Molecular diversity of voltage-gated sodium channel α and β subunit mRNAs in human tissues

European Journal of Pharmacology 541 (2006) 9 – 16 www.elsevier.com/locate/ejphar

Molecular diversity of voltage-gated sodium channel α and β subunit mRNAs in human tissues Luz Candenas, Marian Seda, Pedro Noheda, Helmut Buschmann, Cristina G. Cintado, Julio D. Martin, Francisco M. Pinto ⁎ Instituto de Investigaciones Químicas, Centro de Investigaciones Científicas Isla de la Cartuja, 41092 Sevilla, Spain Instituto de Química Orgánica General, CSIC, Juan de la Cierva, 3, 28006 Madrid, Spain Laboratorios del Dr. Esteve, Avda. Mare de Deu de Montserrat 221, 08041 Barcelona, Spain Received 20 January 2006; received in revised form 20 April 2006; accepted 25 April 2006 Available online 3 May 2006

Abstract Voltage-gated Na+ channels are composed of one α subunit and one or more auxiliary β subunits. A reverse transcription–polymerase chain reaction assay was used to analyse the expression of the nine known α subunits (Nav1.1–Nav1.9) in 20 different human tissues. The mRNA expression of the currently known β subunits (β1, β2, β3 and β4) was also assessed. The mRNAs of voltage-gated Na+ channel α and β subunits were found in a wide variety of human tissues assayed and were present in neuronal and non-neuronal types of cells. These data suggest that, in addition to its well-established role in skeletal muscle, cardiac cells and neurons, voltage-gated Na+ channels might play important, still undetermined local roles in the regulation of cellular functions. These channels could emerge in the next future as potential, new therapeutic targets in the treatment of visceral diseases. © 2006 Elsevier B.V. All rights reserved. Keywords: Voltage-gated Na+ channel; α subunit; β subunit; mRNA expression; Peripheral tissue; (Human)

1. Introduction The propagation of electrical signals in excitable cells is mediated through the gating of membrane-associated ion channels. Among them, voltage-dependent Na+ channels play an essential role in the generation of the rapid depolarisation during the initial phase of the action potential (Catterall, 1992; Tamargo et al., 1998). These complex proteins are composed of an α and one or more auxiliary β subunits (Yu and Catterall, 2003; Catterall et al., 2003). Nine different voltage-gated α subunits and a related, non-voltage-gated atypical isoform have been cloned in mammals and each of them is encoded by a different gene. The names of the human genes are shown in Table 1. In humans, the genes that encode the tetrodotoxin-sensitive α

⁎ Corresponding author. Instituto de Investigaciones Químicas, Avda. Americo Vespucio 49, 41092 Sevilla, Spain. Tel.: +34 95 4489567; fax: +34 95 4460565. E-mail address: [email protected] (F.M. Pinto). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.04.025

subunits Nav1.1, Nav1.2, Nav1.3 and Nav1.7 are located on chromosome 2, while Nav1.4 is located on chromosome 17 and Nav1.6 is located on chromosome 12. The atypical α subunit isoform Nax is also located on chromosome 2. The genes that encode the tetrodotoxin-resistant channels Nav1.5, Nav1.8 and Nav1.9 are located on chromosome 3 (Plummer and Meisler, 1999; Goldin et al., 2000; Catterall et al., 2003). The α subunits are large proteins with a high degree of amino acid sequence identity and consist of four homologous domains, each with six transmembrane segments, being intracellular both the N- and the C-termini. This subunit contains the ion-conducting aqueous pore and is able to function by itself as a Na+ channel (Catterall, 1992, 2000). Four different β subunits, named β1, β2, β3 and β4, are currently known (McClatchey et al., 1993; Makita et al., 1994; Eubanks et al., 1997; Morgan et al., 2000; Yu et al., 2003). The human gene that encodes the β1 subunit (SCN1B) is located on chromosome 19 and the genes encoding β2 (SCN2B), β3 (SCN3B) and the recently discovered β4 subunit (SCN4B) are located on chromosome 11 (McClatchey et al., 1993; Yu et al., 2003). The β subunits contain a single transmembrane domain,

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Table 1 Sequences of forward and reverse primers used to amplify voltage-gated Na+ channel α subunits and the size expected for each PCR-amplified product Na+ channel α subunit

Human gene

Forward primer

Reverse primer

Amplicon size (bp)

Nav1.1 Nav1.2 Nav1.3 Nav1.4 Nav1.5 Nav1.6 Nav1.7 Nav1.8 Nav1.9 Nax

SCN1A SCN2A SCN3A SCN4A SCN5A SCN8A SCN9A SCN10A SCN11A SCN7A

5′-GAAGAACAGCCCGTAGTGGAA-3′ 5′-GAAGGCAAAGGGAAACTCTGG-3′ 5′-AAACCCCAACTATGGCTACACAA-3′ 5′-CAACAACCCCTACCTGACCATAC-3′ 5′-CCGCCATTTACACCTTTGAGT-3′ 5′-AAGGTTGTGTCCAGCGGTTC-3′ 5′-GAATAACCCACCGGACTGGA-3′ 5′-CAAGGGCAACCTCAAAAATAAATG-3′ 5′-TGGCAAGAGGTTTCATTCTGG-3′ 5′-TTCAATGCGGCTTCCATCTT-3′

5′-TTCAAATGCCAGAGCACCA-3′ 5′-CAGTGAGACATCAACAATCAGGAAG-3′ 5′-TCCTAACCCACCTATTCCACTGA-3′ 5′-GCAGAGTCCACCACTTCTTCC-3′ 5′-CGCTGAGGCAGAAGACTGTG-3′ 5′-GGATGGTGCGGATGGTCTT-3′ 5′-TGACTGGATCAAAGCCCCTAC-3′ 5′-CCAGGAAGATTACGAGCACAAA-3′ 5′-CAGGGCAAAGATGCTGAGG-3′ 5′-AACCTCAAACACAGTTACGCTGA-3′

225 297 367 317 294 207 289 347 272 327

with a large extracellular N-terminal domain and a smaller intracellular tail (Isom, 2000; Hanlon and Wallace, 2002). The role of the β subunits is less well established, although they appear to modulate the cellular localization, functional expression, kinetics and voltage-dependence of channel gating (Isom, 2000; Hanlon and Wallace, 2002; Wood et al., 2004). For many years, it was considered that voltage-gated Na+ channels were mostly expressed in nerves, skeletal muscle and heart. Most studies have therefore focused on the analysis of channel function in these tissues. These studies have unravelled that changes in the expression and/or function of these channel proteins represent an important step in the development of different pathologies such as familial generalized epilepsy, hyperkalemic periodic paralysis or the Brugada syndrome (Rojas et al., 1991; Wallace et al., 1998; Escayg et al., 2000; Antzelevitch et al., 2003). On the contrary, little is known about the function of voltage-dependent Na+ channels in other tissues in spite of recent evidences suggesting that they could also be present in other types of cells (Mechaly et al., 2005; Saleh et al., 2005). An analysis of the distribution of Na+ channels in human tissues is essential to assess their functional role at central and peripheral levels. The main objective of the present study was to determine the relative mRNA levels of voltage-dependent Na+ channel α and β subunits in different human tissues. For this purpose, and taken into account the importance of the reference gene in semiquantitative gene expression studies, we first used real-time reverse transcription–polymerase chain reaction (RT-PCR) to quantify the expression of four different widely used housekeeping genes in the human tissues assayed.

2. Materials and methods 2.1. Materials The investigation was approved by the Ethic Committee of Consejo Superior de Investigaciones Científicas (CSIC, Spain) and conforms to the principles outlined in the Declaration of Helsinki. A human total RNA master panel (BD Biosciences Clontech, Palo Alto, CA, USA) was used to assess the mRNA expression of voltage-gated Na+ channel α and β subunits in 20 different human tissues. The expression of all the genes was analysed in three different human panels, in which each mRNA is a pool from different individuals. 2.2. Reverse transcription–polymerase chain reaction (RT-PCR) First-strand cDNA was synthesized from 2 μg of each RNA of the human master panel using Moloney murine leukemia virus reverse transcriptase and random hexamers according to the manufacturer's instructions (First-strand cDNA Synthesis Kit, Amersham Biosciences, Essex, UK). The resulting cDNA samples were amplified by PCR with specific oligonucleotide primer pairs designed with the analysis software Primer 3 (Rozen and Skaletsky, 2000). The sequence of the primers used to amplify the different voltage-dependent Na+ channel α and β subunits is shown in Tables 1 and 2. Table 2 also shows the sequence of the oligonucleotide primers used to amplify β-actin (ACTB), protein phosphatase 1 catalytic subunit β-isoform (PPP1CB), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and polymerase

Table 2 Sequences of forward and reverse primers used to amplify voltage-gated Na+ channel β subunits and the size expected for each PCR-amplified product Na+ channel β subunit

Human gene Forward primer

Reverse primer

Amplicon size (bp)

β1 β2 β3 β4

SCN1B SCN2B SCN3B SCN4B

5′-CGTCTACCGCCTGCTCTTCT-3′ 5′-GCCCACCCGACTAACATCTC-3′ 5′-GCAGGAGTGATTAGTTCGGGTTA-3′ 5′-CTGTCGCTGGAGGTGTCTGT-3′

5′-GGTATTCCGAGGCATTCTCCT-3′ 5′-ATGCGGAACTGGAGGAACA-3′ 5′-ACAGGGAGGGCACACATACA-3′ 5′-TGCGGTCATCGTCTTTCAAC-3′

236 285 346 217

Housekeeping genes β-Actin PP1 β-isoform Glyceraldehyde-3-P dehydrogenase Polymerase IIA

ACTB PPP1CB GAPDH POLR2A

5′-TCCCTGGAGAAGAGCTACGA-3′ 5′-AACCATGAGTGTGCTAGCATCA-3′ 5′-CAATGCCTCCTGCACCAC-3′ 5′-ACATCACTCGCCTCTTCTACTCC-3′

5′-ATCTGCTGGAAGGTGGACAG-3′ 5′-CACCAGCATTGTCAAACTCGCC-3′ 5′-CCTGCTTCACCACCTTCTTG-3′ 5′-GTCTTGTCTCGGGCATCGT-3′

362 472 350 268

Primers for the assayed housekeeping genes are also shown.

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(RNA) II (DNA directed) polypeptide A (POLR2A), which were assayed as housekeeping genes (Cintado et al., 2001; Patak et al., 2003; Radonic et al., 2004). All primers were synthesized and purified by Sigma-Genosys (Cambridge, UK). At the beginning of the experiments, the expression of the housekeeping genes was quantified in all cDNAs by real-time PCR using the iCycler iQ real-time detection apparatus (BioRad Laboratories, Hercules, CA, USA) as previously described (Patak et al., 2003). Three serial dilutions of the cDNA template were prepared from each tissue and each dilution was amplified in triplicate. The PCR reaction mixture contained 0.2 μM primers, 1.5 U of JumpStart Taq DNA polymerase (Sigma Chemical Corporation, MO, USA), the buffer supplied, 2.5 mM MgCl2; 200 μM dNTP and cDNA in 25 μl. The PCR buffer also contained SYBR green I (Molecular Probes, Leiden, The Netherlands; 1:75,000 dilution of the 10,000× stock solution) and fluorescein (diluted 1:100,000) used as a reference dye to normalize any signal fluctuation in the reactions. Control samples without the reverse transcription step and no added RNA were also included in each plate to detect any possible contamination. After a hot start (3 min at 94 °C), the parameters used for PCR amplification were: 10 s at 94 °C, 20 s at 60 °C and 30 s at 72 °C, for 45 cycles. Fluorescence was measured during each extension step. The iCycler software calculated the threshold cycle (CT), defined as the fractional cycle number at which fluorescence reaches 10× the standard deviation of the baseline. Real-time PCR data obtained in one of the kidney samples were arbitrarily chosen as control and this sample was included in all PCR experiments to correct for possible interassay variations. mRNA

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values for each of the four assayed housekeeping genes in the other tissues were then normalized by the levels of ACTB, PPP1CB, GAPDH or POLR2A in the control kidney sample by using the formula: Fold change ¼ 2−DDCT where ΔCT = CTtarget gene (ACTB, GAPDH, PPP1CB or POLR2A) − CThousekeeping gene (ACTB, GAPDH, PPP1CB or POLR2A) and ΔΔCT = ΔCTtest sample − ΔCTcontrol kidney sample. The experimental approach was further validated by the observation that the differences between the CT value for the considered target and reference genes remained essentially constant for each starting cDNA amount. For each gene assayed, we calculated the CT range defined as the difference between the lowest mRNA transcription level (highest CT value) and the highest mRNA transcription level (lowest CT value) in all tissues, as described by Radonic et al. (2004). From these experiments, POLR2A, ACTB and GAPDH gene transcripts were chosen as reference genes and used to normalize and control the efficiency of RT-PCR reactions among the samples. Equal amounts of templates were then amplified by PCR using a DNA thermal cycler (MJ Research, Watertown, USA) and specific primer pairs for each Na+ channel α and β subunits, POLR2A, β-actin and GAPDH. The PCR reaction mixture was identical to that described for real-time RT-PCR excluding SYBR green and fluorescein. Cycle numbers were 35 for Na+ channel α and β subunits, 32 for POLR2A and 26 for βactin and GAPDH. Serial half-dilutions of cDNA were amplified at the indicated number of cycles for each of the

Fig. 1. Real-time RT-PCR quantification of mRNA levels of (A) β-actin, (B) PPP1CB, (C) GAPDH and (D) POLR2A in different human tissues. The plots depict the mRNA/mRNA ratios of the corresponding gene in which the ratio obtained in a human kidney sample was designated as 1. Values are means of three independent experiments, with each experiment carried out in triplicate. S.E.M. is shown by vertical bars. ⁎P b 0.05, significant difference versus mRNA levels of the gene considered in at least 15 tissues. δP b 0.05, significant difference versus mRNA levels of the gene considered in 6–14 tissues, one-way ANOVA.

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amplification products to ensure analysis in the linear range of amplification (Pinto et al., 1999). The PCR products were separated by gel electrophoresis, stained with ethidium bromide, and visualized and photographed under UV transiluminator (Spectronics Corp., New York, USA). Each PCR experiment, with the cDNA from each tissue, was carried out in triplicate and controls containing no reverse transcriptase and no template were included. Amplicon sizes were verified by comparison with a DNA mass ladder and the identity of each PCR product was established by DNA sequence analysis as previously described (Pinto et al., 1999). 2.3. Statistical analysis Quantitative real-time RT-PCR values are means of experiments in three different panels and the value from each panel is the mean of three replicates. Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. A P-value b 0.05 was considered significant. 3. Results 3.1. Expression of housekeeping genes Real-time RT-PCR was used to quantify the expression of four widely used housekeeping genes on a same amount of RNA from each human tissue assayed. Fig. 1 shows the mRNA transcription levels of β-actin, PPP1CB, GAPDH or POLR2A relative to its own expression in a sample of human kidney that was arbitrarily chosen as control. The mRNA levels of all housekeeping genes assayed varied among tissues. The CT range, which gives a good idea of mRNA level variation among tissues, was 5.70 for β-actin, 4.13 for PPP1CB, 5.73 for GAPDH and 3.57 for POLR2A. According with recent findings by Radonic et al. (2004), POLR2A showed the lowest CT range and the most stable expression between tissues, with similar mRNA levels in 17 of the 20 tissues assayed (Fig. 1D). A variation in POLR2A mRNA transcription was only observed in

Fig. 3. Agarose gel showing expression of voltage-gated Na+ channel α subunits in different human tissues. Serial half dilutions of each cDNA were normalized by POLR2A, β-actin and GAPDH expression levels and amplified by PCR for 35 cycles with primers specific for each target gene. The figure shows the results obtained in the most concentrated aliquot. M, molecular weight standards.

testis, trachea and prostate (Fig. 1D). When POLR2A was used as the reference gene, i.e., when mRNA expression levels of βactin (Fig. 2), PPP1CB and GAPDH (not shown) were calculated relative to POLR2A mRNA, it allowed an accurate normalization of mRNA transcription levels in human tissues, with the only exception of the testis, trachea and prostate (see Fig. 2). From these experiments, POLR2A was chosen as reference gene and used in subsequent experiments to control the efficiency of RT-PCR reactions among the samples. Two additional reference genes, β-actin, and GAPDH, were used for normalization of RT-PCR data, particularly in the testis, the trachea and the prostate. 3.2. Expression of voltage-gated Na+ channel α and β subunits By using an end-point RT-PCR assay, we observed the presence of single transcripts corresponding to the sizes predicted

Fig. 2. Real-time RT-PCR quantification of β-actin mRNA levels in different human tissues. The relative levels of β-actin mRNA were normalized by the levels of βactin, PPP1CB, GAPDH or POLR2A transcripts. The plots depict the mRNA/ β-actin, PPP1CB, GAPDH or POLR2A mRNA ratios in which the respective ratios obtained in a sample of human kidney was designated as 1. Values are means of three independent experiments, with each experiment carried out in triplicate. S.E.M. is shown by vertical bars.

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Fig. 4. Agarose gel showing expression of voltage-gated Na+ channel β subunits in different human tissues. Serial half dilutions of each cDNA were normalized by POLR2A, β-actin and GAPDH expression levels, and amplified by PCR for 35 cycles with primers specific for each target gene. The figure shows the results obtained in the most concentrated aliquot. M, molecular weight standards.

for Na+ channel α and β subunits (Fig. 3). Among the tetrodotoxin-sensitive α subunits, Nav1.1 was the only one whose expression was restricted to the brain, in our experimental conditions and in the equal amounts of human cDNAs assayed. Nav1.1 mRNA levels were higher in the adult whole brain than in the fetal whole brain and the adult cerebellum. Nav1.2 mRNAwas strongly expressed in both adult and fetal whole brain and also in the adult cerebellum. Compared to its expression in the brain, this Na+ channel α subunit was present in lower amounts in the testis, kidney and adrenal gland, and a minor expression was detected in the uterus. The mRNA of Nav1.3 was present in considerable amounts in most tissues assayed, with the exception of the adult and fetal liver, heart, bone marrow and salivary gland, where its expression was undetectable. As expected, the highest levels of Nav1.4 mRNA were found in the skeletal muscle. However, this α subunit was detected in virtually all tissues assayed with the exception of the adult liver. In comparison with its mRNA transcription in skeletal muscle, the SCN4A gene was expressed in high levels in the testis, thyroid gland and adrenal gland. The lowest mRNA levels were observed in the lung and uterus. The mRNA of Nav1.6 was widely distributed and expressed in all tissues assayed. The highest expression was detected in the adult and fetal whole brain, the adult cerebellum and the testis and the lowest expression was found in the lung, uterus and bone marrow. The SCN9A gene, which encodes the α subunit Nav1.7, was also present in most tissues assayed. The highest expression was found in the testis, the adult and fetal whole brain, and the placenta. In spite of high expression in the whole brain, Nav1.7 mRNA was undetectable in the cerebellum. Among the tetrodotoxin-insensitive α subunits, Nav1.5 mRNA was detected in most tissues assayed and showed the strongest expression in the heart, according with its consideration as the cardiac Na+ channel. Compared to its mRNA transcription in the heart, the SCN5A gene was expressed in high levels in the adult and fetal brain, the testis and the prostate and in moderate levels in salivary, adrenal and thyroid glands, skeletal muscle, lung, trachea, placenta and uterus. Interestingly, this Na+ channel showed a significant expression in the fetal liver but was undetectable in the adult liver (see Fig. 3), even after amplification of higher amounts of cDNA (data not shown). It is also interesting to note that the SCN5A gene was present in low levels in the

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cerebellum, in comparison with its high expression in the adult and fetal whole brain. In our experimental conditions, the Nav1.8 transcript was undetectable in 19 of the human tissues assayed and a minor expression was only observed in the testis. On the contrary, the mRNA of Nav1.9 was widely distributed and was detected in most tissues, with the exception of salivary gland and bone marrow. The Na+ channel-related isoform Nax was also expressed in many human tissues. Its highest mRNA levels were found in skeletal muscle, lung, trachea, uterus, placenta, testis, prostate, salivary and adrenal gland. The Nax mRNA was undetectable in the fetal and adult liver, bone marrow, cerebellum and spleen. Among the voltage-gated Na+ channel auxiliary β subunits, the mRNA of SCN1B and SCN4B genes, that encode β1 and β4 subunits, respectively, were detected in all (β1) or most (β4) tissues assayed (Fig. 4). Conversely, in our experimental conditions, the mRNAs of β2 and β3 subunits were only detectable in several human tissues (Fig. 4). The SCN2B gene was found in high amounts in the adult whole brain, adult cerebellum and skeletal muscle and in moderate levels in fetal brain and heart. A lower expression was observed in the lung, thyroid gland, prostate and trachea. The highest expression levels of SCN3B were detected in the adult and fetal whole brain. A lower expression was observed in adult cerebellum, skeletal muscle, testis, adrenal and thyroid glands. 4. Discussion The present study shows that mRNAs of voltage-gated Na+ channel α and β subunits are abundantly expressed in different human tissues. This strongly argues for new, important roles of these channels in the regulation of cellular functions at both central and peripheral levels. The appropriate choice of the reference gene is an essential step in semiquantitative gene expression analysis. Real-time RT-PCR, the most accurate actual method for quantification of mRNA transcription levels, was initially used to quantify the expression of four different housekeeping genes in all cDNAs assayed. Previous results from other laboratories and from ours have shown that β-actin is a useful housekeeping gene when comparing mRNA levels within a particular tissue (rat uterus, Pinto et al., 2001; Kamata et al., 2005; rat hippocampal neurons and utricular hair cells, Mechaly et al., 2005; human uterus, Patak et al., 2003; human lymphocytes, Ilani et al., 2001). Conversely, β-actin mRNA levels showed considerable variations among different human tissues (Vandesompele et al., 2002; Radonic et al., 2004; this study). Similar or even higher variations of mRNA transcription levels between tissues were found for PPP1CB and GAPDH. This suggests that the use of β-actin, PPP1CB or GAPDH as a single reference gene is not adequate in the present study. According with recent findings by Radonic et al. (2004), POLR2A showed the most stable expression between tissues. POLR2A mRNA levels were similar in 17 of the 20 tissues assayed and, therefore, it was an accurate housekeeping gene in these tissues. It should however be noted that, in comparison with the other tissues,

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POLR2A levels varied considerably in testis, trachea and prostate and would produce wrong results if chosen as internal control in these tissues. The mRNA levels of β-actin in the testis, and GAPDH in the prostate and trachea, were more stable and each of them would be a better reference gene than POLR2A in this particular case. These data provide evidence of the absolute need of using more than a single housekeeping gene to obtain reliable results in quantitative or semiquantitative gene expression studies (Pinto et al., 1999; Cintado et al., 2001; Radonic et al., 2004). The present study shows that voltage-gated Na+ channels are widely distributed in central and peripheral, human tissues. Similar observations were made in different tissues from cynomolgus monkeys by using DNA microarrays (Raymond et al., 2004). With some exceptions, such as the placenta, which is devoid of nerves (Page et al., 2000; Lecci and Maggi, 2003), many peripheral tissues receive an important innervation. Most Na+ channel α and β subunits are abundantly expressed in nerves where they play an essential role in membrane excitability (Yu and Catterall, 2003). In neurons, translation occurs mainly in the cell soma and the new synthesised proteins are transported through the axon to the nerve terminals. The Na+ channel proteins can be detected in nerve terminals by immunochemistry (Akopian et al., 1996; Caldwell et al., 2000; Wood et al., 2004). Conversely, their precursor mRNAs are present in small amounts in nerve endings and cannot be easily detected by RT-PCR. This suggests that Na+ channel α and β subunit mRNAs presently found in human peripheral tissues might be mainly located in cells of non-neuronal origin. Nevertheless, the existence of dendritic transport of certain RNAs has been demonstrated in neurons (Kohrmann et al., 1999) and the possibility that α and β subunit mRNAs could be present, at least in part, in peripheral ganglia or intrinsic nerves cannot be excluded. Nav1.1, Nav1.2 and Nav1.3 were cloned in the brain and were originally named brain Na+ channel types I, II and III, respectively (Noda et al., 1986; Beckh et al., 1989). Nav1.7 is, evolutionarily, closely related to them and shares many similarities with Nav1.1, Nav1.2 and Nav1.3 in its biophysical properties, sensitivity to tetrodotoxin and broad expression in neurons. It is particularly abundant in the peripheral nervous system (Toledo-Aral et al., 1997; Catterall et al., 2003) where it plays an important role in acute and inflammatory pain (Nassar et al., 2004; Wood et al., 2004). In our experimental conditions, two of these channels (Nav1.1 and Nav1.2) were detected almost exclusively in the brain. Nav1.3 and Nav1.7 mRNAs were expressed in the human brain but were also detected, although in lower levels, in many tissues assayed (Chen et al., 2000; Mechaly et al., 2005; this study). Interestingly, the SCN2A and SCN3A genes were expressed in high amounts in both adult and fetal whole brain and also in the cerebellum. The SCN9A mRNA transcript was expressed in high amounts in the adult and fetal brain but was undetectable in the cerebellum. SCN1A was highly expressed in the adult whole brain, but its expression in the fetal brain and in the adult cerebellum was lower. These findings show that these four α subunits are differentially expressed in distinct regions of the brain and during development (Beckh et al., 1989; this study).

Nav1.6 is similar in its biophysical properties to Nav1.1, Nav1.2 and Nav1.3 channels and as them, is broadly distributed in neurons (Burbidge et al., 2002). This isoform appears to play an essential role in peripheral nerve conduction at both central and peripheral nervous systems (Tzoumaka et al., 2000; Caldwell et al., 2000; Schaller and Caldwell, 2003). In agreement with previous reports (Schaller et al., 1995; Burbidge et al., 2002), we found that Nav1.6 was abundantly expressed in adult and fetal brain with high levels being observed in the cerebellum. In addition, the Nav1.6 transcript was ubiquitously expressed and appeared in all tissues assayed suggesting that it might play a role in modulating local functions in many human tissues. The vital importance of this α subunit was revealed by studies in mice showing that allelic mutation of the Scn8a gene caused alterations in multiple cellular and physiological functions (Meisler et al., 2004), while null mutations produced juvenile lethality (Burgess et al., 1995). Nav1.4 and Nav1.5 are primarily expressed in muscle (George et al., 1992b; Gellens et al., 1992; Goldin et al., 2000). The Nav1.4 channel was previously known as skeletal muscle voltage-gated Na+ channel and is expressed in high levels in this tissue where it mediates action potential initiation and transmission (George et al., 1992b). The “cardiac” Na+ channel, Nav1.5, was strongly expressed in the heart (Gellens et al., 1992). Additionally, our data show that the SCN4A and SCN5A gene transcripts are broadly distributed in most human tissues assayed. These two α subunits might thus participate in the regulation of cellular Na+ homeostasis in a wide variety of human cells. The tetrodotoxin-resistant α subunits Nav1.8 and Nav1.9 were cloned in DRG sensory neurons and appear to play a key role in sensory transmission and pain perception (Akopian et al., 1996; Dib-Hajj et al., 1999; Wood et al., 2004). In our study, the Nav1.8 transcript was undetectable in 19 tissues with only a minor expression being observed in the testis. This is consistent with the concept that Nav1.8 is selectively expressed in C-fibre and Aδ fibre-associated sensory neurons (Wood et al., 2004). Conversely, Nav1.9 mRNA was detected in most human tissues assayed, including non-innervated tissues, such as the placenta. This channel may be primarily expressed in non-neuronal cells in the human periphery and, besides its well established key role in nociception, could play additional, still undefined functions at the peripheral level. The atypical Na+ channel-like isoform Nax was primarily expressed in peripheral tissues and its expression in the CNS was low and probably restricted to specific brain nuclei (George et al., 1992a, Akopian et al., 1997; Watanabe et al., 2000; this study). Studies in Nax knockout mice have shown that it participates in the regulation of salt intake (Watanabe et al., 2000) although its precise functional properties remain unknown. In addition to the α subunit, one or two β subunits form part of the primary structure of most voltage-gated Na+ channels (Catterall et al., 2003). The mRNAs of two of the four known β subunits, β1 and β4, were highly expressed in most human tissues assayed. Moreover, our data do not exclude the possibility that any of the Nav α or β subunits could have a wider expression and be detected if using other experimental

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conditions or higher amounts of cDNA from each tissue (i.e., for the β3 subunit, see Morgan et al., 2000; Stevens et al., 2001). Taken together, these findings strongly argue for an important physiological role of Na+ channels in many types of human cells. Worthy of mention is the high expression of Nav channels in the testis. With the exception of Nav1.1, all voltage-gated Na+ channel α and β subunits are expressed, in different levels, in the human testis supporting a role for these channels in the regulation of male reproductive function. In conclusion, the present study shows that mRNAs of voltage-dependent Na+ channel α and β subunits are widely distributed in central and peripheral human tissues. The differential gene expression profile is consistent with a distinct role of each Nav channel in the regulation of cellular functions and supports the notion that they could behave as important therapeutic targets in the treatment of visceral diseases. The physiological significance of these channels, particularly in nonneuronal cells at the peripheral level, merits further investigation. Acknowledgement This work was supported by Laboratorios del Dr. Esteve (Barcelona, Spain). M. Seda is the recipient of a fellowship from Laboratorios del Dr. Esteve. References Akopian, A.N., Sivilotti, L., Wood, J.N., 1996. A tetrodotoxin-resistant voltagegated sodium channel expressed by sensory neurons. Nature 379, 257–262. Akopian, A.N., Souslova, V., Sivilotti, L., Wood, J.N., 1997. Structure and distribution of a broadly expressed atypical sodium channel. FEBS Lett. 400, 183–187. Antzelevitch, C., Brugada, P., Brugada, J., Brugada, R., Towbin, J.A., Nademanee, K., 2003. Brugada syndrome: 1992–2002 a historical perspective. J. Am. Coll. Cardiol. 41, 1665–1671. Beckh, S., Noda, M., Lubbert, H., Numa, S., 1989. Differential regulation of three sodium channel messenger RNAs in the rat central nervous system during development. EMBO J. 8, 3611–3616. Burbidge, S.A., Dale, T.J., Powell, A.J., Whitaker, W.R., Xie, X.M., Romanos, M.A., Clare, J.J., 2002. Molecular cloning, distribution and functional analysis of the NA(V)1.6.Voltage-gated sodium channel from human brain. Brain Res. Mol. Brain Res. 103, 80–90. Burgess, D.L., Kohrman, D.C., Galt, J., Plummer, N.W., Jones, J.M., Spear, B., Meisler, M.H., 1995. Mutation of a new sodium channel gene, Scn8a, in the mouse mutant 'motor endplate disease'. Nat. Genet. 10, 461–465. Caldwell, J.H., Schaller, K.L., Lasher, R.S., Peles, E., Levinson, S.R., 2000. Sodium channel Na(v)1.6 is localized at nodes of ranvier, dendrites, and synapses. Proc. Natl. Acad. Sci. U. S. A. 97, 5616–5620. Catterall, W.A., 1992. Cellular and molecular biology of voltage-gated sodium channels. Physiol. Rev. 72, S15–S48. Catterall, W.A., 2000. From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26, 13–25. Catterall, W.A., Goldin, A.L., Waxman, S.G., 2003. International Union of Pharmacology. XXXIX. Compendium of voltage-gated ion channels: sodium channels. Pharmacol. Rev. 55, 575–578. Chen, Y.H., Dale, T.J., Romanos, M.A., Whitaker, W.R., Xie, X.M., Clare, J.J., 2000. Cloning, distribution and functional analysis of the type III sodium channel from human brain. Eur. J. Neurosci. 12, 4281–4289. Cintado, C.G., Pinto, F.M., Devillier, P., Merida, A., Candenas, M.L., 2001. Increase in neurokinin B expression and in tachykinin NK (3) receptormediated response and expression in the rat uterus with age. J. Pharmacol. Exp. Ther. 299, 934–938.

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