The discovery of a novel sodium channel in the cockroach Periplaneta americana: Evidence for an early duplication of the para-like gene

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Abstract

Voltage-gated sodium channels (Nav channels) belong to a superfamily of ion channels which play an essential role in membrane excitability. Only one gene encoding Nav channels has been characterized so far in insects. Here, we have cloned one full-length cDNA encoding a conventional insect Nav channel (PaNav1) and two full-length cDNAs encoding putative insect Nav channels (PaFPC1 and PaFPC2) in Periplaneta americana, a model insect for neurophysiological studies. The ORFs of PaFPC1 and PaFPC2 contained 4662 bp and encoded 1553 amino acid residues, and the ORF of PaNav1 contained 6153 bp and encoded 2051 amino acid residues. PaFPC1 and PaFPC2 are two isoforms, which differ by eight single amino acid substitutions. PaFPC1 shares 37.5–55% protein identities with known insect Nav channels, while PaNav1 shares 70–97.5% protein identities with these latter. Both PaFPC1 and PaFPC2 possess the molecular hallmarks of Nav channels except the motif involved in fast inactivation. Contrary to PaNav1 transcripts which are expressed mainly in the central nervous system, those ones of PaFPC are also expressed in non-neuronal tissues (muscles, gut and mushroom-shaped accessory glands). A detailed phylogenetic analysis confirmed that PaNav1 and PaFPC are evolutionarily closely related to insect Nav channel genes.

Introduction

Voltage-dependant sodium channels belong to a superfamily of voltage-gated ion channels (VGICs) consisting in heteromultimeric glycoproteins formed by the association of a pore-forming subunit (α-subunit) and one or several regulatory subunit(s) (Yu et al., 2005). Nav channels are members of the four-P domain channel (FPC) family, in which the α-subunit genes encode a polypeptide with four homologous P domains repeated in tandem (DI–DIV). Each P domain contains six transmembrane segments (S1–S6) and a membrane-inserted hairpin-like P loops between helices S5 and S6 containing the ionic selectivity filter (Maelicke, 1988, Zhorov and Tikhonov, 2004). S4 segments are positively charged and act as the sensor for the voltage sensitivity of the channels due to their positive charges (Noda et al., 1984). Additionally to Nav channels, three other FPC subfamilies are distinguished including voltage-gated calcium channels (Cav), Drosophila sodium channel (DSC1) homologs and Na+ leak channel (NALCN) homologs. DSC1 has been firstly described in Drosophila melanogaster and its homologous channels have been found exclusively in insect orders (Liu et al., 2001, Park et al., 1999, Salkoff et al., 1987). The DSCI-orthologous gene BSCI of Blattella germanica has been characterized as a voltage-gated channel permeable to both Na+ and Ca2+ (Zhou et al., 2004). The fourth subfamily comprises genes encoding proteins homologous to NALCN channels (Snutch and Monteil, 2007). Despite NALCN is highly homologous to Nav and Cav channels, it is voltage-insensitive and was proposed to be a sodium leak channel (Lu et al., 2007). Homologs of this protein were also described in Caenorhabditis elegans (nca1 and nca21) and in D. melanogaster (Dm1α1U/CG1517) genome (Nash et al., 2002, Yeh et al., 2008).

It is widely accepted that Nav channel genes diverged from a primordial Cav channel gene (Strong et al., 1993). Due to the selectivity to both Na+ and Ca2+ ions, the subfamilies of the DCSI are proposed to be the descendants of the primordial Cav channel genes (Zhou et al., 2004). On a functional point of view, Cav channels have contributed to the ability of eukaryotes to use intracellular free calcium as a signalling process. The successive evolution of Nav channels have made fast transmission of the nervous message possible in animals (Hille, 2001, Hille, 1989). As a consequence, Nav channels are the most recent channels of the VGICs family (Goldin, 2002).

In mammalians, nine genes (Nav1 to Nav9) and a tenth one Nax encode different Nav channels isoforms (Catterall et al., 2005). A multigenic family has also been characterized in some invertebrate species such as Hirudo medicinalis (leech) and Holocynthia roretzi (ascidia) with four and two characterized genes, respectively (Blackshaw et al., 2003, Nagahora et al., 2000, Okamura et al., 1994). In insect sequenced genomes, only one confirmed Nav channel gene related to the para gene of D. melanogaster encoding DmNav1 has been identified (Shao et al., 2009). The heterologous expression of DmNav1 and its orthologs (MdNav1 of Musca domestica, BgNav1 of B. germanica) in Xenopus oocyte mediate typical voltage-dependant Na+ currents (Smith et al., 1997, Tan et al., 2002, Warmke et al., 1997). Nevertheless, the expression of this unique gene leads to a large number of isoforms, relying on post-transcriptional events such as alternative splicing and RNA editing in D. melanogaster and B. germanica (Hanrahan et al., 2000, Olson et al., 2008, Song et al., 2004).

Periplaneta americana is widely used as a model to study insect neurophysiology. Because different electrophysiological and pharmacological experiments have demonstrated the existence of Na+ current diversity with specialized functions in, for example, axons and neurosecretory cells identified as dorsal unpaired median neurons (Lapied et al., 1990, Lavialle-Defaix et al., 2006), our main aim was to analyse the diversity of Nav channels in the central nervous system (CNS) at molecular level. Although we searched for different para-like channel isoforms expressed in the CNS of P. americana, we cloned two full-length cDNAs named PaNav1 and PaFPC, which encode a conventional and a putative Nav channel, respectively. By RT-PCR, we detected PaNav1 and PaFPC expression in different tissues. A detailed phylogenetic analysis confirmed that PaNav1 and PaFPC genes are evolutionarily closely related to insect Nav channel genes.

Section snippets

Tissue preparation

Various tissues were isolated from American cockroaches obtained from our laboratory stock colonies maintained under standard conditions (29 °C, photoperiod of 12-h light/12-h dark). Mushroom-shaped accessory glands (MSG), gut, muscles extracted from the coxa of the legs, head and the ventral nerve cord were dissected from adult male cockroaches previously anaesthetised by chilling to 4 °C. Nerve cord preparation included thoracic and abdominal ganglia, together with the connectives.

RNA extraction, RT-PCR, RACE and cloning full-length cDNA sequences

Total RNA

Molecular cloning of two full-length cDNAs: PaNav1 and PaFPC

To clone the cDNA encoding Nav channels of P. americana, we proceeded by conventional RT-PCR strategy (Fig. 1). With degenerate primers corresponding to highly conserved regions of para-related genes, we performed two RT-PCRs using nerve cord tissue. The first RT-PCR was aimed to amplify cDNA fragments encoding a region containing domain I and linker 1. Two cDNA fragments of 1700 bp and 920 bp were cloned and their deduced amino acid sequences were, respectively, 77% and 55% identical to that

Discussion

Our results report the primary structure of one conventional Nav channel named PaNav1 and one putative Nav channel named PaFPC in P. americana. Based on their sequence homology with insect Nav channels, PaNav1 appears to be a classical para-like Nav channels whereas PaFPC1 can be considered as novel members of the four-P domain channel family.

Both primary structure and phylogenetic analysis show that PaNav1 gene is orthologous to para gene. The full-length cDNA of the variant PaNav1 is

Acknowledgements

We thank Pr. Ke Dong and Pr. Maria Stankiewicz for critically reading the manuscript. We also thank Dr Frank Bosmans, Dr Alain Hamon, Dr Laurence Murillo and Dr Hélène Tricoire-Leignel for continuous discussion and supports during the work. B. Moignot was supported by a doctoral fellowship of the Région Pays de la Loire.

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