol Biol 1997 Feb
PMID:A single amino acid change in the para sodium channel protein is associated with knockdown-resistance (kdr) to pyrethroid insecticides in German cockroach. 906 20

The expression of sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1) was examined in developing (E17-P30) hippocampus, cerebellum, spinal cord and dorsal root ganglia using non-isotopic in situ hybridization cytochemistry. The results showed distinct patterns of expression for each of the sodium channel mRNAs with maturation of the nervous system. In the hippocampus, sodium channel mRNA I was not detected at any developmental time, while mRNA II showed increasing hybridization signal between E17 and P30. Sodium channel mRNA III was more prevalent at late embryonic and early postnatal times, and was barely detectable at P30. The transcript for NaG showed transient expression between P2 and P15, being expressed at low levels at E17 and not being detectable at P30. Sodium channel mRNA Na6 exhibited a high level of expression between E17 and P15 in the hippocampal formation, with an attenuation of the signal by P30. hNE (PN1) mRNA was not detected in the hippocampus at any time examined. In the cerebellum, sodium channel mRNA I was not detected at E17 or P2, but became detectable in Purkinje cells at P15 and continued to show a low level of expression in these cells at P30. mRNA I was not detected at any time examined in granule cells of the cerebellum. Sodium channel mRNA II exhibited increasing expression in the developing cerebellum, and showed increasing signal in Purkinge cells beginning on P2 and granule cells on P15. Sodium channel mRNA III was down-regulated with development in the cerebellum, although mRNA III was readily detected at E17, it was not detected in any layers of the cerebellum by P15. NaG mRNA showed a peak of expression at P2, and was present at low levels at E17 and P15 and not detectable at P30. Na6 mRNA was highly expressed in the E17 cerebellum; this mRNA was present at high levels in Purkinje cells throughout development, although in granule cells the signal was attenuated at P15-P30. Sodium channel hNE (PN1) mRNA was not detected in the cerebellum at any time in development. In the spinal cord, sodium channel mRNA I showed increasing expression beginning at P2 and was highly expressed, particularly in ventral motor neurons, by P30. Sodium channel II mRNA was detected at all stages of development in the spinal cord; in contrast, mRNA III was detected at E17 and P2, but showed very low levels of expression by P30. NaG mRNA exhibited a transient expression in spinal cord at P2, but was not detectable at E17 and P30. Na6 mRNA was detectable at very low levels at E17 and became highly expressed at P2, prior to a reduction of the signal at P15 and P30. hNE (PN1) mRNA was not detected in the spinal cord at any time in development. In the dorsal root ganglia, sodium channel I mRNA hybridization signal was detected in DRG neurons at P2, with slightly increased levels at P15 and P30. Sodium channel II mRNA exhibited a relatively constant, moderate level of expression at all developmental ages. Sodium channel III mRNA was highly expressed in DRG neurons at E17 but was down-regulated with further development so that it was not detectable by P30. NaG mRNA was strongly expressed by some DRG neurons at all stages of development from E17 to P30; in general the level of NaG labelling was greater in larger neurons than in smaller neurons. Na6 mRNA showed increasing expression with development in DRG neurons; at E17, low levels of Na6 mRNA were detected and by P15 to P30 high levels of expression were present in some neurons. hNE (PN1) mRNA was present in DRG neurons at P2, and was up-regulated with further development so that by P30 hNE (PN1) was expressed in all DRG neurons sizes. These results demonstrate that sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1) exhibit distinct spatial and temporal patterns of expression in nervous tissue, and suggest that the expression of the sodium channel alpha-subunits is differentially regulated. (ABSTRACT TRUNCATED)
Brain Res Mol Brain Res 1997 Apr
PMID:Sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1): different expression patterns in developing rat nervous system. 910 72


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