Molecular and functional characterization of voltage-gated sodium channel variants from Drosophila melanogaster

Abstract

Extensive alternative splicing and RNA editing have been documented for the transcript of DmNa_V (formerly para), the sole sodium channel gene in Drosophila melanogaster. However, the functional consequences of these post-transcriptional modifications are not well understood. In this study we isolated 64 full-length DmNa_V cDNA clones from D. melanogaster adults. Based on the usage of 11 alternative exons, 64 clones could be grouped into 29 splice types. When expressed in Xenopus oocytes, 33 DmNa_V variants generated sodium currents large enough for functional characterization. Among these variants, DmNa_V5-1 and DmNa_V7-1 channels activated at the most hyperpolarizing potentials, whereas DmNa_V1-6 and DmNa_V19 channels activated at the most depolarizing membrane potentials. We identified an A-to-I editing event in DmNa_V5-1 that is responsible for its uniquely low-voltage-dependent activation. The wide range of voltage dependence of gating properties exhibited by DmNa_V variants represents a rich resource for future studies to determine the role of DmNa_V in regulating sodium channel gating, pharmacology, and neuronal excitability in insects.

1. Introduction

Voltage-gated sodium channels are integral transmembrane proteins responsible for the generation of action potentials across the membranes of excitable cells. Mammalian sodium channels consist of a large pore-forming α-subunit and one or more smaller auxiliary β-subunits. The α-subunit is composed of four homologous domains (I–IV), each containing six hydrophobic transmembrane segments (S1–S6). Mammals have nine α-subunit genes which encode sodium channel isoforms with different gating properties and different expression patterns in various cell types, tissues, and developmental stages, presumably to fulfill unique functions in specific neurons (Catterall, 2000; Goldin et al., 2000; Yu and Catterall, 2003). In contrast, insects appear to have only a single sodium channel gene, such as DmNa_V (formerly para) in Drosophila melanogaster, encoding the equivalent of the α-subunit of the mammalian sodium channels (Loughney et al., 1989). The sodium channel β-subunits in insects are less well understood, except that a unique transmembrane protein, TipE, is required for robust expression of insect sodium channels in heterologous Xenopus oocytes (Feng etal., 1995; Warmke et al., 1997).

Functionally distinct sodium currents have been documented in various insect neurons, indicating existence of heterogeneous sodium channels with distinct gating properties in vivo (Byerly and Leung, 1988; Lapied et al., 1990, 1999; Saito and Wu, 1991, 1993; Schafer et al., 1994; O'Dowd, 1995; Le Corronc et al., 1999; Grolleau and Lapied, 2000; Wicher et al., 2001; Defaix and Lapied, 2005). A fundamental question is how insects produce heterogeneous sodium channels from a single gene. One possibility is that post-transcriptional modifications of the primary sodium channel transcript result in variations of sodium channel gating properties. Indeed, alternative splicing and RNA editing of the DmNa_V transcript have been reported (O'Dowd and Aldrich, 1988; Loughney et al., 1989; Thackeray and Ganetzky, 1994, 1995; O'Dowd, 1995; Warmke et al., 1997; Palladino et al., 2000). The functional consequences of alternative splicing and/or RNA editing of the DmNa_V transcript, however, remain largely unknown. Prior to this study, only one full-length DmNa_V cDNA clone has been functionally characterized in Xenopus oocytes (Warmke et al., 1997).

Recent characterization of the German cockroach BgNa_V sodium channel gene suggests that alternative splicing and RNA editing are two major mechanisms by which cockroaches produce functionally diverse sodium channels (Tan et al., 2002; Liu et al., 2004; Song et al., 2004; Dong, 2007). To determine whether alternative splicing- and RNA editing-mediated generation of diverse sodium channel gating properties is a common phenomenon in insects, we conducted a large-scale functional characterization of alternative splicing and RNA editing variants of the D. melanogaster DmNa_V gene. Here, we report the isolation of 64 full-length DmNa_V cDNA clones from D. melanogaster and functional characterization of these variants in Xenopus oocytes.

2. Materials and methods

2.1. Synthesis of first-strand cDNAs

Total RNA was isolated from about 100 whole adults of D. melanogaster strain W¹¹¹⁸ using the Invitrogen TRIzol Reagent kit (Invitrogen, Rockville, MD). Further isolation of mRNA was performed using the Promega polyA + RNA isolation kit (Promega, Madison, WI). First-strand cDNA was synthesized from mRNA using a DmNa_V-specific primer (D-3′–1: 5′-tac tca tgc taa tac tcg cg-3′) based on the sequence immediately downstream of the stop codon (Genbank accession number: M32078), and Super-Script II RNase H-reverse transcriptase (Invitrogen, Rockville, MD). Conditions for the first-strand cDNA synthesis reaction were: 42°C for 2 min followed by a 60 min incubation at 48°C. RNA was removed by 20 min incubation with RNaseH at 37°C.

2.2. Polymerase chain reaction (PCR), cloning of full-length DmNa_V cDNA and DNA sequencing

To amplify the complete coding region (6 kb) of DmNa_V by PCR, a forward primer (D-DmNa_V-KpnI: 5′-cgg ggt acc gcc acc atg gca gaa gat tcc gac tcg-3′) and a reverse primer (D-DmNa_V-XbaI: 5′-gct cta gat act gct aat act cgc g-3′) were designed based on the 5′ and 3′ end sequences of the DmNa_V open reading frame (ORF). The XbaI and KpnI recognition sequences (bold in the primer sequences) were added to the forward and reverse primers, respectively, to facilitate cloning. A Kozak sequence (bold-italic in the primer sequence) was added to primer D-DmNa_V-KpnI for high-efficiency translation. The reaction mixture (50μl) contained 0.5μl cDNA, 50 pmol each primer, 200μM each dNTP, 1 U eLONGase (Invitrogen), 1.5mM MgCl₂, and 1 × PCR reaction buffer. PCR was carried out as follows: one cycle of 94 °C for 1 min; 33 cycles of 94 °C for 30 s, 58 °C for 30 s, 68 °C for 8 min; and one cycle of 68 °C for 15 min. The PCR products were purified using the QIAEX II Gel Extraction Kit (QIAGEN Sciences, MD) and cloned into pGH19 (a Xenopus expression vector containing the 3′ and 5′ untranslated regions of the Xenopus β-globin gene at the 3′ end of T7 promoter and 5′ end of SP6 promoter, respectively; and was kindly provided by B. Ganetzky, University of Wisconsin). MAX Efficiency Stbl2-competent cells (Invitrogen) were used as host cells. The DNA sequence was determined at the Research Technology Support Facility on campus using primers that anneal to both strands. Overlapping sequences were assembled and all sequence data underwent standard quality control to remove ambiguity. Sequences of DmNa_V inserts in cDNA clones were compared with the Genbank sequence (Accession number: M32078) using DNAStar (DNAStar Inc.).

2.3. Site-directed mutagenesis

Site-directed mutagenesis was performed by PCR using specific primers and Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA). All mutagenesis results were verified by DNA sequencing.

2.4. Expression of DmNa_V sodium channels in Xenopus oocytes

The procedures for oocyte preparation and cRNA injection were identical to those described previously (Tan et al., 2002). Plasmid DNA containing the DmNa_V insert was linearized using the NotI restriction enzyme. The linearized DNA was used for in vitro synthesis of the DmNa_V cRNA using in vitro transcription with T7 polymerase (mMESSAGE mMACHINE kit, Ambion). For robust expression of DmNa_V sodium channels, DmNa_V cRNA (0.25–10 ng) was co-injected into oocytes with the D. melanogaster tipE cRNA (1:1 molar ratio), which enhances the expression of insect sodium channels in oocytes (Feng et al., 1995; Warmke et al., 1997). More than one DmNa_V and TipE cRNA preparations were tested for each clone with similar results. Oocytes carrying peak sodium currents of 0.5–2μA were used for functional analysis using two-electrode voltage clamp.

2.5. Electrophysiological recording and analysis

The voltage dependence of activation and inactivation were measured using the two-electrode voltage clamp technique. Methods for two-electrode recording and data analysis were similar to those described previously (Tan et al., 2002).

3. Results and discussion

3.1. Molecular analysis of 64 full-length cDNA clones

Prior to this study, molecular characterization of partial DmNa_V cDNA sequences resulted in the identification of eleven alternative exons in the DmNa_V transcript (Thackeray and Ganetzky, 1994; O'Dowd et al., 1995; Warmke et al., 1997, Lee et al., 2002). In order to determine the alternative splicing pattern of each full-length DmNa_V transcript and to conduct functional expression of each splice type, it was necessary to isolate and characterize a large number of full-length cDNA clones. Using RT-PCR, we obtained 64 full-length DmNa_V cDNA clones from adult D. melanogaster. PCR and/or sequencing allowed us to group the 64 clones into 29 splice types according to exon usage. Fig. 1 displays the 29 splice types (numbered 1–29) and number of clones (variants) in each splice type. Variants are named according to splice type. For example, variants in splice type 1 are designated as DmNa_V1, and 13 variants belonging to this splice type are named DmNa_V1-1 – DmNa_V1-13.

Fig. 1.

Identification of 29 alternative splice types of the DmNa_V transcript. (A) A schematic diagram of the DmNa_V sodium channel protein topology, with the positions of alternative exons indicated by patterned boxes. Exons, a, b, i, j, e, f and h are optional, whereas exons c/d and l/k are mutually exclusive. Exons l and k correspond to exons G1 and G2, respectively, in the cockroach BgNav sodium channel (Tan et al., 2002). (B) Schematic presentation of the usage of alternative exons in 64 full-length cDNA clones. The number of clones (i.e., variants) of each splice type is indicated.

We found that exons a, b, d, i, j and l were used in greater than 60% of the DmNa_V variants (both D. melanogaster), whereas usage of exons c, e, f, h and k was less frequent (Fig. 1). This observation is consistent with earlier analysis of partial cDNAs from D. melanogaster and D. virilis (O'Dowd et al., 1995; Thackeray and Ganetzky, 1994, 1995). No positive or negative associations of these alternative exons with each other were observed.

3.2. Distinct gating properties of DmNa_V variants expressed in Xenopus oocytes

For functional analysis of DmNa_V variants, we coexpressed each variant with TipE in Xenopus oocytes and recorded sodium current from oocytes. Roughly half of the variants, 33, produced sodium currents large enough (0.7–2.5μA 1–8 days after cRNA injection) for further analysis. Four variants produced currents that were too small (<0.5μA) for functional analysis. Twenty-seven variants did not generate any detectable sodium current even 8 days after cRNA injection.

Analysis of the 33 functional variants revealed a broad range of voltage dependence for channel activation and inactivation (Table 1). Figs. 2A and B display the extent of variation in voltage dependence among functional DmNa_V clones. The voltages for half-maximal activation (V_1/2) of most variants (76%) were within 10 mV range, between −20 and −30 mV. Six variants, DmNa_V19, DmNa_V15, DmNa_V1-6, DmNa_V13-2, DmNa_V1-3 and DmNa_V2-3, required more positive membrane potentials for activation, whereas two variants, DmNa_V5-1 and DmNa_V7-1, activated at more negative membrane potentials (Table 1). In particular, DmNa_V7-1 activated at voltage of <−60 mV (Fig. 2A). The so-called background sodium currents have been reported in cockroach dorsal unpaired median (DUM) neurons in terminal abdominal ganglia, which is believed to play a critical role in providing the depolarizing force needed to maintain pacemaker activity of these neurons (Lapied et al., 1990). The specific sodium channels behind such currents remain elusive. The extremely low-voltage-gating properties of DmNa_V5-1 and DmNa_V7-1 make them strong candidates for conducting background currents.

Table 1. Gating properties of 33 functional DmNa_v variants.

Variant	Activation		Inactivation
	V1/2 (mV)	k (mV)	V1/2 (mV)	k (mV)
DmNaV19	−6.7±0.5	5.2±0.2	−50.1±0.3	6.3±0.1
DmNaV15	−11.4±0.6	7.3±0.2	−34.9±0.9	5.5±0.3
DmNaV1-6	−12.0±2.1	9.8±0.7	−49.9±0.5	5.6±0.9
DmNaV13-2	−15.5±0.4	8.2±0.3	−45.5±0.4	7.5±0.2
DmNaV1-3	−16.2±0.5	8.0±0.2	−42.3±0.6	7.3±0.6
DmNaV2-3	−20.5±1.6	8.0±0.5	−46.7±0.2	4.8±0.2
DmNaV2-2	−20.6±0.4	7.4±0.2	−39.8±0.4	6.1±0.2
DmNaV4-1	−21.6±0.5	6.3±0.2	−41.1±0.2	5.2±0.1
DmNaV3-1	−22.6±0.9	6.6±0.4	−46.4±0.4	5.0±0.1
DmNaV17	−23.1±1.0	7.0±0.3	−47.4±0.6	5.1±0.2
DmNaV28	−23.3±0.8	5.5±0.3	−39.9±0.5	5.4±0.2
DmNaV3-2	−23.4±0.5	6.4±0.2	−43.8±0.4	5.4±0.1
DmNaV1-2	−23.4±0.8	7.5±0.3	−45.1±0.4	5.5±0.1
DmNaV26	−23.6±0.5	3.7±0.2	−39.7±0.2	4.4±0.1
DmNaV10-2	−23.7±0.6	4.5±0.2	−40.1±0.3	4.5±0.2
DmNaV2-1	−23.8±0.9	6.7±0.2	−48.3±0.5	5.3±0.1
DmNaV1-1	−24.6±0.8	7.6±0.3	−47.3±0.3	5.6±0.1
DmNaV23	−24.9±0.7	6.6±0.4	−46.1±0.4	5.2±0.1
DmNaV13-1	−25.3±0.9	4.7±0.3	−42.9±0.3	4.8±0.1
DmNaV2-4	−25.7±0.6	5.6±0.4	−46.1±0.8	4.9±0.1
DmNaV29	−25.9±1.2	4.0±0.3	−38.9±0.3	4.8±0.1
DmNaV14	−26.0±0.7	3.0±0.1	−32.1±0.3	4.0±0.1
DmNaV5-3	−26.1±0.9	5.9±0.3	−35.1±0.6	4.9±0.1
DmNaV20	−26.2±0.9	5.0±0.3	−45.1±0.3	4.9±0.1
DmNaV1-5	−26.7±0.8	4.9±0.2	−43.5±0.2	4.8±0.1
DmNaV10-1	−26.9±1.1	8.1±0.7	−44.8±1.1	4.8±0.8
DmNaV1-4	−27.2±0.7	5.8±0.2	−45.2±0.4	5.4±0.0
DmNaV24	−27.2±0.8	4.9±0.2	−41.9±0.6	5.0±0.1
DmNaV22	−28.4±1.1	4.6±0.2	−42.0±0.3	5.0±0.1
DmNaV27	−29.1±0.6	3.6±0.2	−42.9±0.4	4.5±0.1
DmNaV5-2	−29.5±0.7	10.4±0.4	−49.2±0.5	6.5±0.3
DmNaV5-1	−37.1±0.6	3.4±0.1	−44.4±0.7	4.5±0.1
DmNaV7-1	−47.7±0.6	6.3±0.2	−66.4±0.2	4.5±0.0

Note: A minimum of eight oocytes was used to calculate V_1/2 and SEM values.

Fig. 2.

Gating properties of representative DmNa_V variants. (A) Voltage dependence of activation of DmNa_V1-1, DmNa_V1-6, and DmNa_V7-1. (B) Voltage dependence of steady-state inactivation of DmNa_V1-1, DmNa_V1-6, and DmNa_V7-1. (C) Peak sodium current traces of DmNa_V1-1 and DmNa_V7-1 indicating a persistent current in DmNa_V7-1. (D) Voltage dependence of activation of DmNa_V5-1 and DmNa_V5-1^V260I.

Voltages for half-maximal inactivation (V_1/2) were less variable compared with those for activation, with V_1/2 values ranging from −33 to −66 mV (Table 1 and Fig. 2B). Interestingly, the V_1/2 of inactivation of DmNa_V7-1 was shifted significantly in the hyperpolarizing direction compared to other variants. Inactivation was complete by the end of a 20 ms depolarization for most variants including DmNa_V1-1. However, five variants, DmNa_V22, DmNa_V14, DmNa_V5-1 and DmNa_V7-1 and DmNa_V5-3, exhibited a persistent current about 10% of the peak current remained at the end of the 20 ms depolarizing pulse, as shown for DmNa_V7-1 (Fig. 2C). This persistent current was also evident in the steady-state inactivation curve of DmNa_V7-1, where the foot of the curve is lifted at depolarizing potentials (Fig. 2B).

Surprisingly, among functional DmNa_V variants, there was no apparent correlation between splice types and gating properties (Table 1, Figs. 2A and B). For example, the gating properties of six splice type 1 variants (DmNa_V1-1–DmNa_V1-6) are significantly different (P> 0.05) based on a one-way ANOVA with Scheffe's analysis. This finding suggests that other post-transcriptional modifications (e.g., RNA editing) might play a primary role in determining the voltage dependence of activation and inactivation of DmNa_V variants.

3.3. Nonfunctional DmNa_V variants

Although not characterized further in this study, the 31 “nonfunctional” or poorly functional clones may be a potential source for future in-depth structure–function studies of the DmNa_V channel. Several explanations can be proposed for the production of nonfunctional or poorly functional variants. First, some nonfunctional variants may contain premature stop codons. While the role of truncated, nonfunctional sodium channels are not understood, specific alternative splicing events have been identified in both mammals and insects that result in nonfunctional, truncated two-domain variants (Plummer et al., 1997; Tan et al., 2002; Wang et. al., 2003). Second, some variants may contain exon b, which was previously shown in several cockroach variants to suppress sodium channel expression in Xenopus oocytes (Song et al., 2004). However, our analysis showed that 18 of the 32 functional variants contain exon b (Table 1), suggesting that this exon is not the sole determinant of channel expression in Xenopus oocytes, but that other yet-to-be-identified sequence features are also involved. Identification of these other sequence features should improve our understanding of the mechanisms of intramolecular regulation of sodium channel gating. Third, it is also possible that some of these nonfunctional variants could be artifacts of the cloning, such as introduction of stop codon during RT-PCR cloning, or deletion of a stretch of sequence in the coding region. Finally, we used TipE as an auxiliary protein in the expression of DmNa_V variants in Xenopus oocytes following the protocol of Warmke et al. (1997). It is possible that some variants require an auxiliary protein that is different from TipE for functional expression in this system. Recently, four TipE-homologous genes (TEH1-4) were identified in D. melanogaster (Derst et al., 2006). Interestingly, co-expression of three of these genes (TEH1-3) with a DmNa_V variant in Xenopus oocytes elevated sodium current expression (Derst et al., 2006). In future it would be interesting to determine whether functional expression of some “nonfunctional” DmNa_V variants requires a TEH protein, instead of TipE.

4. Sequence analysis of six DmNa_V variants

A comprehensive sequence analysis of the 64 full-length DmNa_V clones and site-directed mutagenesis of variant-specific amino acid changes are eventually required in order to identify those specific amino acid sequences that are responsible for the gating property differences reported in this paper. Furthermore, it would be important to determine whether these specific changes are caused by RNA editing, alternative splicing, or PCR errors introduced during RT-PCR cloning. Toward this goal, we have sequenced five functional variants (DmNa_V5-1 and DmNa_V7-1, DmNa_V1-1, DmNa_V1-6 and DmNa_V19) that represent a wide range of gating properties (Table 2). As mentioned above, DmNa_V5-1 and DmNa_V7-1 are activated at low (i.e., more hyperpolarizing) voltages, DmNa_V1-6 and DmNa_V19 at high (i.e., more depolarizing) voltages, and DmNa_V1-1 at an intermediate voltage (Fig. 2 and Table 1). Because we isolated the full-length clones by RT-PCR, we expected that some of nucleotide changes would result from random PCR errors. However, such random errors are highly unlikely if a nucleotide change occurs in independent clones that belong to different splice types. Based on this criterion, we could attribute a total of nine amino acid changes (in the upper part of Table 2) to A-to-I RNA editing. Whether these changes are the result of RNA editing remain to be determined because some of the sequence differences could be caused by single-nucleotide polymorphism between the strain (unspecified) in the Genbank and strain W¹¹¹⁸ used in this study.

Table 2. Common and unique amino acid changes in five DmNa_V variants.

DmNaV1-1	DmNaV7-1	DmNaV5-1	DmNaV19	DmNaV1-6	n.t. Δ	Location
	R52Q	R52Q	R52Q		g–a*	N term.
E299Q	E299Q	E299Q	E299Q	E299Q	g–c	IS5–S6
Q471R	Q471R				a–g*	L1
		R1296Q	R1296Q		g–a*	L2
		D1300N	D1300N		g–a*	IIIS1
L1363F	L1363F	L1363F	L1363F	L1363F	g–c	IIIS2–S3
I1661V	I1661V	I1661V			a–g	IVS2
	I1670M	I1670M			a–g	IVS2
I1679V		I1679V		I1679V	a–g	IVS3
I1691V	I1691V	I1691V		I1691V	a–g	IVS3
N1822S		N1822S		N1822S	a–g	IVS5–S6
	I260T				t–c	IS4–S5
		I260V			a–g	IS4–S5
	K303R				a–g	IS5–S6
	W319R				t–c	IS5–S6
				R437K	g–a	L1
L486P					t–c	L1
		H487N			c–a	L1
		D656G			a–g	L1
M846V					a–g	IIS1–S2
				A859T	g–a	IIS2
				F885L	t–c	IIS3
			S922P		t–c	IIS4
V1420A					t–c	IIIS4–S5
				V1436A	t–c	IIIS5
			Q1450R		a–g	IIIS5
				N1463Y	a–t	IIIS5–S6
		I1519V			a–g	IIIS5–S6
			S1587N		g–a*	L3
	H1676R				a–g	IVS2–S3
	A1731V				c–t	IVS3
		H1772Y			c–t	IVS5

Note: Amino acid sequences of these variants were compared with the GenBank sequence (Genbank accession number: M32078). Nucleotide bases that correspond to these in the DmNa_V genomic sequence (FlyBase ID FBgn 003036) are in bold. Asterisks (*) represent the RNA editing events identified by Palladino et al. (2000). L1, L2, and L3 represent the three intracellular linkers connecting domains I and II, domains II and III, and domains III and IV, respectively.

Four of the nine A-to-I editing (Table 2) were previously reported in the DmNa_V transcript (Palladino et al., 2000). However, these RNA editing events are detected in both low-voltage-activated and high-voltage-activated variants and are therefore not likely responsible for the unique gating properties of these variants. In addition to the A-to-I editing, E299Q and L1363F were found in all five sequenced variants; both were caused by C to G changes. Finally, at the amino acid position 260, two separate changes, one from I to V and the other from I to T, were found in DmNa_V5-1 and DmNa_V7-1, respectively. Further sequencing of the corresponding regions in other 59 full-length clones reveal one more clone, DmNa_V4-2, which belongs to a different splice type, and also contains an I260 V change. DmNa_V4-2 was not functional in Xenopus oocytes, presumably caused by another unidentified sequence(s) in this variant. The I260 V amino acid change results from an a(tc) to g(tc) nucleotide substitution. Because it is highly unlikely that two DmNa_V cDNA clones from different splice types would have the PCR errors at the same nucleotide, we concluded that the I260 V change in DmNa_V5-1 is caused by RNA editing. Examination of the genomic sequence of the DmNa_V gene revealed the a(tc) codon at the corresponding position. Therefore, the I260 V change is the result of A-to-I editing. Next, we conducted experiments to determine whether the I260 V change in DmNa_V5-1 is responsible for the low-voltage dependence of activation. We substituted V260 with I260 in DmNa_V5-1. Interestingly, the V260I substitution shifted the voltage dependence of activation by 6 mV in the depolarizing direction (Fig. 2D), demonstrating that this A-to-I editing event is responsible for the low-voltage dependence of activation of DmNa_V5-1.

In summary, we report here the functional characterization of the largest collection of insect sodium channel variants to date. Further site-directed mutagenesis experiments are currently underway to identify potentially all amino acid changes/RNA editing events that are responsible for variant-specific gating properties. This work lays a foundation for further studies to understand the role of diverse DmNa_V variants in regulating neuronal excitability in insects.