Abstract
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.
Highlights
Voltage-gated sodium (Nav) channels are critical for the initiation of action potentials (APs) in excitable cells [1]
Blast analysis indicated that the deduced amino acid sequence of this cDNA shares 51% of sequence identity with the extracellular loop of DmTEH1 and it was identified as TEH1-like protein of P. americana (PaTEH1)
We observed a higher level of membrane relative fluorescence intensity (AlexaH546 for PaTEH1A or PaTEH1B) in oocytes injected with DmNav1-1/PaTEH1B than with DmNav1-1/PaTEH1A RNAs (2.2 fold, p,0.001, Figure 5C). These findings demonstrated that the C-terminal extremity encoded by the intronic sequence of PaTEH1B was responsible for the decrease in the density of DmNav1-1 channels, suggesting that the intron retention mechanism is likely involved in the regulation of Na+ channels expression in neurons of P. americana
Summary
Voltage-gated sodium (Nav) channels are critical for the initiation of action potentials (APs) in excitable cells [1] Because of their crucial role in the regulation of membrane excitability in the nervous system, the Nav channels are major targets of numerous natural toxins and therapeutic compounds, such as local anesthetics (LAs), antiepileptic drugs and antiarrythmics [2]. They constitute a main molecular target of insecticides like pyrethroids and pyrazoline-type insecticides (PTIs) used in pest control [3,4]. Na+ currents elicited by co-expression of DmNav channels and TipE or TEH1-4, display specific inactivation and repriming properties, reminding functional properties of mammalian b-subunits [8,10]
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