J Biol Chem, Vol. 275, Issue 2, 1079-1088, January 14, 2000

Cloning, Localization, and Functional Expression of Sodium Channel 1A Subunits^*

Kristin A. Kazen-Gillespie, David S. Ragsdale^§, Michael R. D'Andrea^¶, Laura N. Mattei, Kathryn E. Rogers^¶, and Lori L. Isom

From the  Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-0632, ^§ Montreal Neurological Institute, McGill University, Montreal, Quebec H3A2B4, Canada, and ^¶ The R. W. Johnson Pharmaceutical Research Institute, Springhouse, Pennsylvania 19477-0776

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Auxiliary 1 subunits of voltage-gated sodium channels have been shown to be cell adhesion molecules of the Ig superfamily. Co-expression of  and 1 subunits modulates channel gating as well as plasma membrane expression levels. We have cloned, sequenced, and expressed a splice variant of 1, termed 1A, that results from an apparent intron retention event. 1 and 1A are structurally homologous proteins with type I membrane topology; however, they contain little to no amino acid homology beyond the shared Ig loop region. 1A mRNA expression is developmentally regulated in rat brain such that it is complementary to 1. 1A mRNA is expressed during embryonic development, and then its expression becomes undetectable after birth, concomitant with the onset of 1 expression. In contrast, 1A mRNA is expressed in adult adrenal gland and heart. Western blot analysis revealed 1A protein expression in heart, skeletal muscle, and adrenal gland but not in adult brain or spinal cord. Immunocytochemical analysis of 1A expression revealed selective expression in brain and spinal cord neurons, with high expression in heart and all dorsal root ganglia neurons. Co-expression of IIA and 1A subunits in Chinese hamster lung 1610 cells results in a 2.5-fold increase in sodium current density compared with cells expressing IIA alone. This increase in current density reflected two effects of 1A: 1) an increase in the proportion of cells expressing detectable sodium currents and 2) an increase in the level of functional sodium channels in expressing cells. [³H]Saxitoxin binding analysis revealed a 4-fold increase in B_max with no change in K_D in cells coexpressing IIA and 1A compared with cells expressing IIA alone. 1A-expressing cell lines also revealed subtle differences in sodium channel activation and inactivation. These effects of 1A subunits on sodium channel function may be physiologically important events in the development of excitable cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sodium channels isolated from brain are composed of a central pore-forming  subunit and two auxiliary subunits, 1 and 2, which do not form the pore yet play critical roles in channel modulation and expression. A mutation in the 1 gene (SCN1B) has been implicated to play a role in febrile seizures and generalized epilepsy, GEFS+ (1). The primary structure of the 1 subunit predicts an integral membrane glycoprotein with type I transmembrane topology as well as an extracellular Ig fold (2, 3). ₁ subunits can be classified as members of the V-set of the Ig superfamily, which includes many cell adhesion molecules. 1 and  subunit co-expression has been well characterized in Xenopus oocytes and in mammalian cells. In oocytes, co-expression of type IIA (SCN2A) or µI (SCN4A)  subunits with 1 increases the proportion of sodium channels that function in a fast gating mode, accelerates the macroscopic rates of activation and inactivation, shifts the voltage dependence of inactivation in the hyperpolarizing direction, and increases the peak current amplitude consistent with increases in channel expression (4-9). In Chinese hamster lung (CHL)¹ cells, stable coexpression of 1 with IIA results in increased channel expression levels at the plasma membrane as well as moderate hyperpolarizing shifts in the voltage dependence of channel activation and inactivation (10).

1 mRNA is expressed only after birth in the developing brain (5, 11). However, previous studies showing the developmental time course of 1 protein expression in rat forebrain suggested that multiple 1 subunit isoforms may be present (12). A 26-kDa 1-immunoreactive protein was observed at embryonic day 18. This protein was also expressed in adult adrenal gland, heart, skeletal muscle, and PC12 cells. After birth, there was a dramatic decrease in the level of this protein in brain, and little if any remained by postnatal day 14. The expression time course of this immunoreactive protein was complementary to that of 1 mRNA. Day 18 embryonic brain membranes also exhibited a low level of an immunoreactive peptide that migrated with an apparent molecular mass greater than 42 kDa. This protein was not detected in rat brain after birth. Other excitable tissues expressed multiple size forms of immunoreactive 1-like subunits as well. Adult rat heart and skeletal muscle membrane preparations exhibited 38- and 41-kDa bands on Western blots in addition to the 26-kDa band. The 41-kDa immunoreactive band observed in these studies was shown to be the adult rat brain isoform and was later identified as C1Aa.1 (4). The immunoreactive peptides identified in the previous study were detected with a polyclonal antibody raised against purified 1 subunits; thus, they could represent 1 subunit isoforms that contained significantly different mRNA sequences from C1Aa.1 that may not have been detected using previous methods.

To test this hypothesis, we screened a rat adrenal cDNA library with a probe encoding only the coding region of 1. The present study reports the molecular cloning and functional expression of 1A, a splice variant of the 1 gene that contains identical amino-terminal and extracellular Ig fold regions as 1 followed by a significantly different extracellular juxtamembrane domain, predicted transmembrane region, and predicted intracellular COOH-terminal domain. 1A mRNA is expressed early in embryonic brain development and then disappears after birth. Western blot analysis of membrane preparations using an antibody to a unique, extracellular region of 1A not found in 1 showed that 1A protein is expressed in adult rat heart, skeletal muscle, and adrenal gland but was not detected in adult rat brain or spinal cord. Immunocytochemical analysis of 1A expression in adult rat tissues revealed high expression in heart and dorsal root ganglion and selective expression in some areas of the brain and spinal cord. 1A functions to increase channel expression at the plasma membrane when coexpressed with IIA subunits in CHL fibroblasts. Unlike 1, however, mean steady state inactivation curves for 1A-expressing cell lines were shifted to more positive potentials than the mean inactivation curves for cells expressing  alone. Previous studies showed that coexpression of  and 1 subunits in CHL cells shifted the voltage dependence of inactivation to more negative potentials compared with  alone (10). Therefore, the novel, carboxyl-terminal domains of 1A may be important for electrophysiological function. It has been shown previously that the extracellular domain of 1 is essential for expression and function of the 1 complex in Xenopus oocytes (13, 14). We propose that the extracellular Ig fold, common to 1 and 1A, is essential for the observed increases in channel expression levels. Thus, this report introduces a novel splice variant of 1, 1A, and adds to our understanding of 1 structure-function relationships in terms of channel expression levels and electrophysiology.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Library Screening-- A cDNA probe encoding nucleotides 345-911 of p1.C1Aa (4) was labeled with digoxigenin following the manufacturer's instructions (Roche Molecular Biochemicals) and used to screen a  Zap Express rat adrenal cDNA library prepared by Stratagene (La Jolla, CA). pBK plasmids containing cDNA inserts that hybridized strongly to the probe were rescued from the  phage according to the manufacturer's instructions, confirmed by Southern blot analysis, and sequenced using ThermoSequenase (Amersham Pharmacia Biotech).

Reverse Transcriptase-PCR from Rat Adrenal RNA-- To confirm independently that the 1A transcript identified by library screening was expressed by rat adrenal gland, we amplified a region of 1A from the amino terminus past the region in which the amino acid sequence changed from identity to nonidentity to 1, or the putative splice site, by reverse transcriptase-PCR using adult rat adrenal gland total RNA as template and 1A-3 (5'-GAAGATGAGCGCTTTGAGG-3', primer sequence common to 1 and 1A) and 1A-5 (5'-GAGAGACACAGCAAGC, primer sequence unique to 1A) as oligonucleotide forward and reverse primers, respectively. Rat adrenal gland cDNA was synthesized from total RNA using Superscript II (Life Technologies, Inc.) according to the manufacturer's instructions in a total volume of 20 µl. 2.3 µg of total rat adrenal RNA (purified using Trizol reagent; Life Technologies) was used in the reaction. The PCR conditions were as follows: 1 µl of cDNA, 0.5 µM concentration of each primer, 200 µM concentration of each dNTP (Roche Molecular Biochemicals), 5 µl of Mg²⁺-free 10× PCR buffer (Perkin-Elmer), and 1.5 mM MgCl₂ were mixed in a total volume of 50 µl. Following a hot start at 94 °C, 0.25 µl of AmpliTaq DNA polymerase (Perkin Elmer) were added to the reaction tube, and the amplification cycle was started. The cycling parameters were as follows: 40 cycles of 45 s at 94 °C, 20 s at 60 °C, 90 s at 72 °C. This was followed by 10 min at 72 °C and then 4 °C until the tubes were removed from the thermocycler (GeneAmp 2400, Perkin-Elmer). Analysis of the PCR products on a 1% agarose gel revealed a 750-base pair band (data not shown). The band was excised form the gel, subcloned into pCR2.1 (Invitrogen, Carlsbad, CA), and analyzed using ThermoSequenase (Amersham Pharmacia Biotech). The sequence obtained from this PCR clone was identical to that obtained from the original 1A clone plaque-purified from the adrenal cDNA library.

Rat 1 Gene-- Intron 3 of the rat 1 gene (15) was amplified by PCR using rat genomic DNA as the template; oligonucleotides that encode 1 coding sequence flanking intron 3, VVDK (5'-AGATCCACCTGGAGGTGGTGGACAAGG-3') and ANRD (5'-ACACGATGGATGCCATATCTCTGTTGG-3'), as forward and reverse primer, respectively; and the Expand Long Template PCR System (Roche Molecular Biochemicals). All oligonucleotide primers were synthesized by Life Technologies. The amplification conditions were as follows. 300 ng of rat genomic DNA (CLONTECH Laboratories, Inc., Palo Alto, CA), 250 ng of each primer, 1 mM concentration of each dNTP (Roche Molecular Biochemicals), and 5 µl of Expand Buffer 3 were mixed in a total reaction volume of 25 µl. Following a hot start at 95 °C, 0.5 µl of Expand DNA polymerase were added, and the amplification cycle was started. 40 cycles of the following regimen were performed: 94 °C for 10 s, 58 °C for 30 s, and 68 °C for 4 min plus 20 s added to each successive cycle. The samples were then held at 4 °C until removal from the thermocycler (GeneAmp 2400, Perkin-Elmer). The 5-kilobase pair PCR product (data not shown) was gel-purified and sequenced directly using oligonucleotide VVDK as the sequencing primer.

RNase Protection Analysis-- A plasmid containing a RNase protection probe template (pRPA-1) was constructed corresponding to nucleotides 364-533 in the 1A sequence. Briefly, a 169-nucleotide AluI/AccI fragment was excised from pBK.1A and ligated into the SmaI and AccI sites of pBluescript (Stratagene). The resulting plasmid was then sequenced using ThermoSequenase. To synthesize labeled cRNA, a 10-µg aliquot of pRPA-1 was linearized with XhoI, ethanol-precipitated, resuspended in RNase-free water, and labeled with digoxigenin using the T3 MAXIscript kit (Ambion, Austin, TX) according to the manufacturer's instructions. Following a 2-h incubation at 37 °C, the reaction was incubated at 95 °C for 2 min, chilled on ice, and then treated with RNase-free DNase (2 units) for 15 min at 37 °C. EDTA (final concentration 30 mM) was added to stop the reaction. Free nucleotides were removed by ethanol precipitation with 0.5 M ammonium acetate, and the final pellet was resuspended in 20 µl of RNase-free water. The probe (RPA-1) was quantitated by comparison of serial dilutions of the labeled probe with serial dilutions of control digoxigenin-labeled RNA following the manufacturer's instructions. Typical labeled probed concentrations were 10 ng/µl throughout our experiments.

RNase protection experiments were performed using the HybSpeed RPA kit (Ambion). Briefly, 20 µg of rat embryonic day 18 brain RNA were mixed with 1 µl of digoxigenin-labeled RPA-1 probe and 30 µg of yeast tRNA in 0.5 M ammonium acetate plus 2.5 volumes of ethanol. The reaction tubes were left at 20 °C for 15 min, and the RNA was precipitated by centrifugation in a microcentrifuge at top speed. The RNA was resuspended in 10 µl of HybSpeed hybridization buffer that had been preheated to 95 °C and vortexed vigorously, and the tubes were placed at 95 °C for 3 min. The samples were then hybridized for 10 min at 68 °C and digested with a mixture of RNase A and T1 (10 units/ml RNase A and 400 units/ml RNase T1) for 30 min at 37 °C. 150 µl of HybSpeed Inactivation/Precipitation mix were added to each reaction, and the RNA was precipitated and resuspended in 10 µl of gel loading buffer 1. The reactions were separated on a 1.5-mm-thick 6% acrylamide TBE denaturing gel containing 7 M urea in the Mini-Protean gel format (Bio-Rad), transferred to nylon (Roche Molecular Biochemicals), and UV-cross-linked using a Stratalinker (Stratagene). Hybridization of the digoxigenin-labeled probe was detected with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (1:10,000 dilution) and CSPD chemiluminescent substrate solution (Roche Molecular Biochemicals) according to the manufacturer's instructions.

Preparation of RNA and Northern Blot Analysis-- Time-mated pregnant female Harlan Sprague Dawley rats were anesthetized with 60 mg/kg Beuthanasia-D intraperitoneal (Schering-Plow Animal Health Corp., Kenilworth, NJ), and the fetuses were surgically removed. Embryonic day 9 rats were homogenized in their entirety in Trizol reagent (Life Technologies) to purify total RNA according to the manufacturer's instructions. Whole fetal brains were dissected at the remaining embryonic time points, and total RNA was purified using Trizol reagent. RNA was also subsequently prepared from the brains and adrenal glands of the adult female rats. Postnatal rats at the indicated ages were anesthetized with Beuthanasia-D, brains were dissected, and total RNA was purified with Trizol reagent. Northern blot analysis of 20 µg of each sample was performed as described previously using a digoxigenin-labeled 1A antisense cRNA probe encoding nucleotides 428-850 or a digoxigenin-labeled antisense cRNA probe specific to the 3'-untranslated region of 1 (nucleotides 1053-1508 of p1.C1Aa (4).

Construction of 1A Expression Vector-- A plasmid containing 1 cDNA including an in-frame amino-terminal hemagglutinin (HA) epitope tag was obtained as a generous gift from the laboratory of R. A. Maue (Dartmouth University) (16). This construct has been shown to express functional 1 subunits in Xenopus oocytes. The HA-tagged 1 cDNA was recloned into the EcoRI and NotI sites of the mammalian expression vector pCIneo (Promega, Madison, WI) to create pCI.1-HA. pCI.1-HA was subsequently digested with AccI and NotI and agarose gel-purified to remove the 3' end of 1. The AccI restriction endonuclease site is common to 1 and 1A. pBK.1A cDNA was digested with AccI and NotI and gel-purified. The 3' end of 1A was then ligated into AccI/NotI-digested pCI.1-HA to create pCI.1A-HA. The junctions were then sequenced to confirm that the segments of 1 and 1A were successfully ligated in frame.

Transfection of SNaIIA Cells with HA-tagged 1A-- SNaIIA cells were transfected with pCI.1A-HA using DOTAP as described previously (10). Because SNaIIA cells are resistant to G418 as a result of the original transfection of the IIA subunit, pCI.1A-HA was cotransfected with pSV2*Hyg to confer resistance to the antibiotic hygromycin. Drug selection with hygromycin (400 µg/ml) required approximately 1 week; clonal cell lines were selected, analyzed by Northern blot, and expanded for further analysis.

[³H]Saxitoxin Binding Analysis-- Whole cell saturation binding analysis of SNaIIA and SNaIIA-1A cells was performed as described previously (10) over a concentration range of 0.1-10 nM [³H]saxitoxin ([³H]STX) with the addition of 10 µM unlabeled tetrodotoxin (TTX; Calbiochem) to assess nonspecific binding. [³H]STX (28 Ci/mmol) was obtained from Amersham. Binding data were normalized to protein concentration using the BCA Protein Assay kit (Pierce). Saturation binding data were analyzed by nonlinear regression using Prism (GraphPad Software, La Jolla, CA) to obtain K_D and B_max values.

Antibodies-- A multiple antigenic peptide with amino acid sequence RWRDRWKEGDRLVSHRGQ, encoded by nucleotides 160-177 of 1A, was synthesized by the Protein and Carbohydrate Structure Facility at the University of Michigan. Rabbit polyclonal antibodies were subsequently generated in two separate animals and tested by enzyme-linked immunosorbent assay against the 1A peptide to determine the antibody titer (Research Genetics, Inc., Huntsville, AL).

Western Blot Analysis of 1A Protein Expression-- Adult female Harlan Sprague Dawley rats were sacrificed by decapitation. Brain, spinal cord, heart, skeletal muscle, and adrenal gland tissues were immediately removed, minced, and briefly stored on ice. SNa1A-16 cell line cells were washed with PBS and scraped into 50-ml conical tubes. Membranes were prepared as described previously (10), and the final pellets were resuspended in 50 mM Tris, pH 8, 10 mM EGTA containing Complete-Mini protease inhibitor tablets according to the manufacturer's instructions (Roche Molecular Biochemicals). The total protein in each membrane preparation was quantitated with the BCA Protein Assay Kit (Pierce) using bovine serum albumin as the standard. 250 µg of each membrane preparation were separated by SDS-PAGE as described previously (10), transferred to nitrocellulose (HyBond ECL; Amersham Pharmacia Biotech), and stained with Ponceau-S prior to immunodetection. Western blot analysis was performed as follows. The blot was washed for 10 min in TBS-T (10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20) at room temperature and then blocked for 1 h in 5% nonfat dry milk in TBS-T at room temperature. Primary anti-1A antibody (1:750 dilution) was applied in blocking solution for 30 min at room temperature. The blot was then washed five times for 15 min each in TBS-T. Secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG, ICN) diluted to 1:100,000 in blocking solution was applied for 30 min at room temperature. The blot was then washed five times for 15 min each in TBS-T. SuperSignal WestFemto chemiluminescent substrate solution (Pierce) was applied according to the manufacturer's instructions, and the blot was placed between plastic sheet protectors and exposed to Hyperfilm-ECL (Amersham Pharmacia Biotech) for the indicated times (typically 10-30 s) at room temperature.

Immunohistochemical Analysis of 1A Expression-- Sprague Dawley rats (4-6 weeks of age; Harlan Industries, Indianapolis, IN) were perfused with 4.0% neutral buffered formalin for approximately 20 min, and tissues were removed for further processing. Tissues were postfixed overnight in 10% neutral buffered formalin, processed, embedded in paraffin blocks, and sectioned onto slides (5-µm thickness). Tissues were processed for immunohistochemistry as described previously (17). Briefly, slides were blocked with normal goat serum, incubated with rabbit anti-rat 1A antibody at a dilution of 1:600 and then incubated with biotinylated goat anti-rabbit IgG (Vector Labs, Burlingham, CA). All incubation were performed for 30 min at room temperature. Detection was accomplished using the ABC-horseradish peroxidase system (Vector Labs) followed by 3'-diaminobenzidine (Biomeda, Foster City, CA) as the chromogen, stained in Mayer's hematoxylin, and coverslipped with Permount (Fisher).

Electrophysiological Analysis-- Electrophysiological recordings on SNaIIA and SNaIIA1A cells were performed by the patch clamp technique in the whole cell configuration (18), using an Axopatch 200B patch clamp amplifier and pCLAMP software (Axon Instruments, Foster City, CA). Data were filtered at 5 kHz and digitally sampled at 50 kHz. Series resistance was compensated 60-80%. Capacitive transients, elicited by voltage steps, were partially canceled using the internal clamp circuitry. Additional subtraction of transients and leak currents was obtained using the P/4 procedure (19). For whole cell recordings, recording pipettes were filled with 105 mM CsF, 10 mM CsCl, 10 mM NaCl, 10 mM EGTA, 10 mM HEPES, pH 7.4, with CsOH. Pipette resistances were 1-3 megaohms. The bath solution consisted of 130 mM NaCl, 4 mM KCl, 1.5 mM CaCl₂, 1 mM MgCl₂, 5 mM glucose, 10 mM HEPES, pH 7.4, with NaOH. As described previously (10), the voltage dependence of sodium current activation and inactivation progressively shifted to more negative potentials over the first few minutes of experiments with fluoride-based intracellular solutions. Thus, all experiments were begun 10 min after break in, at which point the shifts in channel gating had stabilized.

For each cell, we examined the voltage dependence of current activation and steady state inactivation. Activation was assessed by applying test pulses to potentials from 50 to +70 mV in 5-mV steps, following a 100-ms prepulse to 100 mV. Peak current amplitude (I_peak) was measured at each test potential and converted to conductance (g) according to the equation, g = I_peak/(V_rev  V_test), in which V_test is the test potential and V_rev is the current reversal potential, determined by linear extrapolation of the straight line portion of the falling phase of the current-voltage relationship. The conductance values were normalized with respect to the maximal conductance, plotted as a function of V_test, and fit with the Boltzman equation: 1/(1 + exp((V_test  V_1/2)/k), in which V_1/2 is the midpoint of the curve and k is a slope factor. Steady state inactivation was examined by applying 100-ms-long prepulses to potentials ranging from 100 to 10, in 5-mV steps, followed by a test pulse to 0 mV. The peak amplitude of currents evoked by the test pulses were normalized with respect to the largest currents, plotted as a function of prepulse potential, and fit with the Boltzman equation.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Molecular Cloning and Analysis of 1A-- A rat adrenal gland cDNA library prepared in the  Zap Express vector was screened with a digoxigenin-labeled cDNA probe encoding nucleotides 345-911 of p1.C1Aa (4). A clone encoding a protein with a 5' region of identity to 1 and a novel 3' region was identified by DNA sequencing. The identity of this clone was then confirmed independently by reverse transcriptase-PCR from rat adrenal cDNA using the oligonucleotides 1A-3 and 1A-5 followed by DNA sequencing, as described under "Experimental Procedures." This clone, designated 1A, encoded a novel 253-amino acid protein of 29,055 daltons (predicted molecular mass of the mature protein with the signal sequence removed), which contains a predicted amino-terminal region of identity to 1, residues Met¹ through Lys¹²⁹, followed by a novel carboxyl-terminal region (Fig. 1, A and B). Hydrophobicity analysis of the novel, carboxyl-terminal region revealed an apparent 66-amino acid extension of the extracellular region of 1 followed by a 19-amino acid transmembrane domain and short, 39-amino acid intracellular carboxyl terminus (Fig. 1, A and C). The novel 3' region of 1A is structurally homologous to 1 in that it predicts a transmembrane domain and short intracellular region, yet it contains little to no homology at the amino acid level (Fig. 1B). Interestingly, the amino-terminal region common to 1 and 1A contains the extracellular immunoglobulin fold. 1A can thus be characterized structurally as a cell adhesion molecule (17).

View larger version (41K): [in this window] [in a new window]   Fig. 1.   Sequence analysis of 1A. A, comparison of 1A and 1 amino acid sequences. Upper sequence, 1A; lower sequence, 1. B, putative membrane topologies of 1A and 1. 1A is predicted to encode a type I membrane protein with similar topology to 1. The disulfide-linked immunoglobulin fold is common to both 1 and 1A. Following the alternate splice site (arrow), 1A contains a novel 66-amino acid juxtamembrane region, 19-amino acid transmembrane segment, and 39-amino acid intracellular domain. C, sequence homology to known proteins. Results of BLAST-P search of the Swiss-Prot data base using the novel, carboxyl-terminal domain of 1A beginning at residue 130 as the query sequence. Accession numbers for LRP2 and tensin are indicated. D, schematic structure of the 1/1A gene. As reported previously, the 1 gene contains five introns, indicated as I1 through I5, and six exons. The 1A cDNA is the result of retention of I3, creating a novel 3' end.

Analysis of the novel 3' region of 1A by BLAST-P search of the Swiss-Prot data base revealed a 55-residue region of 1A with 32% identity to an extracellular low density lipoprotein receptor class A domain of human low density lipoprotein receptor-related protein 2 (LRP2), also called megalin or glycoprotein 330 (Fig. 1D) (20-23). This region of homology in 1A is predicted to be located extracellularly, just proximal to the plasma membrane followed by the transmembrane region. LRP2 has been shown to be a cysteine-rich type I membrane protein that forms a multimeric complex with receptor-associated protein. LRP2 binds clusterin with high affinity and is localized to clathrin-coated pits, suggesting that it may be an endocytic receptor. Interestingly, LRP2 has been shown to interact with extracellular matrix components, similar to sodium channel 1 and 2 subunits (24). The BLAST-P search also revealed a 63-residue region of 1A with 26% identity to tensin, a protein that has been implicated as the anchor for actin filaments at focal adhesions and is thought to act as a link between the cytoskeleton and signal transduction proteins (25). The region of homology to 1A is located in the insertion domain of tensin. This domain has been shown to permit polymerization of actin filaments.

1A Is Encoded by a Retained Intron in the 1 Gene-- The genomic organization of the human sodium channel 1 subunit gene has been reported previously (15). Using this information, we determined that the site of divergence between the 1 and 1A cDNAs was located precisely at the exon 3-intron 3 boundary of the 1 gene (Fig. 1E). Furthermore, a consensus sequence for exon-intron boundaries in genomic DNA was readily identified at this location. Amplification of intron 3 (approximately 5 kilobase pairs; data not shown) from rat genomic DNA by PCR followed by sequencing showed that the sequence of 1A beyond the amino acid sequence VVDK was indeed that of intron 3. We next performed a series of RNase protection experiments using a probe that was designed to span the exon 3-intron 3 boundary in the rat sequence. Fig. 2 shows that this 169-nucleotide probe was fully protected by rat embryonic day 18 brain mRNA. Thus, we propose that the novel extracellular, transmembrane, and carboxyl-terminal domains of 1A are encoded by alternative splicing of a retained intron within the 1 gene that includes an in-frame termination codon. These data are in agreement with the previously reported observation that 1 is represented only once in the rat and human genomes (26, 27).

View larger version (31K): [in this window] [in a new window]   Fig. 2.   RNase protection analysis of 1A. 20 µg of rat embryonic day 18 brain RNA was mixed with 1 µl of digoxigenin-labeled RPA-1 probe and 30 µg of yeast tRNA in 0.5 M ammonium acetate plus 2.5 volumes of ethanol. A control reaction contained embryonic brain RNA and yeast tRNA with no added probe. The reaction tubes were left at 20 °C for 15 min, and the RNA was precipitated by centrifugation in a microcentrifuge at top speed. The RNA was resuspended in 10 µl of HybSpeed hybridization buffer that had been preheated to 95 °C and vortexed vigorously, and the tubes were placed at 95 °C for 3 min. The samples were then hybridized for 10 min at 68 °C and digested with a mixture of RNase A and T1 (10 units/ml of RNase A and 400 units/ml RNase T1) for 30 min at 37 °C. 150 µl of HybSpeed inactivation/precipitation mix were added to each reaction, and the RNA was precipitated and resuspended in 10 µl of gel loading buffer 1. The reactions were electrophoresed on a 1.5-mm-thick 6% acrylamide TBE denaturing gel containing 7 M urea in the Mini-Protean gel format, transferred to nylon, and UV-cross-linked. Hybridization of the digoxigenin-labeled probe was detected with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (1:10,000 dilution) and CSPD. The blot was then exposed to Hyperfilm-ECL at room temperature. Lane 1, embryonic brain RNA plus RPA-1 probe digested with RNase mixture; lane 2, embryonic brain RNA digested with RNase mixture; lane 3, undigested RPA-1 probe; lane 4, RNA ladder. The lower diagram indicates predicted results of undigested, fully protected, and partially digested probes.

1A mRNA Is Expressed in Embryonic Brain and Adult Adrenal Gland-- A comparison of the developmental time courses of 1A and 1 mRNA expression in developing rat brain was determined using specific, noncross-hybridizing antisense cRNA probes for 1A and 1, respectively. Fig. 3 compares the developmental time course of expression of 1A (upper panel) versus 1 (lower panel) in total rat brain RNA from embryonic day 9 through postnatal day 21. The transcript size of 1A is approximately 4.4 kilobase pairs and reflects the retention of a portion of intron 3. Interestingly, the expression time course of 1A parallels that of the 26-kDa 1-immunoreactive band described previously (12) and complements the expression pattern of 1 (5) Thus, 1A is expressed early in development and disappears after birth. In contrast, 1 expression is not detectable during embryonic brain development and becomes detectable as 1A mRNA expression is decreased.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Developmental time course of 1A and 1 mRNA expression. Time-mated pregnant female Harlan Sprague Dawley rats were anesthetized with 60 mg/kg Beuthanasia-D intraperitoneally, and the fetuses were surgically removed. Embryonic day 9 fetuses were homogenized in their entirety in Trizol reagent to purify total RNA according to the manufacturer's instructions. Whole fetal brains were dissected at the remaining embryonic time points, and total RNA was purified using Trizol reagent. RNA was also prepared from the brains and adrenal glands of the adult female rats. Postnatal rats at the indicated ages were anesthetized with Beuthanasia-D, brains were dissected, and total RNA was purified with Trizol reagent. Northern blot analysis of 20 µg of each sample was performed using a digoxigenin-labeled 1A antisense RNA probe encoding nucleotides 428-850 or a digoxigenin-labeled antisense RNA probe specific to the 3'-untranslated region of 1 (nucleotides 1053-1508 of p1.C1Aa (4)). Upper panel, 1A-specific probe. Lane 1, embryonic (E) day 9 brain; lane 2, embryonic day 9 brain; lane 3, embryonic day 13 brain; lane 4, embryonic day 15 brain; lane 5, embryonic day 15 brain; lane 6, embryonic day 18 brain; lane 7, embryonic day 20 brain; lane 8, postnatal (P) day 2 brain; lane 9, postnatal day 7 brain; lane 10, postnatal day 14 brain; lane 11, postnatal day 20 brain; lane 12, postnatal day 60 brain; lane 13, adrenal. Lower panel, 1-specific probe. Lane 1, embryonic day 9 brain; lane 2, embryonic day 13 brain; lane 3, embryonic day 15 brain; lane 4, embryonic day 18 brain; lane 5, embryonic day 20 brain; lane 6, postnatal day 2 brain; lane 7, postnatal day 7 brain; lane 8, postnatal day 14 brain; lane 9, postnatal day 20 brain; lane 10, postnatal day 60 brain; lane 11, adrenal.

Analysis of 1A Protein Expression-- To determine whether alternative splicing of the 1 gene resulted in expression of a novel protein, a polyclonal antibody was generated against a multiple antigenic peptide containing the amino acid sequence RWRDRWKEGDRLVSHRGQ encoded by the retained portion of intron 3 found in the 1A cDNA clone. Aliquots of membrane preparations from adult rat brain, heart, skeletal muscle, spinal cord, and adrenal gland were separated by SDS-polyacrylamide gel electrophoresis and blotted to nitrocellulose, and Western blot analysis was performed using the 1A antibody described above (1:750 dilution). As shown in Fig. 4, 1A immunoreactive bands migrating at approximately 45-50 kDa were observed in heart, skeletal muscle, and adrenal gland but were not detected in brain or spinal cord. An immunoreactive doublet was observed in adrenal gland. The absence of immunoreactive 1A protein bands in adult brain and spinal cord is consistent with the Northern blot results shown above.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Western blot analysis of 1A expression. Brain, spinal cord, heart, skeletal muscle, adrenal, and SNaIIA1A-16 cell line membranes were prepared as described previously (10). The total protein in each membrane preparation was quantitated with the BCA protein assay kit using bovine serum albumin as the standard. 250 µg of each membrane preparation were separated by SDS-polyacrylamide gel electrophoresis as described previously (10), transferred to nitrocellulose, and stained with Ponceau-S prior to immunodetection. Western blot analysis was performed as follows. The blot was washed for 10 min in TBS-T (10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20) at room temperature to remove acetic acid from the Ponceau-S stain and then blocked for 1 h in 5% nonfat dry milk in TBS-T at room temperature. Primary 1A antibody (1:750 dilution) was applied in blocking solution for 30 min at room temperature. The blot was then washed five times for 15 min each in TBS-T. Secondary antibody (horse radish peroxidase-conjugated goat anti-rabbit IgG, ICN) diluted to 1:100,000 in blocking solution was applied for 30 min at room temperature. The blot was then washed five times for 15 min each in TBS-T. SuperSignal WestFemto chemiluminescent substrate solution was applied according to the manufacturer's instructions, and the blot was placed between plastic sheet protectors and exposed to Hyperfilm-ECL for 10 s at room temperature. B, adult brain; H, adult heart; SM, adult skeletal muscle; A, adult adrenal; SC, adult spinal cord; TC, transfected cells (SNaIIA1A-16).

Immunohistochemical Analysis of 1A Expression-- Positive 1A protein immunoreactivity was detected as brown product (Figs. 5 and 6). Product was not detected in the negative controls, which included the replacement of the primary antibody with similar species isotype serum (data not shown). Fig. 5 represents various central and peripheral neuronal populations that contained 1A. Fig. 5, A and B, demonstrates that 1A was found in most but not all Purkinje cells in the cerebellum. In addition, some 1A-positive neurons in the dentate nucleus of the cerebellum were observed (Fig. 5C). 1A was absent from the granular layer (G) and the molecular layer (M) of the cerebellum. In Fig. 5D, a distinct population of pyramidal neurons of the cerebral cortex contained 1A, while glial cell populations remained negative. Spinal cord also contained several distinct populations of 1A-containing neurons. Fig. 5E shows 1A in a small population of motor neurons, while 1A neurons were also observed in laminae II-V of the dorsal horn. All neuronal cell types of the dorsal root ganglia contained 1A (Fig. 5F), while processes and glial cells were negative for the 1A product.

View larger version (131K): [in this window] [in a new window]   Fig. 5.   Immunocytochemical analysis of 1A expression in neurons. A, × 10 magnification of the rat cerebellum shows 1A immunolabeling in both Purkinje cells (large arrow) and a population of neurons in the cerebellar white matter (small arrow). M, molecular layer; G, granular layer; W, white matter. B, × 60 magnification of the cerebellar Purkinje cell layer, demonstrating that most of the Purkinje cells contain 1A (black arrow), but some adjacent Purkinje cell neurons remain negative for 1A (blue arrow). C, ×60 magnification of the cerebellar dentate nucleus, indicating positive neurons (black arrow) and negative glial cells (blue arrow). D, 1A-positive pyramidal neurons in the rat cerebral cortex (black arrow) and negative glial cells (blue arrow). E, × 40 magnification of 1A positive spinal cord motor neurons (black arrow). F, × 60 1A-positive c-fiber neuron (black arrow) adjacent to 1A-positive large A neurons.

View larger version (97K): [in this window] [in a new window]   Fig. 6.   Nonneuronal immunohistochemical analysis of 1A expression. A, × 40 magnification of 1A immunoreactivity in cells and at junctions between individual muscle fibers in the right atria of the rat heart (arrowhead). B, × 40 magnification of endothelial cells in rat lung alveoli (arrowheads). C, distal areas of bronchus columnar epithelial cells and endothelial cells in a pulmonary arteriole contain 1A immunoreactivity (arrowheads). D, × 60 magnification of the pulmonary artery demonstrates 1A-positive endothelial cells (arrowhead).

Fig. 6, A-D, shows examples of nonneuronal sites of 1A localization. In Fig. 6A, the membranes of individual muscle fibers in rat atria were positive for 1A. Additionally, other areas of the rat heart such as ventricles contained 1A product (data not presented). 1A immunoreactivity was also observed in the alveoli (Fig. 6B) and in some bronchus columnar epithelial cells (Fig. 6C). Finally, labeling of endothelial cells in the pulmonary artery is shown in Fig. 6D.

Mammalian Cell Expression-- To investigate the functional role of 1A, we constructed a HA epitope-tagged version of the 1A cDNA. We included the epitope tag for potential use in the event that our polyclonal antibody production was unsuccessful. We were successful in raising an anti-1A antibody and thus did not use the HA tag in these experiments. We created stably transfected cell lines expressing 1A in the previously characterized SNaIIA cell line. SNaIIA cells are a stable line expressing type IIA sodium channel  subunits in CHL cells (10). Because SNaIIA cells are G418-resistant, we co-transfected pcDNA3-1A with pSV2*Hyg in a 10:1 ratio (pcDNA3-1A:pSV2*Hyg) so that transfected clones could be selected with the antibiotic hygromycin. A number of hygromycin-resistant colonies were analyzed by Northern blot for 1A mRNA expression. Positive clones were expanded and analyzed further by [³H]STX binding. Western blot analysis of one of these cell lines, SNaIIA1A-16, is shown in Fig. 4, in which an immunoreactive band of approximately 45 kDa was observed.

[³H]STX binding analysis revealed a significant increase in the expression levels of functional sodium channels at the plasma membrane of SNaIIA1A-16 cells as compared with the parent line, SNaIIA (Table I). Nonlinear regression analysis of saturation binding showed a 4.4-fold increase in B_max as compared with SNaIIA with no significant change in the K_D (0.8 nM for SNaIIA versus 0.9 nM for SNaIIA1A-16). Our results are similar to those of a previous study showing that coexpression of IIA and 1 resulted in a 2-4-fold increase in the level of [³H]STX binding compared with cells expressing IIA alone (10). The K_D values obtained in the present study were very similar to those reported values as well. Our data suggest that a function common to 1 and 1A is to increase the level of sodium channel expression at the plasma membrane. We hypothesize that 1 and 1A may stabilize the conformation of channels such that they become more resistant to degradation and/or target newly synthesized channels to the plasma membrane from intracellular stores. Because 1 and 1A contain a common cell adhesion molecule domain, we propose that the extracellular Ig loop may be necessary for this function.

View this table: [in this window] [in a new window]   Table I [3H]Saxitoxin binding analysis of IIA- and IIA/1A-expressing cell lines Whole cell saturation binding analysis of SNAIIA and SNaIIA-1A cells was performed as previously described (10) over a concentration range of 0.1-10 nM [3H]STX with the addition of 10 µM unlabeled tetrodotoxin to assess nonspecific binding. [3H]STX (28 Ci/mmol) was obtained from Amersham Pharmacia Biotech. Binding data were normalized to protein concentration using the BCA protein assay kit. Saturation binding data were analyzed by nonlinear regression using Prism to obtain KD and Bmax values. Values are shown ±S.E.

To determine whether coexpression of 1A subunits affected the functional properties of type IIA sodium channels in CHL cells, we used whole cell electrophysiological recording to compare sodium currents in the parent SNaIIA cell line and in three different SNaIIA1A cell lines. Fig. 7A shows currents, elicited by depolarizations to varying test potentials, recorded in a typical SNaIIA cell and a typical SNaIIA1A cell. As is evident from these traces, coexpression of 1A did not dramatically alter the properties of voltage-activated sodium currents. Nevertheless, currents in 1A-expressing cell lines were subtly different from currents in SNaIIA cells. For example, mean steady state inactivation curves for SNaIIA1A cell lines were shifted to more positive potentials than the mean inactivation curve for SNaIIA cells (Fig. 7, B and D). Although this difference was quite small, it was observed in all three SNaIIA1A cell lines and was statistically significant in two of the three 1A-containing lines (SNaIIA1A-7, p = 0.037; SNaIIA1A-8, p = 0.024). Thus, one effect of 1A association with IIA may be a small positive shift in the voltage dependence of steady state inactivation. For activation, mean voltage-conductance curves for two of the three 1A-expressing lines were statistically indistinguishable from SNaIIA (Fig. 7, B and D); however, for SNaIIA1A-16, the voltage dependence of activation was significantly more negative than for SNaIIA (p = 0.001). Thus, data from one of the three 1A cell lines suggest that 1A may also alter sodium channel activation. For comparison, Fig. 7D also shows the effects of the adult 1 isoform on the voltage dependence of sodium channel activation and inactivation. As has been shown previously (10), when coexpressed with IIA in CHL cells, 1 shifted the voltage dependence of steady state inactivation approximately 10 mV negative compared with IIA alone (Fig. 7D). Thus, 1 and 1A have clearly different effects on steady state inactivation. SNaIIA1 cells also exhibited a small negative shift in the voltage dependence of activation compared with SNaIIA cells, as was previously reported (10).

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Effects of 1A on the functional properties of whole cell sodium currents. A, voltage-dependent sodium currents recorded in a SNaIIA cell (top traces) and a SNaIIA1A cell (bottom traces). Currents were elicited by depolarizations to 40, 30, 20, 10, 0 and +10 mV, from a prepulse potential of 100 mV. B, mean activation (filled symbols) and inactivation (open symbols) curves for cell lines SNaIIA (circles), SNaIIA1A-7 (squares), SNaIIA1A-8 (triangles), and SNaIIA1A-16 (inverted triangles). For each cell, activation and inactivation were analyzed as described under "Experimental Procedures." The symbols show means of the activation and inactivation data for the different cell lines. In this figure and Fig. 8, error bars indicate S.E. The smooth lines were generated with the Boltzman equation (see "Experimental Procedures"), using mean values of V1/2 and k determined for each cell line from fits of individual experiments. Mean values for V1/2 and k and the number of experiments for each cell line are as follows: activation, SNaIIA, V1/2 = 11.0 ± 0.96, k = 8.3 ± 0.28, n = 15; SNaIIA1A-7, V1/2 = 8.1 ± 2.13, k = 7.9 ± 0.48, n = 6; SNaIIA1A-8, V1/2 = 11.6 ± 1.21, k = 7.4 ± 0.31, n = 11; SNaIIA1A-16, V1/2 = 17.0 ± 1.41, k = 7.18 ± 0.39, n = 11; inactivation: SNaIIA, V1/2 = -48.3 ± 0.77, k = 6.5 ± 0.20, n = 13; SNaIIA1A-7, V1/2 45.2 ± 1.25, k = 6.8 ± 0.22, n = 6, SNaIIA1A-8, V1/2 = 45.9 ± 0.63, k = 6.5 ± 0.21, n = 11; SNaIIA1A-16, V1/2 = 47.2 ± 0.50, k = 6.5 ± 0.15, n = 10. C, inactivation time constants (inactivation) determined from fits of current decay for SNaIIA (), SNaIIA1A-7 (), SNaIIA1A-8 (), SNaIIA1A-16 (), and SNaIIA1 () cells, plotted as a function of test potential. D, mean V1/2 values for activation (filled symbols) and inactivation (open symbols) for cell lines SNaIIA, SNaIIA1A-7, SNaIIA1A-8, SNaIIA1A-16, and SNaIIA1.

To determine whether 1A affected the time course of macroscopic sodium currents, we fit the decaying phase of whole cell currents, elicited over a range of test potentials, with single exponential functions (Fig. 7C). For SNaIIA, the inactivation time constants determined from these fits were progressively less at progressively more positive test potentials, approaching a minimum of approximately 0.5 ms at the most positive test potentials examined. Inactivation time constants were significantly less for SNaIIA1, suggesting that 1 may slightly speed inactivation time course (Fig. 7C). For two of the three SNaIIA1A cell lines, the rate of current inactivation was virtually identical to SNaIIA (Fig. 7C). However, for SNaIIA1A-16, inactivation was similar to SNaIIA1 at all test potentials examined (Fig. 7C). Because sodium channel inactivation is coupled to activation (19, 28), it is likely that the faster decay rates for SNaIIA1 and SNaIIA1A-16 currents were, at least in part, secondary to the negative shifts in activation that were observed in these two cell lines. In addition, it is also possible that 1 and 1A subunits have direct effects on the rate of sodium channel inactivation.

The most dramatic effect of 1A, detected electrophysiologically, was a large increase in the amplitudes of macroscopic sodium currents. This is illustrated in Fig. 8A, which shows current densities for depolarizations to +10 mV in SNaIIA, SNaIIA1A, and SNaIIA1 cell lines. Current densities for SNaIIA cells were 69 pA/picofarads, whereas current densities were approximately 2.5 times greater for the three SNaIIA1A cell lines (Fig. 8A). The increase in current density observed with coexpression of 1A was similar to that seen with coexpression of the adult 1 isoform (Fig. 8A). This increase in mean current density reflected two distinct effects of 1A on sodium channel expression. First, 1A greatly increased the proportion of cells with measurable whole cell sodium currents. This effect is shown in Fig. 8B, which plots the number of SNaIIA (black bars) or SNaIIA1A (white bars) cells with peak currents within different amplitude ranges. For SNaIIA, this amplitude-frequency distribution was bimodal. In 40% of the cells (16 of 40), currents were indistinguishable from the small inward currents recorded in untransfected CHL cells (i.e. <100 pA). In the remaining 60% of the cells, currents ranged from 500 pA to 5 nA and thus were clearly due to expression of cloned type IIA channels. In contrast, all SNaIIA1A cells expressed large sodium currents (Fig. 8A). The frequency histogram for SNaIIA1A followed a normal distribution with a modal current range of 2-3 nA.

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Effect of 1A on the level of expression of functional sodium channels. A, current densities for SNaIIA, SNaIIA1A, and SNaIIA1 cell lines. Currents were elicited by depolarization to +10 mV from a prepulse potential of 100 mV. Peak current amplitude was divided by cell capacitance to give current density. Cell capacitance was determined by integrating the area under transients elicited by 3-mV voltage steps applied before series resistance compensation and capacitive transient cancellation. Mean capacitance measurements for the four different cell lines were not significantly different, indicating that coexpression of 1A or 1 did not alter cell surface area. B, amplitude-frequency histogram for SNaIIA (black bars) and SNaIIA1A (white bars; data for all three SNaIIA1A cell lines were combined). Currents were evoked by depolarization to +10 mV. The bars indicate the number of cells with peak currents that fell within different amplitude ranges.

To determine whether the lower mean current density of SNaIIA cells was solely due to its large proportion of low expressing cells, we recalculated the mean current density for SNaIIA, excluding these low expressers. Eliminating low expressing cells increased the mean current density for SNaIIA cells from 69 to 116 pA/picofarads (Fig. 8A); however, this value was still significantly smaller than the mean current density of cells expressing 1A (p = 0.014). Thus, even when comparing only those cells that expressed measurable sodium currents, 1A still increased the density of functional sodium channels on the cell surface.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A number of cases of intron retention have been reported in the literature, including alternative splicing of the genes encoding leukocyte-common antigen-related protein tyrosine phosphatase, CD44, effector cell protease receptor-1, the microtubule-associated protein tau, thyrotropin-releasing hormone receptor, and bovine growth hormone (29-35). In many cases, the retained intron contains an alternate, in-frame termination codon as well as a polyadenylation signal. Alternative splicing that results in retention of the intron in the primary transcript thus results in an isoform of the protein containing a novel carboxyl terminus. Interestingly, this is not the first report of intron retention in the 1 gene. Waxman and co-workers (36, 37) have previously reported that intron 5 of 1 can be retained, creating a novel isoform that is expressed in rat brain, optic nerve, sciatic nerve, and skeletal muscle. This isoform contains an 86-nucleotide insert encoded by intron 5 in the 3'-untranslated region. While the intron retention reported previously does not alter the 1 coding sequence, our present data describe a very significant coding sequence change resulting in a novel carboxyl terminus.

Effects of 1A on sodium currents and [³H]STX Binding Levels in CHL Cells-- The most striking functional consequence of IIA and 1A coexpression in CHL cells was a 2.5-fold increase in sodium current density compared with cells expressing IIA subunits alone. This increase in current density reflected two distinct effects of 1A: 1) an increase in the proportion of cells expressing detectable sodium currents and 2) an increase in the level of functional sodium channels in expressing cells. Increases in sodium channel expression with 1A are similar to previous results obtained with the adult 1 isoform in both mammalian cells (10) and Xenopus oocytes (4). These observations are consistent with the hypothesis that 1 and 1A subunits facilitate the expression of sodium channels and/or stabilize the channels in the plasma membrane and that the molecular basis for this function resides in the extracellular cell adhesion molecule domain common to the two isoforms. The results of our [³H]STX binding experiments support this hypothesis. We propose that interaction of the extracellular cell adhesion molecule domain (Ig fold) common to 1 and 1A with  may be responsible for the observed effects on channel expression levels. Consistent with this interpretation, two previous studies have shown that the extracellular domain of 1 is essential for modulation of both brain and skeletal muscle