Molecular and functional characterization of a novel sodium channel TipE-like auxiliary subunit from the American cockroach Periplaneta americana.

In Drosophila melanogaster, the functions of voltage-gated sodium (Nav) channels are modulated by TipE and its orthologs. Here, we describe a novel TipE homolog of the American cockroach, Periplaneta americana, called PaTipE. Like DmTipE, PaTipE mRNAs are ubiquitously expressed. Surprisingly, PaTipE mRNA was undetectable in neurosecretory cells identified as dorsal unpaired median neurons. Phylogenetic analysis placed this new sequence in TipE clade, indicating an independent evolution from a common ancestor. Contrary to previous reports, our data indicate that the auxiliary subunits of insect Nav channels are very distant from the mammalian BKCa auxiliary subunits. To decipher the functional roles of PaTipE, we characterized the gating properties of DmNav1-1 channels co-expressed with DmTipE or PaTipE, in Xenopus oocytes. Compared to DmTipE, PaTipE increased Na(+) currents by a 4.2-fold. The voltage-dependence of steady-state fast inactivation of DmNav1-1/PaTipE channels was shifted by 5.8 mV to more negative potentials than that of DmNav1-1/DmTipE channels. DmNav1-1/PaTipE channels recovered 3.2-fold slower from the fast-inactivated state than DmNav1-1/DmTipE channels. In conclusion, this study supports that the insect Nav auxiliary subunits share functional features with their mammalian counterparts, although structurally and phylogenetically distant.

Similar Comparative Low and High Doses of Deltamethrin and Acetamiprid Differently Impair the Retrieval of the Proboscis Extension Reflex in the Forager Honey Bee (Apis mellifera).

In the present study, the effects of low (10 ng/bee) and high (100 ng/bee) doses of acetamiprid and deltamethrin insecticides on multi-trial learning and retrieval were evaluated in the honey bee Apis mellifera. After oral application, acetamiprid and deltamethrin at the concentrations used were not able to impair learning sessions. When the retention tests were performed 1 h, 6 h, and 24 h after learning, we found a significant difference between bees after learning sessions when drugs were applied 24 h before learning. Deltamethrin-treated bees were found to be more sensitive at 10 ng/bee and 100 ng/bee doses compared to acetamiprid-treated bees, only with amounts of 100 ng/bee and at 6 h and 24 h delays. When insecticides were applied during learning sessions, none of the tested insecticides was able to impair learning performance at 10 ng/bee or 100 ng/bee but retention performance was altered 24 h after learning sessions. Acetamiprid was the only one to impair retrieval at 10 ng/bee, whereas at 100 ng/bee an impairment of retrieval was found with both insecticides. The present results therefore suggest that acetamiprid and deltamethrin are able to impair retrieval performance in the honey bee Apis mellifera.

Pharmacological profile of zacopride and new quaternarized fluorobenzamide analogues on mammalian α7 nicotinic acetylcholine receptor.

From quaternarization of quinuclidine enantiomers of 2-fluoro benzamide LMA10203 in dichloromethane, the corresponding N-chloromethyl derivatives LMA10227 and LMA10228 were obtained. Here, we compared the agonist action of known zacopride and its 2-fluoro benzamide analogues, LMA10203, LMA10227 and LMA10228 against mammalian homomeric α7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We found that LMA10203 was a partial agonist of α7 receptor with a pEC50 value of 4.25 ± 0.06 µM whereas LMA10227 and LMA10228 were poorly active on α7 homomeric nicotinic receptor. LMA10227 and LMA10228 were identified as antagonists of acetylcholine-induced currents with IC50 values of 28.4 µM and 39.3 µM whereas LMA10203 and zacopride possessed IC50 values of 8.07 µM and 7.04 µM, respectively. Moreover, despite their IC50 values, LMA10227 was the most potent inhibitor of nicotine-induced current amplitudes (65.7 ± 2.1% inhibition). LMA10203 and LMA10228 had the same inhibitory effects (26.5 ± 7.5% and 33.2 ± 4.1%, respectively), whereas zacopride had no significant inhibitory effect (4.37 ± 4%) on nicotine-induced responses. Our results revealed different pharmacological properties between the four compounds on acetylcholine and nicotine currents. The mode of action of benzamide compounds may need to be reinterpreted with respect to the potential role of α7 receptor.

Intron retention in mRNA encoding ancillary subunit of insect voltage-gated sodium channel modulates channel expression, gating regulation and drug sensitivity.

Insect voltage-gated sodium (Nav) channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation "temperature-induced-paralysis locus E." The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na(+) currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1) strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3'UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1) co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280) in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280). PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be modulated by an intron retention process in the transcription of the neuronal TEH1-like ancillary subunits of P. americana.

Ca²âº/calmodulin-dependent protein kinase II in the cockroach Periplaneta americana: identification of five isoforms and their tissues distribution.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key kinase that transduces Ca²âº signals into downstream effects acting on a range of cellular processes in nervous system and muscular tissues. In insects, different CaMKII isoforms have been reported in Drosophila melanogaster, Apis florae, Bombus terrestris, and Bombus impatiens but little is known on the organization and tissue-specific expression of these isoforms with the exception of Drosophila. The present study reports the cloning of five CaMKII splice variants issued from a single gene and their tissue-specific expression in the cockroach Periplaneta americana. Each CaMKII isoform shared 82-90% identity with Drosophila CaMKII isoforms and accordingly were named PaCaMKII-A, PaCaMKII-B,PaCaMKII-C,PaCaMKII-D, and PaCaMKII-E. PaCaMKII-A and PaCaMKII-D isoforms are ubiquitously expressed in all tissues, but some such as PaCaMKII-B andPaCaMKII-C are preferentially expressed in the nerve cord and muscle. In addition, using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR), we found a tissue-specific expression of PaCaMKII-E in the dorsal unpaired median neurons. Alternative splicing of PaCaMKII transcripts is likely a common mechanism in insects to control the pattern of isoform expression in the different tissues.