Báo cáo khoa học: Molecular cloning and functional expression of the human sodium channel b1B subunit, a novel splicing variant of the b1 subunit potx

Here, we report the cloning and characterization of a novel, splicing variant of the human b1 subunit by rapid amplification of cDNA end polymerase chain reaction RACE-PCR based on the hu

Molecular cloning and functional expression of the human sodium

Ning Qin1, Michael R D’Andrea1, Mary-Lou Lubin1, Navid Shafaee2,*, Ellen E Codd1and Ana M Correa2 1

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, Spring House, PA, USA;

2

Department of Anesthesiology, University of California, Los Angeles, CA, USA

The voltage gated sodium channel comprises a pore-forming

a subunit and regulatory b subunits We report here the

identification and characterization of a novel splicing variant

of the human b1 subunit, termed b1B The 807 bp open

reading frame of the human b1B subunit encodes a 268

residue protein with a calculated molecular mass of

30.4 kDa The novel human b1Bsubunit shares an identical

N-terminal half (residues 1–149) with the human b1subunit,

but contains a novel C-terminal half (residues 150–268) of

less than 17% sequence identity with the human b1subunit

The C-terminal region of the human b1Bis also significantly

different from that of the rat b1Asubunit, sharing less than

33% sequence identity Tissue distribution studies reveal that the human b1Bsubunit is expressed predominantly in human brain, spinal cord, dorsal root ganglion and skeletal muscle Functional studies in oocytes demonstrate that the human b1Bsubunit increases the ionic current when coex-pressed with the tetrodotoxin sensitive channel, NaV1.2, without significantly changing voltage dependent kinetics and steady-state properties, thus distinguishing it from the human b1and rat b1Asubunits

Keywords: sodium channel; b1Bsubunit; splicing variant

By mediating the rapid entry of sodium ions into excitable

cells in response to voltage changes across the plasma

membrane, voltage gated sodium channels (VGSCs) play a

fundamental role in the control of neuronal excitability in

the central and peripheral nervous systems The VGSC is a

heteromeric protein complex that comprises at least a large

(200–300 kDa) pore-forming a subunit and several smaller

(30–40 kDa) regulatory b subunits [1–4] It is well known

that sodium channel a subunits determine the basic

properties of the channel, while b subunits modulate the

channel properties Functional studies in a heterologous

system have demonstrated that, depending on the type of

coexpressed a subunit, b subunits are able to modulate

almost all aspects of the channel properties, including

voltage dependent gating, activation and inactivation, as

well as greatly increasing the number of functional channels

present on the plasma membrane [5,6] Currently, at least

nine different a subunits, three b subunits, and a splicing

variant of the b1subunit, rat b1A[7], have been cloned and

characterized

The rat b1A subunit is a splicing variant of the b1 subunit via intron retention The N-terminal half of the

b1A subunit is identical to that of the rat b1 subunit, whereas its C-terminal half, encoded by a retained intron with an in-frame stop codon, is completely different from that of the rat b1 subunit (to which it shows less than 17% identity) Coexpression of the rat b1A subunit with the pore forming alpha subunit, NaV1.2, in Chinese hamster lung 1610cells, increased the sodium current density and produced subtle changes in voltage depend-ent activation and inactivation [7] To further explore the function and physiological relevance of the sodium channel b1 splicing variant, we first tried to clone the same splicing variant from human tissue Here, we report the cloning and characterization of a novel, splicing variant of the human b1 subunit by rapid amplification

of cDNA end polymerase chain reaction (RACE-PCR) based on the human b1 sequence The novel b1 subunit splicing variant, named b1B, is produced via extension of exon 3 with an in-frame stop codon The human b1B subunit is significantly different from the rat b1A subunit

in sequence, expression pattern and regulatory properties, although they share a similar splicing pattern Functional studies indicate that the human b1B subunit performs a physiological function distinct from that of the human b1 subunit when it is coexpressed with NaV1.2 in Xenopus oocytes

Experimental procedures

Molecular cloning of the human sodium channel b1B

subunit Full-length human b1B cDNA was cloned using a strat-egy that combined reverse transcription polymerase chain

Correspondence to N Qin, Drug Discovery, Johnson & Johnson

Pharmaceutical Research and Development, PO Box 776, Welsh and

McKean Roads, Spring House, PA 19477-0776, USA.

Fax: + 1 215 628 3297, Tel.: + 1 215 5404886,

E-mail: nqin@prdus.jnj.com

Abbreviations: DRG, dorsal root ganglia; RACE-PCR, rapid

ampli-fication of cDNA end-polymerase chain reaction; RT-PCR, reverse

transcription–polymerase chain reaction; VGSC, voltage gated

sodium channel.

*Present address: Royal College of Surgeons, Dublin, Ireland.

(Received 28 July 2003, revised 6 October 2003,

accepted 15 October 2003)

reaction (RT-PCR) and RACE-PCR Marathon-ReadyTM

human adrenal gland and fetal brain cDNA libraries were

purchased from Clontech (Palo Alto, CA, USA) The

RACE-PCR was performed according to the supplier’s

instructions The reaction mixture (50 lL final volume)

contained 5 lL of Marathon-ReadyTM human adrenal

gland cDNA, 200 lM dNTP, 200 nMAP1 primer

(Clon-tech), 200 nM human b1 subunit specific primer (SB1-10:

5¢-TGGACCTTCCGCCAGAAGGGCACTG-3¢), and

1 lL of 50· Advatage2 DNA polymerase mixture

(Clon-tech) The thermal cycling parameters for RACE–PCR were

as follows: an initial denaturation at 94C for 30s; five cycles

of 94C for 5 s and 72 C for 4 min; five cycles of 94 C for

5 s and 70C for 4 min; and 20cycles of 94 C for 5 s and

68C for 4 min The RACE–PCR product was then cloned

into the PCR-ScriptTM Amp Cloning vector (Statagene,

La Jolla, CA, USA), according to the protocol provided

by the supplier

The full-length b1Bsubunit was cloned from the

Mara-thon-ReadyTMhuman fetal brain cDNA library based on

the C-terminal sequence of human b1B subunit obtained

using the RACE-PCR The PCR was performed in a final

volume of 50 lL, containing 5 lL of Marathon-ReadyTM

human fetal brain cDNA, 5 lL of 10· reaction buffer,

200 lM dNTP, 200 nM SB1-6 primer (5¢-GCCATGGG

GAGGCTGCTGGCCTTAGTGGTC-3¢) and SB1-19

pri-mer (5¢-GTGTGCCTGCAGCTGCTCAA-3¢), and 1 lL

of 50· HF2 DNA polymerase mixture (Clontech) Four

independent clones were selected and subjected to double

stranded DNA sequencing analysis All four independent

clones from the human fetal brain were found to contain

sequences identical to that of the RACE-PCR cloned b1B

subunit from human adrenal gland

Generation of polyclonal antibody

A peptide (RWRDRWQAVDRTGC), derived from the

C terminus of the human b1B subunit, was synthesized

and used for raising polyclonal antibodies in rabbits

(This peptide was chosen because the seqeunce shows

the highest homology between human and rat b1A

subunits.) The antibody was raised and affinity purified

by BioSource International, Inc The resulting affinity

purified antibody was used for immunohistochemical

analysis

Northern blot analysis

Human Multiple Tissue Northern blot (MTNTM) and

human Brain II MTNTM blot were purchased from

Clontech The cDNA fragment encoding residues 217–

268 of the human b1Bsubunit was used as a probe The

antisense single stranded DNA probe was synthesized

using the Strip-EZTM PCR kit (Ambion, Austin, TX,

USA), in the presence of antisense primer SB1-20(5¢-TC

AAACCACACCCCGAGAAA-3¢) and [32P]dATP[aP]

(3000 CiÆmmol)1; Amersham Pharmacia Biotech.),

follow-ing the manufacturer’s instructions The labeled probe was

then separated from free [32P]dATP[aP] using a

Micro-SpinTMG-50column (Amersham Pharmacia Biotech.) The

cDNA fragment encoding the human b1subunit from amino

acids 150to 218 was used as a human b subunit specific

probe The single stranded antisense b1specific probe was labeled and purified as described above A 2 kb human b-actin cDNA fragment was used as the control probe and labeled with Ready-To-GoTM DNA Labelling Bead (–dCTP) (Amersham Pharmacia Biotech.), followed by purification as described above

The blots were prehybridized with 5 mL of UltraHyb Solution (Ambion), at 42C for 2 h, and then hybridized

in the presence of 1· 106c.p.m.ÆmL)1probe (b1B, b1and b-actin separately) at 42C overnight The blots were washed with 2· 200 mL of 0.2· NaCl/Cit/0.1% SDS, at

65C for 2 h Finally, the blots were exposed to X-ray film in a )80 C freezer for 2–18 h The same blots were used for all three probes (b1B, b1 and b-actin) after stripping at 68C for 15 min and reconstitution at room temperature for 15 min using the Strip-EZTMremoval kit (Ambion)

Immunohistochemistry Protocols for immunohistochemistry have been described previously [8] All incubations were performed at room temperature After microwaving the slides in Target (Dako, Carpenturia, CA, USA), the slides were placed

in NaCl/Piand then in 3% H2O2, rinsed in NaCl/Piand then the appropriate blocking serum was added for 10min Subsequently, primary antibody, rabbit polyclonal anti-(human b1B), at a titer of 1 : 200, was applied to the slides for 30min After several washes in NaCl/Pi, a biotinylated secondary antibody (Vector Laboratories) was placed on the slides for 30min Subsequently, the slides were washed in NaCl/Pi and the avidin–biotin complex (ABC; Vector Laboratories) was applied to the cells for 30min The presence of the primary antibody was detected after two 5 min incubations in 3¢-diaminobenzi-dine-HCl (Biomeda, Foster City, CA, USA) Slides were briefly exposed to Mayer’s hematoxylin for 1 min, dehy-drated and coverslipped Antibody specificity controls included (a) replacement of the primary antibody with nonimmune serum, (b) omission of the primary antibody with the antibody dilution buffer (Zymed Laboratories Inc., San Francisco, CA, USA), and (c) preincubation with specific antigen (preabsorption) Preabsorption was carried out using a 10-fold titer excess of the antigen peptide preincubated with antibody overnight at 4C This mixture was then used as the primary antibody b1B subunit immunolabeling was not detected in the preab-sorption controls or in other negative controls Specimens were examined and photographed using an Olympus BX-50microscope

In vitro synthesis of cRNA The expression constructs of b1Band NaV1.2 were linearized following digestion with restriction enzymes The cRNAs were synthesized in vitro with T7 RNA polymerase using reagents and protocols from the mMESSAGE mMACHINETM transcription kit (Ambion), except that the LiCl precipitation was repeated twice To ensure full-length clones of the NaV1.2 a subunit, the reaction mixture was supplemented, halfway through transcription, with additional enzyme and nucleotides The cRNAs were

suspended in diethylpyrocarbonate-treated H2O at a final

concentration of 1–2 mgÆmL)1

Oocyte preparation and RNA injection

Conventional methods were followed for oocyte isolation

and removal of the follicular membrane [9] Adult female

Xenopus laevis(Xenopus One, Ltd, Dexter, MI, USA) were

anesthetized by immersion in 0.1% tricaine Ovaries

were removed through an abdominal incision Ovarian sacs

were rinsed in Ca2+-free medium and teased apart to expose

the oocytes The follicular layer was removed by

treat-ment with collagenase (200 UÆmL)1; Gibco) in Ca2+-free

medium, followed by rinsing and storage in saline medium

containing Ca2+and 50 lgÆmL)1gentamicin Stage V–VI

oocytes were separated for injection the following day

Normally, 20–25 oocytes per RNA sample were

micro-injected, each with 50nL of 1 mgÆmL)1cRNA

Combina-tion of subunits was obtained by injecting the premixed

cRNAs Microinjection was performed under sterile,

RNase-free conditions After injection, oocytes were

maintained at 18C

Recording solutions

External and internal recording solutions contained mostly

impermeant anions and were made iso-osmolar to the

oocyte media (120mM; 220–240 mOsmÆkg)1) Sodium

currents were recorded in external 120mMsodium methane

sulfonate, 1.8 mM CaCl2, 10 mM Hepes-sodium, pH 7.2;

and internal 120mM cesium methane sulfonate, 10mM

sodium methane sulfonate, 10mM Hepes-sodium, 1 mM

EGTA-sodium, pH 7.2 Voltage electrodes were filled with

2.7M TMA, which comprised 2.7M tetra

methyl-ammo-nium, 10mMNaCl, and 10mMHepes-sodium, pH 7.0

Recording and analysis of macroscopic ionic

and gating currents

The cut open oocyte Vaseline gap technique [10] was used

to record macroscopic ionic currents This technique,

described previously [11], greatly improves the temporal

resolution over that of the conventional two-electrode

voltage clamp The currents were recorded from an area of

the oocyte equivalent to 20–25% of the total surface

Voltage electrodes had resistances of 0.2–0.5 MW

Custom-made software and hardware were used for acquisition and

analysis of data Leakage and linear capacity currents were

subtracted by using P/4 protocols Data were sampled once

every 5 ls and were filtered at 1/5 of the sampling frequency

Conventional pulse protocols were used to record

mac-roscopic sodium currents in response to changes in

mem-brane voltage Test pulses of 15 ms were applied from

holding potentials of )80or )100 mV; the range of test

potentials used to cover the whole activation curve was

typically)60mV to 100mV, at 5 mV intervals For

steady-state inactivation curves, a 15 ms test pulse to 0mV was

preceded by a preconditioning 100 ms pulse spanning

)140mV to 20mV, at intervals of 10mV Conductance

vs voltage (G-V) curves were obtained from the I-V plots

fitting the data to: I¼ G(V)Æ(Vm–Vrev), where I is the

current amplitude, G(V) is the voltage-dependent

conduct-ance, Vmis the membrane voltage, and Vrevis the voltage for current reversal Once Vrevwas determined from the I-V fits, the individual I-V curves were divided by Vm–Vrevto obtain the G(V) The G-V plots were fitted to: G¼ Gmax/ (1 + exp[–zÆ(V-V½)/25]), where Gmax is the maximum conductance, z is the valence of the process and V½is the midpoint voltage of activation

Ionic current expression levels were determined from batches of oocytes injected with NaV1.2 alone or with

NaV1.2 combined with b1Bsubunit at an a : b ratio of 1 : 5

or 1 : 20 Only data from batches expressing all three a : b combinations were included in the analysis Unpaired t-test statistics were used to compare the different current amplitude data sets

Results

Cloning and analysis of the human VGSC b1Bsubunit

In order to clone the human b1 splicing variant, we first used RT-PCR with a forward primer based on the human b1subunit and degenerated reverse primers based

on the rat b1A C-terminal sequence However, these attempts failed to clone the human b1A subunit from cDNA libraries of adrenal gland, brain and fetal brain

BLASTsearches of the human genomic sequence with the cDNA encoding the rat b1A subunit C terminus also failed to identify any homologous region in the human

b1 gene These results suggested that, if the human b1 gene also undergoes alternative splicing, it might have a very different sequence from that of the rat b1A subunit Therefore, a RACE-PCR technique was applied to clone

a novel, splicing variant of the human b1 subunit To perform RACE-PCR of a novel C terminus of the human

b1subunit, we designed a forward primer (SB1-10) based

on the N-terminal cDNA sequence The resulting RACE-PCR product was directly cloned into the PCR cloning vector As the human b1 subunit specific primer was used for 3¢ extension, both the b1 subunit and its splicing variant would be amplified and cloned

To exclude the b1 subunit clones, each individual clone was characterized by PCR using a pair of primers for specific amplification of the b1 subunit C terminus All PCR negative, non-b1 subunit clones were subjected to further sequencing analysis, which revealed that one of the clones had a continuous reading frame from the N-terminal sequence of the human b1 However, the C-terminal sequence was significantly different from that

of the human b1, suggesting that it might encode a novel splice variant of the human b1subunit.BLASTsearches of the National Institutes of Health (NIH) database with this sequence also identified a shorter, but identical, unannotated EST clone (accession number: AI742310) in the human EST database, which was cloned from a pool

of five normalized cDNA libraries

Based on the novel C-terminal sequence of the human b1 subunit obtained by RACE-PCR, a full-length splice variant was cloned from the human fetal brain cDNA library The full-length cDNA contained a 979 bp sequence encoding 268 amino acids and a 172 bp 3¢ untranslated region (GenBank accession number: AY391842) The amino acid sequence deduced from the cDNA sequence is

Fig 1 Sequence analysis and genomic

struc-ture of the human sodium channel b 1B subunit.

(A) Amino acid sequence comparisons

between human b 1B and b 1 subunits The

signal peptide sequence and transmembrane

domain (TM) are indicated in (A), and an

IgG-like motif is located between residues 22

and 150 (B) C-terminal amino acid sequence

comparisons of human b 1B, rat b 1A and

putative mouse b 1A , which is predicated based

on the mouse genomic sequence Conserved

residues are underlined (C) Genomic

struc-ture of the human b 1 gene, SCN1B The

SCN1B gene spans  9 kb on chromosome 19

across six exons Exon 3A is an extended exon

3 (retention of part of intron 3) via alternative

splicing Exons 1, 2, 3, 4 and 5 (solid boxes)

encode the b 1 subunit, while exons 1, 2 and 3A

(solid and diagonally shaded boxes) encode

the b 1B subunit The 5¢ and 3¢ untranslated

regions are indicated by solid thin lines (b 1 )

and shaded thin lines (b 1B ), and the

unidenti-fied 3¢ untranslated region of b 1B is indicated

using the thin interrupted broken line The

stop codon is indicated by an asterisk The

RACE-PCR primer, SB1-10(indicated by an

arrow), is located at the end of exon 2.

Table 1 Intron–exon boundary sequence of the sodium channel b 1 /b 1B gene (SCN1B) UT, untranslated.

Exon (bp)

cDNA

Exon 1 (> 136) ) 89 to +22 1–14 GCA CTG G– – gtgagt Intron 1 (1.67)

Exon 2 (166) 23–207 14–69 ccacag –TG TCC TCA TTT GTC AAG gtgtgc Intron 2 (1.80)

Exon 3 (240) 208–458 70–149 ccctag ATC CTG CGC GAC AAA G– – gtgagt Intron 3 (5.38)

Exon 3A (> 770) 208–978 70–268 ccctag ATC CTG CGC GTG GTT TGA xxxxxx Intron 3A (?)

Exon 4 (141) 459–580150 –197 ctgcag –CC AAC AGA GAG AAT GC– gtgagt Intron 4 (0.38)

Exon 5 (71) 581–662 198–218 ccacag – –C TCG GAA TAG CCC TG– gtaagg Intron 5 (0.09)

Exon 6 (> 641) 663–1307 3¢ UT (b1) cttcag GCC CTG GGC

shown in Fig 1A The open reading frame, designated b1B,

is related to the sodium channel b1 subunit Conserved

motifs of the sodium channel b subunit family were also

presented in the human sodium channel b1B subunit,

including a signal peptide sequence, the extracellular

immunoglobulin fold domain and the C-terminal

trans-membrane domain The predicted peptide contained a

hydrophobic N-terminal residue (1–16 residues) with

sequences highly predictive of signal cleavage sites that

would result in mature proteins initiating at amino acid 17

(alanine) The hydrophobic C-terminal region (residues

243–262) may serve as a transmembrane domain The

estimated protein molecular mass was 30.4 and 28.9 kDa

before and after removing the signal peptide from the N

terminus, respectively The in vitro translated human b1B

subunit migrated with an apparent molecular mass of

30kDa (with signal peptide) when analyzed by 8–20%

SDS/PAGE (data not shown) Peptide sequence

compar-ison revealed that the predicted peptide was 72% identical

to both that of human (Fig 1A) and rat sodium channel b1

subunits and rat b1A subunit (Fig 1B) Like the rat b1A

subunit, the human b1Bsubunit contained an N-terminal

region (residues 1–149) of 100% identity to the b1subunit

and a novel C-terminal region (residues 150–268) with an

identity to the b1subunit of less than 17% (Fig 1A) The

C-terminal region of the human b1B subunit was also

significantly different from the rat b1Aand putative mouse

b1A subunits (The amino acid sequence of mouse b1A

subunit is deduced from mouse genomic sequence The presence of such a splicing variant has not been confirmed

by any experiment.) The C-terminal portion of human b1B

shares less than 33% and 36% peptide sequence identity with rat and mouse b1A subunits, respectively, while the same region of rat and mouse b1A shares at least 77% identity (Fig 1B)

A genomic organization study of the human sodium channel b1subunit gene, SCN1B [12], revealed that the gene spans 9 kb over six exons and five introns on chromo-some 19 (19q13.1-q13.2) BLAST searches of the human genomic database, using the cDNA sequence of human b1B, revealed that the N-terminal region of the human b1B subunit (residues 1–149) was encoded by exons 1–3, whereas the novel C-terminal region was encoded by the part of intron 3 adjacent to exon 3 (Fig 1C and Table 1) As the site of divergence between the b1and b1Bsubunit cDNAs was located precisely at the exon 3/intron 3 boundary of the SCN1B gene, the human sodium channel b1B subunit should be considered as a splicing variant of the b1subunit via the extension of exon 3 to intron 3 (or partial intron 3 retention) with an in-frame stop codon

Tissue distribution of the human b1Bsubunit Northern blot analysis, using a human b1Bspecific probe, showed that the b1Btranscript is abundant in human brain and skeletal muscle (Fig 2A), and present at a very low level

Fig 2 Northern blot analysis of the gene expression of human b 1B (A,B) and b 1 (C,D) subunits, using human b-actin mRNA level as the control (E,F) (A), (C) and (E) are human multiple tissue blots; (B), (D) and (F) are human brain II blots The cDNA fragment encoding residues 217–268 of the human b 1B subunit, and the cDNA fragment encoding the human b 1 subunit from amino acids 150to

218, were used as probes for detecting the messages of human b 1B and b 1 subunits, respectively A 2 kb human b-actin cDNA fragment was used as the control probe The blots were incubated at 42 C overnight and washed with 0.2· NaCl/Cit/0.1% SDS at

65 C for 2 h Finally, the blots were exposed

to X-ray film in a )80 C freezer for 2–18 h.The same blots were used for all three probes in the order b 1B , b 1 and b-actin, after they were stripped at 68 C for 15 min and reconstituted at room temperature for 15 min using the Strip-EZTMremoval kit provided by Ambion.

in heart, placenta, lung, liver, kidney and pancreas In

human brain, the b1Btranscript was most abundant in the

cerebellum, followed by the cerebral cortex and occipital

lobe (Fig 2B) The overall expression pattern of human b1B

was very similar to that of human b1(Fig 2C,D), except

that human b1was more abundant in cerebral cortex than in

cerebellum If the transcript of the human b1B subunit is

spliced only from exon 1 (111 bp), exon 2 (185 bp), exon 3

(250bp) and either partial or entire intron 3 (5.3 kb), the

calculated size of the transcript should be less than 6 kb

However, the major transcript of b1B, as determined by

Northern blot, is 7.5 kb This suggests that an additional

unidentified splicing event must be present to generate a

longer 3¢ untranslated region, which needs to be identified

by further experiments In addition, a second transcript of

the human b1B, of  1.5 kb, was observed in skeletal

muscle

Expression of the novel b1Bsubunit was further

investi-gated by immunohistochemistry with affinity purified

anti-b1B (see Experimental procedures) The anti-b1B was

generated against a peptide derived from the retained intron

3 in the human cDNA clone As shown in Fig 3,

immunohistochemical analyses revealed that the b1B sub-unit was expressed in many different regions in the human brain, including cerebellar Purkinje cells (Fig 3A), cortex pyramidal neurons, and many of the neuronal fibers throughout the brain (data not shown), consistent with the results of Northern blot analysis Strong immunolabe-ling was also observed in human dorsal root ganglion (DRG) (Fig 3C), in fibers (arrowheads) of the spinal nerve (Fig 3D) and in cortical neurons (large arrowheads) and their processes (small arrowheads) (Fig 3E) The specific

b1Blabeling in the Purkinje cells (arrowhead) was abolished when the primary antibody was preabsorbed with the specific peptide

Functional expression of the human b1Bsubunit with NaV1.2 inXenopus oocytes

To explore the regulatory function of the human b1B subunit, we injected cRNA of human b1B, as well as cRNA

of the sodium channel pore forming subunit NaV1.2, into Xenopus oocytes As shown in Fig 4A–D, the rates of activation and inactivation of the sodium current via

Fig 3 Immunohistochemical analysis of b 1B subunit expression in human tissues (A) The presence of b 1B in Purkinje cells (large arrowheads) and in their processes (small arrowheads) of the cerebellum (B) Specific b 1B labeling was abolished in the Purkinje cells (arrowhead) when the primary antibody was preabsorbed with the specific peptide (C) Human b 1B was also detected in dorsal root ganglia (large arrowheads) as well as in the surrounding capsule cells (small arrowheads) (D) b 1B was present in fibers (arrowheads) of the spinal nerve (E) b 1B was present in cortical neurons (large arrowheads) and their processes (small arrowheads) Bar ¼ 25 lm (A, B, D, E); 50 lm (C).

Fig 4 The effect of b 1B and b 1 subunits on the function and expression levels of the Na V 1.2 channel expressed in Xenopus oocytes Representative sodium current traces from oocytes expressing the sodium channel a subunit, Na V 1.2, in the absence (A) and presence of b 1 (B) and b 1B (C) subunits Sodium currents were evoked by 15 ms long depolarizing pulses (as indicated) from a holding potential of )80mV (D) The effect of b 1B and b 1 subunits on current time courses Representative currents at )10 mV in the presence or absence of b 1B or b 1 subunits are shown normalized

to their individual peak value (E) Inset: current-voltage relationship (I-V curve) for Na V 1.2 alone (s) and Na V 1.2 : hb 1B (1 : 5) (d) Sodium currents were evoked by 15 ms long depolarization steps, ranging from )60to 80mV, at 10mV increments, from a holding potential of )100 mV The peak magnitude of the currents, elicited by test depolarizations to the various potentials, were measured and used to construct I-V curves Data represent average currents from a single batch of oocytes Main panel: voltage dependence of the conductance (G-V curve) Data from the average G-V curves for Na V 1.2 alone (s), Na V 1.2 : hb 1B (d), and Na V 1.2 : hb 1 ( d ) were fitted to G ¼ G max /(1 + exp[–zÆ(V-V ½ )/25]) with parameters

G max , z, and V, as described in the text Curves were normalized by dividing by G max (F) Voltage dependence of inactivation (steady-state inactivation curves) Channels were inactivated by 100 ms conditioning pulses ranging from )140to 20mV, at 10mV increments, then activated by

a 15 ms test pulse to 0mV (symbols as in part E: main panel) The relative fraction of channels available for activation was measured as the peak current during the test pulse to 0mV Data from individual oocytes were fitted by I ¼ I max /G ¼ G max /(1 + exp[–zÆ(V-V ½ )/25]) + I min and normalized by I m /I max obtained from the fit All data points (E, F) correspond to the mean ± SEM of the averaged normalized currents for the number of oocytes indicated (G) Effect of b 1B on the current amplitude of Na V 1.2 Each individual data point in the histogram represents the peak inward current for a single oocyte Also shown are the mean (j) and standard deviation (bars) for each cRNA a:b ratio, and the sample size per cRNA combination is shown in parenthesis Data are from five different batches of oocytes, each batch injected with cRNA for the a subunit alone

or with the human b 1B subunit at ratios of 1 : 5 and 1 : 20 Unpaired t-test statistical analysis resulted in P-values of 0.056 (a vs a:b 1B ; 1 : 5), 1.3e-5 (a vs a:b ; 1 : 20 ) and 0 0 35 (a:b 1 : 5 vs a:b ; 1 : 20 ).

NaV1.2 did not change significantly in the presence or

absence of the b1B subunit, whereas, under the same

conditions, the rate of inactivation was increased in the

presence of the b1subunit The effects of b1Bwere further

assessed by studying the current-voltage (I-V) relationships,

the voltage-dependence of the conductance (G-V curve),

and the voltage dependence of steady-state inactivation

Except for a minor negative shift (3–4 mV) in the

voltage-dependence of activation (Fig 4E), no significant effect of

the human b1Bsubunit on the regulation of NaV1.2 sodium

channel properties was observed (Fig 4E,F, and Table 2)

Under the same conditions, the human b1 subunit also

shifted the G-V relationship left, to a similar extent (Fig 4E

and Table 2), but caused a significant shift of the steady

state inactivation curve by 10mV, towards more negative

potentials (Fig 4F and Table 2) Although no significant

modulatory effect of the b1Bsubunit on channel kinetics and

steady-state properties was observed, we found that the b1B

subunit increased the ionic current conducted by NaV1.2

sodium channels (e.g Fig 4E, inset) At cRNA ratios of

1 : 5 and 1 : 20 (NaV1.2 : b1B), the average (n¼ 16–22)

peak ionic current densities were increased by two- and

threefold, respectively (Fig 4G) Despite significant

vari-ability in current densities within and between batches of

oocytes, statistical analysis indicated that the difference

between NaV1.2 expressing oocytes and those expressing

NaV1.2 : b1B at a ratio of 1 : 20was significant

(P < 0.0001)

Discussion

We report here the cloning and characterization of the

human VGSC b1B subunit The human b1Bsubunit is a

novel splicing variant of the b1 subunit via alternative

intron 3 retention The retained intron encodes a novel

extracellular, a transmembrane, and an intracellular region,

sharing little homology with the human and rat b1(17%

identity) and the rat b1A(33% identity) subunits Although

the novel b1Bsubunit has a structure similar to other sodium

channel b subunits, it exhibits regulatory properties in

Xenopus oocytes that distinguish it from the b1 and b1A

subunits

It is interesting that the only regulatory function of the

b1Bsubunit, observed in this study, was its ability to increase

the sodium current density when coexpressed with the

tetrodotoxin sensitive channel, NaV1.2, in oocytes without

affecting any of its voltage dependent properties Several

previous studies have shown that the b1subunit not only

increases the levels of functional sodium channel on the cell

surface, but that it also changes voltage dependent

activa-tion and inactivaactiva-tion [3,4,13] In the present study, we also observed that the simultaneous injection of b1with NaV1.2 into oocytes resulted in an increase of the inactivation rate and a shift of the steady state inactivation curve to a more negative potential ( 10mV), as well as an increase in ionic current amplitude (not shown), consistent with other studies

in oocytes [3] However, under the same conditions, the b1B subunit had little effect on the properties of NaV1.2 The increase in ionic currents induced by coexpression of the human b1Bcould result from an increase in the number of channels present in the membrane, No, an increase in the probability of opening of the channels, Po, and/or an increase in the single channel conductance, co Discrimin-ation among these options, however, requires evaluDiscrimin-ation of single channel parameters by other means (single channel recording or mean-variance analysis)

The modulatory property of the b1B subunit is also different from that of the b1Asubunit reported by Isom’s group In their studies [7], the coexpression of b1A with

NaV1.2 in Chinese hamster lung cells resulted in a 2.5-fold increase in the sodium current density, slightly shifted the steady state inactivation curve to a more positive potential (which also distinguishes it from the b1subunit) and had no effect on channel activation However, we are unable to rule out the possibility that the different regulatory properties observed between rat b1Aand human b1Bresults from the use of different expression systems in the two studies Although b1, b1Aand b1Bhave different effects on NaV1.2 channel properties (b1affects both activation and inactiva-tion, b1Aaffects inactivation only, and b1Bhas no effect on either), the subunits all share a common regulatory prop-erty, i.e they increase the sodium current density regardless

of expression system These results suggest that alteration of channel kinetics and steady state properties may be a function distinct from the increase in current density on the cell surface induced by b1 subunits The functional differ-ences of the b1, b1A and b1B subunits suggest that the C-terminal half of b1 and its splicing variants play an important role in the modulation of sodium channel properties Based on the sequence differences between b1 and its splicing variants, there are three regions on the human b1Bsubunit that may alter its regulatory properties (a) the additional extracellular region ( 90residues in the human b1Bvs. 55 residues in rat b1A), (b) the transmem-brane domain, and (c) the intracellular region The trans-membrane region of the human b1B is located at the C terminus (241–262 residues) with five intracellular residues This unique structure of the human b1B subunit is very similar to that of the calcium channel a2d subunit, which also has six residues downstream from the transmembrane

Table 2 Steady-state properties of the sodium channel Na V 1.2 in the presence or absence of the b 1B or b 1 subunit.

Activation:

G = G max /(1 + [exp( )z *

(V )V 1/2 )/25])

Inactivation:

I = I max /(1 + [exp( )z *

(V )V 1/2 )/25]) + I min

domain serving as an intracellular segment [14] To date, no

regulatory function related to the single transmembrane

domain and the short, five-residue intracellular segment of

the calcium channel a2d subunit has been reported, except

that the transmembrane domain is essential for anchoring

the protein into the membrane [14] Therefore, the

addi-tional extracellular region is probably responsible for the

differences of regulatory functions between the b1Band b1

subunit or rat b1Asubunit Recently, Meadows et al [15]

reported that the intracellular segment of the b1subunit is

required for the interaction with the a subunit, probably a

crucial step for the regulation of channel properties

The tissue distribution of the human b1Bsubunit is similar

to that of the human b1subunit Its message was detected in

skeletal muscle and in a variety of subregions in the brain

(Figs 2 and 3) More interestingly, the human b1Bsubunit is

also expressed in DRG neuron and fibers (Fig 3) It is well

known that the number of functional sodium channels and

magnitude of sodium currents are differentially changed

following peripheral nerve injury [16–19] Our observations

of the existence of the b1Bsubunit in human DRG, and its

ability to increase sodium current density when coexpressed

with NaV1.2a in Xenopus oocytes, suggest that the human

b1Bsubunit may be another candidate useful for studying

the mechanism of upregulation of functional sodium

channels on the cell surface and increasing the rate of

spontaneous firing in peripheral neurons after nerve injury

It will be interesting to determine whether the human b1B

subunit is up-regulated, and whether its up-regulation is

correlated with the increase of sodium channel activity, in

injured human DRG neuron

Acknowledgements

We thank Drs Mike X Zhu, Rich R Ryan and Yi Liu for their critical

discussion of the manuscript, Ms S Yagel for her help in subcloning,

and Ms Patti A Reiser, Norah A Gumula, Brenda M Hertzog and

Debbie Polkovitch for their histological and immunohistochemical

expertise.

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