©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Co-expression of the 1 and Type IIA Subunits of Sodium Channels in a Mammalian Cell Line (*)

(Received for publication, July 27, 1994; and in revised form, November 7, 1994)

Lori L. Isom Todd Scheuer Alice B. Brownstein David S. Ragsdale Brian J. Murphy William A. Catterall

From the Department of Pharmacology, SJ-30, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Brain sodium channels are a complex of alpha (260 kDa), beta1 (36 kDa), and beta2 (33 kDa) subunits. alpha subunits are functional as voltage-gated sodium channels by themselves. When expressed in Xenopus oocytes, beta1 subunits accelerate the time course of sodium channel activation and inactivation by shifting them to a fast gating mode, but alpha subunits expressed alone in mammalian cells activate and inactivate rapidly without co-expression of beta1 subunits. In these experiments, we show that the Chinese hamster cell lines CHO and 1610 do not express endogenous beta1 subunits as determined by Northern blotting, immunoblotting, and assay for beta1 subunit function by expression of cellular mRNA in Xenopus oocytes. alpha subunits expressed alone in stable lines of these cells activate and inactivate rapidly. Co-expression of beta1 subunits increases the level of sodium channels 2- to 4-fold as determined from saxitoxin binding, but does not affect the K for saxitoxin. Co-expression of beta1 subunits also shifts the voltage dependence of sodium channel inactivation to more negative membrane potentials by 10 to 12 mV and shifts the voltage dependence of channel activation to more negative membrane potentials by 2 to 11 mV. These effects of beta1 subunits on sodium channel function in mammalian cells may be physiologically important determinants of sodium channel function in vivo.


INTRODUCTION

Voltage-sensitive Na channels are the membrane proteins responsible for initiation of the action potential in most excitable cells(1) . Na channels isolated from rat brain are heterotrimeric, composed of a 260-kDa alpha subunit, a 36-kDa beta1 subunit, and a 33-kDa beta2 subunit(2) . Multiple cDNAs encoding Na channel alpha subunit isoforms have been cloned and sequenced (reviewed in (2, 3, 4) ). The primary structure of the rat brain beta1 subunit deduced from cDNA sequence predicts a membrane glycoprotein with a type 1 transmembrane topology including a single transmembrane segment(5) , in agreement with previous biochemical analyses(2) . Rat brain beta1 subunit mRNA is expressed in brain, spinal cord, heart, and skeletal muscle tissues(5, 6) . Although the Na channel alpha subunit is sufficient for expression of functional Na channels(7, 8, 9, 10, 11, 12) , co-expression of rat brain beta1 subunit and rat brain type IIA alpha subunit mRNAs in Xenopus oocytes results in acceleration of the macroscopic rates of activation and inactivation, a hyperpolarizing shift in the voltage dependence of inactivation, and an increase in peak current amplitude(5, 13) . Furthermore, the functional effects of beta1 are not limited to the adult central nervous system because similar functional effects of beta1 subunits have been reported on skeletal muscle µ1 alpha subunits and on embryonic brain type III alpha subunits(13, 14, 15) .

Na channel alpha subunits expressed alone in Xenopus oocytes function in two gating modes characterized by fast and complete inactivation versus slow and incomplete inactivation(16, 17, 18) . Expression of beta1 subunits in Xenopus oocytes shifts the proportion of Na channels gating in the fast mode as compared to the slow(13, 14) . This mode shift is responsible for the acceleration of activation and inactivation. In contrast to Xenopus oocytes, Na channel alpha subunits expressed alone in transfected mammalian cells gate primarily in the fast mode(12, 19, 20) , and no functional effect of beta1 subunits on Na channels expressed in mammalian cells has been defined. An essential step in understanding the possible physiological significance of beta1 subunits, therefore, is to investigate the functional effects of alpha and beta1 co-expression in transfected mammalian cells and to determine whether the fast gating observed in these cells is caused by endogenous beta1 subunit expression or by other factors contributed by the genetic background of mammalian somatic cells. In the present study, we show that functional co-expression of rat brain type IIA alpha and beta1 subunits in Chinese hamster cells results in hyperpolarizing shifts in both the voltage dependence of Na channel activation and inactivation and an increase in Na channel expression at the plasma membrane as compared to alpha alone. Furthermore, we show that fast gating of Na channel alpha subunits expressed alone in these cells is not due to the presence of endogenous beta1 subunits, but may be due instead to differences in biosynthesis, post-translational modification, or protein-protein interactions in mammalian somatic cells.


EXPERIMENTAL PROCEDURES

Materials

Na channel was purified from rat brain as described previously(21) . The catalytic subunit of cyclic AMP-dependent protein kinase was purified from bovine heart as described previously(22) .

Synthetic Na channel peptides were synthesized using the solid phase method of Merrifield(23) . The identities of the synthetic peptides were confirmed by amino acid analysis and/or mass spectrometry. The synthetic peptide, SP1, corresponds to residues 554-563 in the primary sequence of the rat brain type IIA Na channel alpha subunit(9) . The synthetic peptide, beta1-1, corresponds to amino acid residues 1-18 of the beta1 subunit(5) . Anti-peptide antibodies were prepared as described by Gordon et al.(24) .

Construction of Expression Vectors Containing beta1 Subunit cDNA

pcDNA3.beta1

The plasmid pcDNA3.beta1 was constructed from pcDNA3 (Invitrogen) by blunt-end insertion of nucleotides 291 (ApaI site) to 1255 (StuI site) of pbeta1.C1Aa (5) into the EcoRV site of pcDNA3, 3` to the cytomegalovirus promoter. pbeta1.C1Aa was digested with the restriction endonucleases ApaI and StuI. The ApaI site was then made blunt by treatment of the DNA with T4 DNA polymerase (Boehringer Mannheim) in the presence of dNTPs. The resulting 964-nucleotide fragment was then excised from a 1% Sea-Plaque agarose gel (FMC BioProducts) and treated with Agarase (Boehringer Mannheim) to digest the agarose. This fragment was then ligated into the EcoRV site of pcDNA3. The expression vector pcDNA3.beta1 was then used to transfect 1610 and SNaIIA cell line cells as described below.

pCDM8.beta1

The expression vector pCDM8.beta1 was constructed from the plasmid pCDM8 (Invitrogen) by blunt-end insertion of the ApaI-StuI fragment of pbeta1.C1Aa described above into the HindIII site of pCDM8 that was made blunt by treatment with T4 DNA polymerase. The expression vector pCDM8.beta1 was then used to transfect CNaIIA cells as described below.

Transfection of SNaIIA, 1610, or CNaIIA Cells with beta1 Expression Vectors

The Chinese hamster lung 1610 cell line SNaIIA stably expresses rat brain type IIA Na channel alpha subunits, as described previously(25) . pcDNA3.beta1 was stably transfected into this cell line or the parent cell line, 1610, using N-[1-(2,3-dioleoxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP, (^1)Boehringer Mannheim). For this procedure, 5 times 10^5 SNaIIA cells were seeded onto a 35-mm culture dish and grown overnight in RPMI containing 5% fetal bovine serum, 200 µg/ml G418, 25 units/ml penicillin, and 25 µg/ml streptomycin in 5% CO(2) at 37 °C. On day 2, the medium was replaced with fresh solution. A suspension containing 1 mg/ml DOTAP reagent, 10 µg of pcDNA3.beta1, and 1 µg of pSV2*Hyg in 20 mM HEPES, pH 7.4, 150 mM NaCl was then added dropwise to the cells and swirled to mix. The cells were allowed to grow overnight in 5% CO(2) at 37 °C. Because SNaIIA cells were already resistant to G418(25) , the intrinsic resistance marker in pcDNA3, the plasmid pSV2*Hyg containing a gene which confers resistance to hygromycin was co-transfected into the SNaIIA cells. On day 3, the cells were trypsinized, washed with fresh medium, and replated at a 1:40 surface area dilution (one 35-mm dish into four 100-mm dishes). Selection was started immediately by inclusion of 400 µg/ml hygromycin B (Calbiochem) in the culture medium. After 10-15 days of selection for hygromycin resistance, 35 colonies were isolated and transferred to individual cell culture dishes for expansion and analysis. Colonies that expressed high levels of beta1 subunit mRNA as assessed by Northern blot were propagated for further pharmacological and electrophysiological studies.

The Chinese hamster ovary (CHO) cell line CNaIIA, which stably expresses rat brain type IIA alpha subunits(20) , was transfected with pCDM8.beta1 using DOTAP reagent as described above. pSV2*Hyg was co-transfected with pCDM8.beta1 to confer resistance to hygromycin B.

Preparation and Northern Blot Analysis of Total Cellular RNA

Total RNA from each transfected cell line and from the parent 1610 cell line was isolated by an acid-phenol method as described previously(26) . The RNA pellet was washed with 70% ethanol, dried, and resuspended in diethylpyrocarbonate-treated 10 mM Tris, pH 8.0, 0.1 mM EDTA.

Northern blot analysis of 10 µg of each RNA sample was carried out as described previously (13) except that Boehringer Mannheim Nylon membrane was substituted for nitrocellulose. An antisense cRNA probe was transcribed from the plasmid pbeta1.C1Aa, incorporating digoxigenin-11-UTP (Boehringer Mannheim) and hybridized to the blot as described in the product literature. Hybridization and washing of the blot were performed at 65 °C. Wash solutions contained 0.5% SSC and 0.5% SDS. Chemiluminescent detection of the digoxigenin-labeled probe was accomplished using LumiPhos (Boehringer Mannheim) according to the manufacturer's instructions. The blot was then exposed to Kodak X-Omat AR film at room temperature for 30 min.

Isolation and Immunoblotting of Membranes from Cultured Cells

Membranes were prepared from the transfected SNaIIAbeta1-16 cell line and the parent 1610 cell line as described previously(20) . Protein concentrations were determined by the method of Peterson(27) . Membrane proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the highly porous gels described by Doucet et al.(28) using the Mini-PROTEAN II gel system (Bio-Rad Laboratories). Protein was then electrophoretically transferred to nitrocellulose (0.45 µm) by the method of Towbin et al.(29) . Nitrocellulose blots were incubated for 2 h at 20 °C in 5% nonfat dry milk in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl (TBS) to saturate nonspecific binding sites. Blots were then incubated with protein A-Sepharose-purified IgG or preimmune IgG for 2 h at 20 °C in TBS containing 0.05% (v/v) Tween 20 (TBST) and 5% milk. Blots were subsequently washed three times for 10 min each in TBST to remove unbound antibodies and incubated for 1.5 h with protein A coupled to horseradish peroxidase in TBST containing 5% milk. Blots were washed 3 times for 10 min each in TBST, and bound protein A was visualized with the Enhanced Chemiluminescence Detection System (Amersham) using Kodak X-Omat AR film.

Phosphorylation and Immunoprecipitation of Na Channels Isolated from Cultured Cells

SNaIIAbeta1-16 and 1610 cells in confluent 150-mm dishes were harvested and lysed as described previously(30) . Soluble cell extract was then subjected to a double immunoprecipitation protocol using 0.15 mg of protein A-Sepharose-purified anti-SP1 IgG (26) or nonimmune IgG per ml of cell solubilisate as described previously(30) . Immunoprecipitated material was phosphorylated in the presence of 50 µM purified cAMP-dependent protein kinase and 20 µM [-P]ATP and analyzed by SDS-PAGE as described previously(30) . Gels were subjected to autoradiography against Kodak X-Omat AR film at -70 °C with a DuPont CRONEX Lightning Plus intensifying screen for 2 h.

Poly(A) Selection of Total Cellular RNA

Total RNA was isolated from 1610, SNaIIA, and SNaIIAbeta1-16 cell line cells as described above. Poly(A) selection was performed using the Oligotex-dT mRNA Kit (QIAGEN Inc., Chatsworth, CA) according to the manufacturer's instructions.

cRNA Transcription

Na channel type IIA alpha subunit cRNA was synthesized from the linearized plasmid ZemRVSP6-2580(20) , using the Ambion SP6 mMessage mMachine kit according to manufacturer's instructions. Na channel beta1 subunit cRNA was synthesized from the EcoRI-linearized plasmid pSP64T.beta1 (13) using the Ambion SP6 mMessage mMachine kit according to manufacturer's instructions.

[^3H]Saxitoxin Binding

Intact 1610 and SNaIIA Cells

[^3H]Saxitoxin ([^3H]STX, 20 Ci/mmol) was obtained from Amersham. Each cell line to be analyzed was typically plated in 3-150-mm tissue culture dishes and grown past confluency under the conditions described above. Each dish was washed one time with binding buffer (50 mM HEPES-Tris, pH 7.5, 130 mM choline chloride, 5.4 mM KCl, 0.8 mM MgSO(4), 5.5 mM dextrose), and the cells were scraped into 50-ml conical tubes (Falcon, Lincoln Park, NJ, No. 2070) and pelleted by centrifugation at 200 times g for 10 min at room temperature. The cells were then resuspended in 4 ml of ice-cold binding buffer, triturated with a 10-ml pipette, and vortexed, and 200 µl was aliquoted into 15-ml polypropylene tubes (Falcon No. 2018) on ice, containing 25 µl of a 10-fold concentration of the indicated final [^3H]STX concentration plus 25 µl of 100 µM tetrodotoxin (Calbiochem) or binding buffer to assess nonspecific binding. All tubes were incubated on ice in a 4 °C cold room for 1 h. Binding was stopped by first vortexing the cells, pouring over a Whatman GF/C filter under vacuum, and washing 3 times with ice-cold wash buffer (163 mM choline chloride, 5 mM HEPES-Tris, pH 7.5, 1.8 mM CaCl(2), 0.8 mM MgSO(4)). [^3H]STX bound to the filters was assessed by scintillation counting. Specific binding was normalized to protein by Peterson analysis(29) . Scatchard analysis of saturation binding (0.1 to 10 nM [^3H]STX) was performed using the program, LIGAND/EBDA(31) .

Intact CNaIIA Cells

[^3H]STX binding to the CHO cell-derived lines CNaIIA and CNaIIAbeta1-B6 was performed as described previously(20) .

Membrane Preparations

[^3H]STX binding to membrane preparations of 1610 or SNaIIAbeta1-16 cells was carried out as described above with the following modifications. 250 µg of membrane protein was added per tube in a total volume of 800 µl of ice-cold binding buffer with added protease inhibitors. 100 µl of 50 nM [^3H]STX was added along with 100 µl of 100 µM TTX or binding buffer to assess nonspecific binding.

Electrophysiological Recording

Xenopus oocytes were injected with mRNA, and currents were recorded 2 to 3 days later by two-microelectrode voltage clamp as described(32) .

Whole cell voltage clamp experiments on mammalian cells were performed as described previously(20) . The extracellular solution contained: 130 mM NaCl, 4 mM KCl, 1.5 mM CaCl(2), 1.0 mM MgCl(2), 10 mM HEPES, and 5 mM glucose, adjusted to pH 7.4 with NaOH. For one series of experiments comparing CNaIIA cells to CNaIIAbeta1-B6 cells, a low Na solution containing 13 mM NaCl and 117 mM choline Cl was used. Intracellular solutions contained either fluoride or aspartate as the dominant intracellular anion. The fluoride-based solution contained: 105 mM CsF, 40 mM CsCl, 10 mM NaCl, 10 mM CsEGTA, and 10 mM HEPES. The aspartate-based intracellular solution contained: 140 mM cesium aspartate, 5 mM NaCl, 3 mM MgCl(2), 10 mM HEPES, and 5 mM CsEGTA. When the fluoride-based solution was included in the pipette, the voltage-dependent parameters underwent a slow shift in the hyperpolarizing direction after achieving the whole cell configuration that was largely complete within 10 min. Therefore, at least 10 min were allowed before recording voltage-dependent properties when using this solution. Such shifts were not observed with the aspartate-based intracellular solution.

The voltage dependence of channel activation was determined from the peak current recorded during 16-ms-long prepulses to a range of potentials. Chord conductance (G) was calculated from peak current (I) according to G = I/(V - V) where V is the test pulse potential and V is the measured reversal potential. Inactivation curves were measured using a 200-ms-long prepulse to a range of potentials followed by a test pulse to 0 mV. Conductance-voltage curves and inactivation curves were fit with a Boltzmann relationship, G = 1/(1 + exp((V - V)/k))) where V is the midpoint of the curve, and k is a slope factor.


RESULTS

Transfection of CNaIIA Cells with beta1 Expression Vectors

Our initial experiments were aimed at stable co-expression of beta1 subunits with type IIA alpha subunits in CHO cells using the cell line CNaIIA which stably expresses alpha. Preliminary experiments utilizing the expression vector pCDM8.beta1 showed that beta1 subunits increased Na channel expression by approximately 4-fold, as determined by [^3H]STX binding (Table 1). Physiological studies in the whole cell voltage clamp configuration also showed substantially increased, but highly variable, peak sodium currents compared to the parental CNaIIA cells. Occasional cells exhibited peak sodium currents exceeding 50 nA, far in excess of any individual cells in the extensively studied parental line CNaIIA (mean I = 4.5 ± 0.6 nA (S.E.)(20) ). Co-expression of beta1 subunits also consistently shifted the voltage dependence of Na channel inactivation by approximately 12 mV in the hyperpolarizing direction (Table 1). These initial results strongly suggested that beta1 subunits do substantially modify sodium channel expression and function in mammalian cells. However, perhaps because of the unusually high and variable levels of sodium channel expression in individual cells, we were unable to isolate any cell lines which stably expressed both alpha and beta1 subunits and could be analyzed in detail.



Isolation and Characterization of SNaIIA Cells Expressing beta1 Subunit mRNA

Because we were unable to produce a stable cell line expressing both alpha and beta1 subunit polypeptides in CHO cells, we chose an alternative expression vector and cell line. The SNaIIA cell line, which stably expresses rat brain Na channel type IIA alpha subunit mRNA, was derived from Chinese hamster lung 1610 cells as described previously(25) . SNaIIA cells or parental 1610 cells were transfected with the expression vector pcDNA3.beta1, using DOTAP reagent (Boehringer Mannheim) as described under ``Experimental Procedures.'' Total cellular RNA isolated from the resulting hygromycin-resistant clonal cell lines was analyzed by Northern blot using an antisense cRNA probe corresponding to the entire beta1 subunit gene contained in the plasmid pbeta1.C1Aa(5) . Chemiluminescent detection of the digoxigenin-labeled probe revealed a number of clones expressing mRNA encoding the beta1 subunit. Fig. 1compares mRNA from parental 1610 cells and SNaIIA cells to mRNA from cell lines 16, D1, 15, 19, and D3 that were stably transfected with beta1 subunit cDNA. In addition, we analyzed the cell line beta1-B3 as an example of 1610 cells that were transfected with pcDNA3.beta1 only. All of the transfected cell lines expressed a beta1 mRNA with the expected size of 1.4 kb. In addition, several cell lines (for example, lines 16, 15, and D3 in Fig. 1) also expressed a 3-kb mRNA encoding beta1 subunits. The nature of this larger mRNA was not investigated further because the properties of sodium channels in cell lines with only the 1.4-kb mRNA were similar to those which also expressed the 3-kb mRNA (see below). In contrast to the beta1 subunit-transfected cell lines, neither 1610 nor SNaIIA cells express detectable levels of beta1 subunit mRNA. Overexposure of the Northern blot did not reveal endogenous beta1 subunit mRNA in these two cell lines.


Figure 1: Northern blot analysis of RNA from parental and transfected cell lines. Total cellular RNA was isolated from the parent cell lines 1610 and SNaIIA and the beta1 subunit-transfected cell lines. 10 µg of each RNA sample was electrophoresed on a 7.4% formaldehyde gel as described under ``Experimental Procedures.'' Following electrophoresis, the RNA was transferred to Boehringer Mannheim Nylon membrane, UV cross-linked, hybridized with a cRNA probe synthesized from pbeta1.C1Aa, and detected by chemiluminescence as described under ``Experimental Procedures.'' Lane 1, SNaIIA; lane 2, SNaIIAbeta1-16; lane 3, SNaIIAbeta1-D1; lane 4, SNaIIAbeta1-15; lane 5, SNaIIAbeta1-19; lane 6, beta1-B3; lane 7, SNaIIAbeta1-D3; lane 8, 1610.



Immunochemical Identification of Na Channel beta1 Subunits in Transfected SNaIIA Cells

Na channel beta1 subunit polypeptide was identified in membrane preparations of the transfected cell line SNaIIAbeta1-16 but not in the nontransfected line 1610 (Fig. 2A). Membrane proteins from each cell line were separated in adjacent lanes of an SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with the anti-peptide antibody anti-beta1-1 or, as a negative control, anti-beta1-1 antibody that had been preincubated with the peptide against which the antibody was directed. Anti-beta1-1 is directed against a peptide contained in the amino terminus of the beta1 subunit protein as described previously (5) . Anti-beta1-1 recognized a 41-kDa beta1 subunit in membranes from the transfected SNaIIAbeta1-16 cell line, but not in membranes from the nontransfected line. Anti-beta1-1 that had been preincubated with peptide did not recognize any proteins in membrane preparations from either cell line, showing that the protein band recognized by anti-beta1-1 is specific. These results show that beta1 subunits of mature size are synthesized in SNaIIAbeta1-16 cells but not in the non-beta1-transfected cell line. Thus, the parent 1610 cell line used in these experiments does not express endogenous beta1 subunits that we could identify at either the nucleotide or peptide levels.


Figure 2: Detection of beta1 and alpha subunit polypeptides in membrane fractions or cell extracts isolated from parent and transfected cell lines. A, Na channel beta1 subunit polypeptide was identified in membrane preparations of SNaIIAbeta1-16 (indicated as 16) or 1610 cells. Membrane proteins (125 µg/lane) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-beta1-1 antibody (lanes 1 and 2) or anti-beta1-1 antibody that had been preincubated with beta1-1 peptide (lanes 3 and 4) as described under ``Experimental Procedures.'' Visualization of the blot was performed with the Enhanced Chemiluminescence Detection System (Amersham) using Kodak X-Omat AR film. B, Na channel alpha subunit polypeptide was identified in cell extracts prepared from 1610 or SNaIIAbeta1-16 (indicated as 16) cells using a combination of immunoprecipitation, phosphorylation with cAMP-dependent protein kinase and [-P]ATP, and SDS-PAGE as described under ``Experimental Procedures.'' Lane 1, 1610 cell extract immunoprecipitated with anti-SP1 antibody followed by phosphorylation; lane 2, SNaIIAbeta1-16 cell extract immunoprecipitated with anti-SP1 antibody followed by phosphorylation; lane 3, SNaIIAbeta1-16 cell extract immunoprecipitated with nonimmune serum followed by phosphorylation. Gels were subjected to autoradiography against Kodak X-Omat AR film at -70 °C with a DuPont CRONEX Lightning Plus intensifying screen for 2 h.



Identification of Na Channel alpha Subunits in SNaIIAbeta1-16 Cells

Initial experiments attempting to identify type IIA alpha subunits in SNaIIA and SNaIIAbeta1-16 cells by immunoblot analysis were unsuccessful, in spite of our ability to immunologically detect beta1 subunits in SNaIIAbeta1-16 cells, as shown in Fig. 2A. Further analysis of membrane preparations by [^3H]STX binding, however, revealed that membrane preparations from SNaIIAbeta1-16 cells contained as little as 10 fmol/mg of protein of STX receptor (data not shown), making it very difficult to detect Na channel alpha subunits by immunoblot analysis. As an alternative approach, we used the method of immunoprecipitation followed by phosphorylation by cAMP-dependent protein kinase to detect transfected Na channel alpha subunit protein in membrane preparations (Fig. 2B). Using this method, we were able to detect a specific immunoreactive protein band corresponding to 260-kDa Na channel alpha subunits in transfected but not in non-transfected cells. Replacement of alpha subunit-directed antiserum with nonimmune serum resulted in no signal, indicating that the observed immunoreactive band was specific.

Analysis of Cell Lines for the Presence of Endogenous beta1 Subunit-like Activity

The experiments we have described to this point have shown that the nontransfected 1610 cell line does not express Na channel beta1 subunits that are detectable either at the nucleic acid or amino acid levels based on Northern blot and immunoblot analyses. It is possible, however, that 1610 cells could express Chinese hamster beta1 subunits which are not recognized by any of our probes from rat brain. Because this is a critical point in the interpretation of our experiments, we checked for endogenous beta1 subunit expression at the functional level to be sure that all observed beta1 subunit effects were indeed due to transfected beta1 subunit cDNA. We prepared poly(A)-selected mRNA from 1610 cells and from SNaIIAbeta1-16 cells as described under ``Experimental Procedures'' and co-injected this mRNA into Xenopus oocytes in various combinations along with alpha subunit cRNA transcribed from the plasmid ZemRVSP6-2580 or beta1 subunit cRNA transcribed from the plasmid pbeta1.C1Aa. Fig. 3(dotted traces) confirms previous data(5) , demonstrating that co-expression of alpha subunit cRNA and beta1 subunit cRNA results in a 5-fold increase in the macroscopic rate of inactivation as compared to alpha alone. Co-expression of alpha subunit cRNA with poly(A)-selected mRNA from the SNaIIAbeta1-16 cell line gave results virtually identical with those from oocytes injected with alpha plus beta1 cRNA (Fig. 3, solid trace). Significantly, however, co-expression of alpha subunit cRNA with poly(A)-selected mRNA from the parent 1610 cell line resulted in a current trace that was no different from that produced by alpha alone, confirming the results of our previous experiments that 1610 cell lines do not express an endogenous beta1 subunit detectable with our cDNA and antibody probes (Fig. 3, dotted trace). More importantly, this experiment also showed that 1610 cells do not express an endogenous beta1 subunit-like activity. Thus, we can be confident that any functional effects observed in co-expression experiments are due to the exogenous beta1 subunit.


Figure 3: Functional effects of mRNA from parental and transfected cell lines on Na channels expressed in Xenopus oocytes. Na currents were recorded from Xenopus oocytes injected with type IIA alpha subunit cRNA (5 ng/µl) alone or with pbeta1.C1Aa cRNA (5 ng/µl), SNaIIAbeta1-16 cell poly(A)-selected mRNA, or 1610 cell poly(A)-selected mRNA. Currents were evoked by applying a depolarizing pulse to 0 mV from a holding potential of -90 mV. The records were normalized with respect to the peak current amplitudes.



Effect of Co-expression of beta1 Subunits on [^3H]Saxitoxin Binding

Saxitoxin (STX) and tetrodotoxin (TTX) bind at neurotoxin receptor site 1 on the neuronal Na channel with nanomolar affinity and block the Na current(1) . These toxins also bind with high affinity to the Na channel type IIA alpha subunit expressed in CNaIIA cells(20) . Results of our initial CHO cell transfection experiments showed that the presence of beta1 subunits caused an approximate 4-fold increase in [^3H]STX binding as compared with alpha alone (Table 1). In a more thorough study using 1610 cells, the parent cell lines as well as hygromycin-resistant clonal cell lines that expressed high levels of beta1 subunit mRNA (Fig. 1) were subjected to further analysis by [^3H]STX binding. Specific binding of [^3H]STX to intact SNaIIA and SNaIIAbeta1-16 cells was measured after incubation of the cells for 1 h at 4 °C (Fig. 4A). Scatchard analysis of saturation binding revealed that the K(d) for STX remained constant in the absence and presence of beta1 subunit expression (0.55 ± 0.12 nM in SNaIIA versus 0.50 ± 0.16 nM in SNaIIAbeta1-16). However, the number of functional Na channels expressed at the plasma membrane, B(max), increased by approximately 5-fold in the presence of beta1 in this experiment. Specific binding of a saturating concentration (5-10 nM) of [^3H]STX, to Na channels in 1610 cells, SNaIIA cells, and representative cell lines expressing beta1 subunit mRNA is shown in Fig. 4B. In all cases, beta1 subunit-expressing cells bound at least twice as much [^3H]STX as SNaIIA, suggesting that one functional consequence of beta1 expression in mammalian cells is an increase in the number of functional Na channel alpha subunits at the plasma membrane of at least 2-fold. This increase is comparable in size to the increase in sodium current recorded in Xenopus oocytes co-expressing beta1 subunits(5) . These observed binding properties were stable through 25 subcultures in all cell lines examined.



Figure 4: Analysis of [^3H]STX binding to parental and transfected cell lines. A, saturation binding. SNaIIA and SNaIIAbeta1-16 cells were grown past confluency, suspended, and incubated with increasing concentrations of [^3H]STX (0.1, 0.2, 0.5, 1, 2, 5, or 10 nM, 10 nM point not shown) (20 Ci/mmol) in the presence and absence of 10 µM TTX for 1 h at 4 °C as described under ``Experimental Procedures.'' Specific binding data were normalized to protein using Peterson analysis(18) . Scatchard analysis of the saturation binding data was performed using LIGAND/EBDA to calculate K (SNaIIA = 0.55 ± 0.12 nM, SNaIIAbeta1-16 = 0.50 ± 0.16 nM) and B(max) (SNaIIA = 3.2 ± 0.4 fmol/mg of protein, SNaIIAbeta1-16 = 14.5 ± 1.3 fmol/mg protein). These data are representative of three separate experiments. Closed squares, SNaIIAbeta1-16; open diamonds, SNaIIA. B, comparison of [^3H]STX binding at saturation. Each indicated cell line was grown past confluence, suspended, and incubated with 5 or 10 nM [^3H]STX in the presence and absence of 10 µM TTX as described under ``Experimental Procedures.'' Specific binding data were normalized to protein using Peterson analysis(18) . The mean and standard deviation values for each cell line were analyzed using Sigma Plot (Jandel Scientific). The error bars shown reflect n independent culture experiments as follows: SNaIIA, n = 6; SNaIIAbeta1-16, n = 6; SNaIIAbeta1-15, n = 5; SNaIIAbeta1-D1, n = 5; SNaIIAbeta1-D3, n = 5; SNaIIAbeta1-19, n = 4.



Electrophysiological Properties of Na Currents in Cells Expressing alpha and beta1 Subunits

Whole cell voltage clamp recording of Na currents from SNaIIA cells and SNaIIAbeta1 cells was performed to detect the electrophysiological consequences of alpha and beta1 subunit co-expression. Currents recorded during depolarizations to positive potentials were similar in time course in cells without or with expression of the beta1 subunit (Fig. 5A). In those beta1-expressing cell lines where a negative shift in activation was observed (see below), currents activated and inactivated more rapidly at a given potential as expected for the negatively shifted voltage dependence of activation, but the time course of activation and inactivation was similar when corrected for the shift in the voltage dependence of activation.


Figure 5: Na currents in cells expressing alpha subunits or alpha + beta1 subunits. A, currents in response to a 16-ms pulse from a holding potential of -100 mV to -20 mV in an SNaIIA cell and in an SNaIIAbeta1-16 cell. B, voltage dependence of Na channel activation and inactivation in cells expressing alpha subunits alone and in four independent cell lines expressing alpha + beta1 subunits. Mean conductance-voltage curves (open symbols) and inactivation curves (filled symbols) for control cells (bullet), SNaIIAbeta1-16 cells (), SNaIIAbeta1-D1 cells (), SNaIIAbeta1-15 cells (), and SNaIIAbeta1-D3 cells (). Conductance-voltage and inactivation curves for individual experiments were fit with Boltzmann functions by a least squares technique as described under ``Experimental Procedures.'' Mean curves were calculated from the mean values of V and k obtained from the fits. Mean values for fits to inactivation curves for each cell line are reported in Table 2. Mean values of V and k for fits to conductance-voltage curves for each cell line are reported in Table 3.







The voltage dependence of Na channel inactivation was determined using 200-ms-long prepulses in 4 cell lines co-expressing alpha and beta1 subunits and compared to the parent cell line expressing alpha alone. Measurements were obtained after 10 min of recording to minimize effects of the spontaneous negative shift in the voltage dependence of activation and inactivation in fluoride-containing intracellular solution that had largely stabilized by this time. In each cell line expressing beta1 subunits, the voltage dependence of inactivation was shifted 10 to 13 mV negative to the parent cell line (Fig. 5B and Table 2). Effects on the voltage dependence of activation were more variable with activation being shifted 2 to 11 mV negative relative to the parent cell line in beta1-expressing lines (Fig. 5B and Table 3). Voltage dependences of activation and inactivation were also studied using an aspartate-based intracellular solution. With this intracellular solution, as opposed to the fluoride-based intracellular solution, shifts in voltage-dependent parameters during intracellular dialysis in the whole cell voltage clamp are minimal. However, it was much more difficult to obtain and maintain high resistance seals on these transfected cells without fluoride, preventing collection of a large set of recordings under these conditions. In the aspartate-based intracellular solution, voltage dependences of activation and inactivation were more positive than in the fluoride-based solution ( Fig. 6and Table 4). However, the half-activation and inactivation voltages were shifted -6 mV and -11 mV, respectively, in the SNaIIabeta1-16 cell line expressing the beta1 subunit. Thus, in this solution, as in the fluoride-based solution, the voltage dependences of activation and inactivation for SNaIIAbeta1-16 were more negative than for the parent SNaIIA cell line. These results confirm the more complete data in the fluoride-containing intracellular solution ( Table 2and Table 3), demonstrating a significant negative shift in activation and inactivation in this cell line due to co-expression of the beta1 subunit of the Na channel.


Figure 6: Voltage dependence of Na channel activation and inactivation in cells expressing alpha subunits alone or alpha + beta1 subunits measured using aspartate intracellular solution. Mean conductance-voltage curves (open symbols) and inactivation curves (filled symbols) for control cells (circles) and SNaIIAbeta1-16 cells (squares). Conductance-voltage and inactivation curves for individual experiments were fit with Boltzmann functions by a least squares technique as described under ``Experimental Procedures.'' Mean curves were calculated from the mean values of V and k obtained from the fits. Mean values of V and k for fits to conductance-voltage curves and mean values for fits to inactivation curves for each cell line are reported in Table 4.






DISCUSSION

Co-expression of Na channel alpha and beta1 subunits in Xenopus oocytes demonstrated that beta1 subunits play a modulatory role in Na channel function in that cell type (5) . beta1 subunits increased peak current amplitude, shifted the voltage dependence of Na channel inactivation in the hyperpolarizing direction, and accelerated the rates of Na channel activation and inactivation through a mechanism involving an increase in the proportion of channels gating in the fast mode(5, 13) . In the present study, we observed similar modulatory effects for alpha and beta1 co-expression in mammalian somatic cell lines. beta1 subunits cause a hyperpolarizing shift in the voltage dependence of inactivation and increase the functional expression of Na channels at the plasma membrane. In addition, beta1 subunits cause a variable shift in the voltage dependence of Na channel activation in the hyperpolarizing direction. Our results provide the first evidence for modulation of the functional properties of sodium channels expressed in mammalian somatic cells by beta1 subunits and support the hypothesis that beta1 subunits have important modulatory effects on sodium channels in vivo.

Previous reports have demonstrated the efficient expression of functional Na channels in various mammalian cell lines by transfection of alpha subunits of rat brain sodium channels alone(12, 20) . Similar results have been obtained with transient expression of skeletal muscle sodium channels (19) and stable expression of cardiac sodium channels (33) in mammalian cell lines. In each case, transfection of alpha subunits alone resulted in expression of Na channels which activated and inactivated with a rapid time course. It was possible that this fast gating could have resulted from endogenous expression of beta1 subunits by each of the several cell lines used in these previous experiments. Alternatively, the rapid time course of gating could be explained by differences in protein synthesis and processing between mammalian cells and Xenopus oocytes. In the present study, we have demonstrated through Northern blot and immunoblot analysis and through expression of poly(A)-selected mRNA in Xenopus oocytes that the parent 1610 cell line does not express endogenous beta1 subunits or beta1 subunit-like activity. Thus, our results support the conclusion that beta1 subunit expression has direct physiological effects on Na channel activation, inactivation, and expression levels in mammalian cells. The fast gating mode observed for Na channel alpha subunits expressed in mammalian cells is likely to be caused by factors in addition to beta1 subunit expression such as post-translational glycosylation, acylation, or processing, association of Na channel alpha subunits with other proteins such as cytoskeletal components, or the higher temperature at which these processes take place in a mammalian cell.

In Xenopus oocytes, where some of the post-translational events observed in mammalian cells are altered or absent, beta1 subunits appear required to stabilize the alpha subunit in a conformation that gates in the fast mode(13) . While beta1 subunits most likely also contribute to stabilization of the functional state of sodium channels in mammalian cells, other elements are evidently present which exert a sufficient stabilizing effect to ensure Na channel gating in the predominant fast mode in the absence of beta1 subunits. Efficient, stable co-expression of Na channel alpha and beta1 subunits now provides a system in which the role of the auxiliary beta1 subunits in Na channel biosynthesis and processing can be assessed, and other cellular factors which influence Na channel functions such as gating mode can be identified.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: DOTAP, N-[1-(2,3-dioleoxy)propyl]-N,N,N-trimethylammonium methyl sulfate; PAGE, polyacrylamide gel electrophoresis; STX, saxitoxin; TTX, tetrodotoxin; kb, kilobase(s).


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