Molecular Brain Research, 7 (1990) 105-113
Elsevier
105
BRESM 70180
Toxin and kinetic profile of rat brain type III sodium channels
expressed in Xenopus oocytes
Rolf H. Joho 1, J. Randall Moorman 2, Antonius M.J. VanDongen 1, Glenn E. Kirsch 1,
H a n n a Silberberg 1, Gabriele Schuster I and A r t h u r M. B r o w n 1
1Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030 (U.S.A.) and 2Departrnent of
Medicine, University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.)
(Accepted 15 August 1989)
Key words: Sodium channel; Xenopus oocyte; Toxin
Sodium (Na ÷) channels are members of a multigene family and are responsible for generation and propagation of the action potential in
excitable cells. We have assembled, in a transcription-competent vector, a full-length cDNA clone encoding the rat brain type III Na ÷ channel.
Xenopus oocytes microinjected with in vitro synthesized mRNA expressed functional rat brain Na ÷ channels from such 'cloned' RNA
transcripts, We found that type III Na ÷ currents in whole cell microelectrode voltage clamp and in cell-attached patch recordings decayed much
more slowly than any other reported Na ÷ current. In addition, we saw typical and additive effects of a- and fl-scorpion toxins, suggesting that
the Na + channel a-subunit itself contains functional and distinct toxin binding sites.
INTRODUCTION
Voltage-gated Na ÷ channels are large transmembrane
glycoproteins whose functional behavior has been studied
extensively. Although the primary amino acid sequence
is known, virtually nothing is known about the precise
structures responsible for the two characteristic properties of these proteins: voltage sensitivity and ionic
selectivity. Several c D N A clones encoding different rat
brain Na + channels have been isolated using a c D N A
probe for the Na ÷ channel of Electrophorus electroplax
to detect cross-hybridizing sequences in a rat brain c D N A
library 3'18'28. The sequences of two clones (types Ia and
Ib) are identical with the exception of a 33 nucleotide
insert/deletion corresponding to 11 amino acids in the
coding region. Two other c D N A clones (types II and III)
have been isolated from the rat brain. They are approximately 90% homologous to type I and to each other. The
most recently reported clone differs from type II by 6
amino acid changes and has been named I I A 3. Northern
blot analyses of m R N A from rat brain using Na ÷
channel c D N A clones as radioactively labeled probes
show several large m R N A species migrating at 9-10
kb28, 37.
Full-length c D N A clones covering the entire coding
region of the 3 brain channel types have been assembled
in transcription-competent vectors and the resulting in
vitro R N A transcripts have been injected into Xenopus
oocytes 3'29'37. Currents through expressed voltage-gated
Na ÷ channels could be detected with voltage-dependence
and tetradotoxin (TI'X) sensitivity similar to native brain
Na ÷ channels. However, only oocytes injected with Na +
channel type II-, IIA-, and Ill-specific m R N A s showed
substantial inward sodium currents upon depolarization;
oocytes injected with type Ia- and Ib-specific m R N A had
only very small currents or none at all 29. The functional
differences and the physiological significance of the
different Na ÷ channel types encoded by these 3 m R N A s
in the rat brain are not yet clear.
To begin studies of the structure-function relationship
of the Na ÷ channel, we have isolated and assembled the
rat brain type III Na ÷ channel in a transcriptioncompetent vector, and we have used in vitro synthesized
R N A transcripts to express type III Na + currents in
microinjected Xenopus oocytes 8. We show that the decay
of macroscopic type III Na ÷ currents is much slower than
for native neuronal Na ÷ currents or those expressed by
oocytes injected by total brain R N A , or type II- or
IIA-specific R N A . In addition, we show that both a- and
fl-scorpion toxins from Tityus serrulatus have characteristic effects on the gating of the expressed type III Na ÷
channel.
Correspondence: R.H. Joho, Dept. of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX
77030, U.S.A.
0169-328X/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
106
MATERIALS AND METHODS
Recombinant DNA technology
Unless otherwise specified, standard techniques were used to
handle RNA, recombinant DNA, library screening and subcloning6'
24. After cloning in bacteriophage lambda vectors, the DNA inserts
were subcloned either into M13 mpl8 or 19 for DNA sequencing
purposes, or into pBluescript (Stratagene) for bulk growth and
assembly of full-length cDNA clones that could be used for in vitro
transcription.
Construction of rat brain cDNA libraries enriched for long inserts
Library construction is described in detail elsewhere9. Briefly,
total cellular RNA was isolated from frozen brains from adult
Wistar rats using the urea/LiCl method 2. Poly(A) + RNA was
selected by one cycle of oligo(dT)-cellulose chromatography,
precipitated with ethanol, dissolved in water, and stored at -80 °C
for subsequent use. To produce a cDNA library with large inserts,
we size-fractionated poly(A) + RNA on a 10-30% sucrose gradient.
Starting with an oligo(dT) primer and an RNA template bigger than
5 kb, we were able to obtain cDNA up to 6-9 kb in length although
the bulk of the first strand product migrated between 2 and 4 kb on
an alkaline agarose gel. Second strand synthesis was done according
to Gubler and Hoffman ~2, and after methylation and linker
addition, long cDNA was selected by preparative electrophoresis
through a neutral agarose gel (BioRad). Using 50 ng cDNA of
2.5-6.5 kb, we obtained 50,000 recombinants in 2 gtl0.
Partial cDNA libraries enriched for Na + channel inserts were
generated in 2 ZAP (Stratagene) using two Na + channel-specific
oligonucleotides as primer. Forty/~g of poly(A) + RNA were mixed
with 1.0/@ of each specific primer in 20/~1 10 mM HEPES-HCI (pH
6.9), 0.2 mM EDTA. The mixture was heated for 2 min to 90 °C,
then cooled on ice. First and second strand syntheses were done as
described9, except that EcoRI linker were used for cloning into the
EcoRI site of 2 ZAP. cDNA larger than 1000 bp was selected on an
agarose gel, and 50 ng of purified cDNA yielded 106 pfu in 2 ZAP.
To isolate Na + channel III-specifie 5" end clones, we used an
oligonucleotide specific for the type III sequence as a 3zP-labeled
screening probe.
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Fig. 1. Construction of Na + channel cDNA clones. The cDNA
isolates (clones 34, 14, 5 and 3) and the strategy used to generate
a full-length construct are shown in the top part of the figure. The
coding region is depicted with a box, the 5" and 3" untranslated
regions are indicated by the heavy line. The zigzag lines at the
beginnings and at the ends represent the polylinker of pBluescript
(SK(-) (not to scale). The relevant restriction endonuclease sites
used in the construction are shown and are abbreviated as: A =
ApaI; E = Eco47III; S =SalI; X = XhoI. Clone 5 contains 3 inserts
illustrated by the shaded areas on the map. The synthetic 78 bp
XhoI fragment including part of the polylinker sequence is shown
below clone 3. The construct used for in vitro RNA transcription is
depicted in the lower part of the figure. For RNA transcription
purposes, the construct in pBluescript was linearized at the NotI site
shown.
Assembly of full-length cDNA clones for in vitro transcription
The cDNA clones and the important restriction sites that were
used to generate full-length constructs are depicted in Fig. 1. All
isolates and constructs were propagated in pBluescript-SK(-). Clone
3 stopped 14 nucleotides short of the presumptive ATG initiation
codon. To complete the missing 5" end and part of the 5"
untranslated region, we synthesized two complementary oligonucleotides of 78 residues each. The resulting short double-stranded
DNA fragment begins with part of the DNA sequence of the
pBluescript polylinker starting at the Xhol site and including the
following Sail site. The next 14 nucleotides match the 5" untranslated region of channel III with the exception of a T to C transition
(position -7) in order to change an ATG immediately upstream and
out of frame with the initiation triplet. The synthetic fragment
extends from the ATG initiation codon for another 53 nucleotides
encoding the channel III sequence and ends with a XhoI site. This
synthetic 5" end fragment was ligated into XhoI cut and phosphatase
treated clone 3, and a hybrid clone X/3 carrying the 78 bp Xhol
fragment in the correct orientation was selected. The SalI-NsiI
fragment (2203 bp) from X/3 was isolated, ligated into SalI and NsiI
opened clone 34 to generate the construct $34. $34 contains about
700 nucleotides of 3" untranslated region, followed by a unique Notl
site in the polylinker of the cloning vector.
In vitro RNA synthesis and microinjection of Xenopus oocytes
Plasmid templates used for RNA transcription were linearized by
digestion with NotI. The DNA was treated with proteinase K,
extracted with phenol, phenol/chloroform, and concentrated by
precipitation with ethanol. Transcription reactions were performed
in 25 pl volumes containing 40 mM Tris-HCl (pH 8.0), 80 mM NaCI,
8 mM MgCI 2, 30 mM DTT, 2 mM spermidine, 1.0 mM ATP, CTP
and UTP, 0.4 mM GTP, 2 mM m7G(5")ppp(5")G, 40 U RNasin, 25
U T7 RNA polymerase, and 150 ng°of linearized DNA template.
After 30 min at 37 °C, 10 U of RNAase-free DNase I (Boehringer)
were added, and the samples were incubated for an additional 30
min at 37 °C. The transcripts were extracted twice with phenol/
chloroform, and once with chloroform. The samples were adjusted
to 2 M ammonium acetate and precipitated twice with ethanol,
dissolved in water, and stored at -80 °C. The integrity of the
transcripts was tested on a glyoxal gel, and the appropriate
concentrations of RNA were diluted into 100 mM KCI, and 75 nl
samples were injected into Xenopus oocytes.
Oocyte preparation and injection
Our methods for oocyte preparation and injection have been
described previously26. Briefly, laboratoy-reared adult female Xenopus laevis (Xenopus One, Ltd., Ann Arbor, MI) were anesthetized by immersion in 0.1% tricaine methanesulfonate and portions
of ovaries were removed aseptically. Stage V - V I oocytes were
isolated manually and injected with RNA solutions using a
micrometer-driven 10/~1 micropipettor (Drummond Scientific Co.,
Broomall, PA). After 3-6 days incubation in modified Barth's
solution at 19 °C, the follicular layer of cells was removed manually,
and oocytes were tested for Na ~ channel expression. Uninjected
oocytes from the same ovary were assayed for endogenous Na +
channels3°. TTX was obtained from Calbiochem, and scorpion
toxins were the gracious gift of Dr. L.D. Possani.
Electrophysiological recording
Expression of voltage-dependent Na* currents in injected oocytes
were monitored using a two-microelectrode voltage clamp (Axoclamp-2A, Axon Instruments, Burlingame, CA). De-folliculated
oocytes were placed in a recording chamber and perfused with a
solution containing 145 mM NaCI, 2 mM KCI, 1.8 mM CaCI2, 10
mM glucose, and 10 mM HEPES (pH 7.35) and had resistances of
1-5 MQ. Experiments were carried out at room temperature
(20-22 °C). Macroscopic currents were acquired and analyzed using
pCLAMP (Axon Instruments, Burlingame, CA).
For patch clamp recording, the vitelline membrane was removed
manually in a hyperosmolar solution 25. Patch-clamp electrodes
107
(borosilicate glass, Corning 7052 and 8161) were pulled in two
stages, polished, and coated with Sylgard near the tip to reduce the
capacitance. The pipette solution contained 145 mM NaCI, 2 mM
KCI, 1.8 mM CaCI2, 10 mM glucose, and 10 mM HEPES (pH 7.35).
The bath solution contained 150 mM potassium aspartate, 10 mM
EGTA, and 10 mM HEPES (pH 7.35). Gigaohm seals were
obtained in the cell-attached configuration~s, Recordings of macroscopic currents from large electrodes were acquired and analyzed
using pCLAMP. Single-channel recordings were stored on videotape
and analyzed offiine. The analog signal was filtered at 5-10 kHz (-3
dB, 4-pole Bessel), digitized with 12-bit resolution at 20 kHz, and
digitally filtered at 1 kHz (-3 dB, 4-pole Bessel). The technique for
single-channel idealization has been previously described23.
nucleotide insert in clone 5. Both inserts maintain the
reading frame. Therefore, the two isoforms could have
arisen by alternate splicing and differ by 11 or 32 extra
amino acids in channel I and III, respectively. In the
channel models that have been put forward 11'13'14'2°,
these additional amino acids are located in the cytoplasmic region between domains 1 and 2. This interdomain
region contains several sites that undergo cAMP-dependent phosphorylation in vitro and in vivo 32 and therefore
may be of particular importance in channel function.
RESULTS
Construction of full-length Na + channel c D N A s
Fig. 1 depicts the strategy followed to assemble a
full-length cDNA ($34) encoding the rat brain type III
Na ÷ channel. The construct was assembled in pBluescript-SK(-) with the NotI restriction site at the 3" end of
the coding region. R N A transcripts were made in vitro
using T7 R N A polymerase, and the 'cloned' m R N A was
microinjected in Xenopus oocytes for functional studies.
The m R N A sequence obtained from $34 corresponds to
the D N A sequence shown in Fig. 2. The first 37
nucleotides (small letters in Fig. 2) are derived from the
polylinker present in pBluescript, and are followed by an
additional 14 nucleotides preceding the A U G initiation
codon.
Isolation of overlapping sodium channel c D N A clones
A Na + channel clone bearing a nearly full-length
coding region (clone 34 in Fig. 1) was isolated. The
sequence of the 5.8 kb insert of clone 34 was identical
except for 7 nucleotides to the Na ÷ channel type III gene
recently isolated 18 (Fig. 2). The D N A sequence starts in
the first domain (after the $3 segment), shows a long
open reading frame that ends at a position corresponding
to the carboxyl-terminus in channels I, II, and III, and
extends for approximately 700 nucleotides into the 3"
untranslated region.
To isolate the missing 5" end of the cDNA, we
synthesized two oligonucleotides complementary to the
sense sequence of Na ÷ ch,annel type III starting 661 base
pairs (bp) and 1530 bp downstream from the beginning of
clone 34. These oligonucleotides were used to prime
c D N A synthesis, and a partial library, enriched for the 5"
ends of Na ÷ channel m R N A was constructed in 2 ZAP.
Using a type Ill-specific radiolabeled probe, the missing
5" ends were cloned (Fig. 1). Two of the isolates (clones
5 and 14) carried 5" untranslated regions of 223 bp and
625 p, respectively. The third isolate (clone 3) lacked the
first 14 nucleotides of the coding region. In the areas of
overlap the D N A sequences of clones 3 and 14 were
identical to clone 34 (Fig. 2). There are ten base changes
in our completed sequence compared to the previously
reported sequence of Na ÷ channel Ill 18. Two of the
nucleotide changes lead to two conservative amino acid
changes (Fig. 2). Comparison of clone 5 to the known
channel III sequence showed three inserts. The two
inserts closest to the 5" end are 182 and 818 nucleotides
in length (position shown in Fig. 2). Both inserts
interrupt the open reading frame of channel III and lead
to premature termination of translation of the corresponding m R N A . The third insert is 96 nucleotides long
and maintains the reading frame (position and sequence
shown in Fig. 2). Previously, Na ÷ channel I had been
isolated as two isoforms that were identical to one
another with the exception of a 33 nucleotide insert 28.
This insert is at the same position in channel 1 as the 96
Characterization of Na + currents in oocytes injected with
Na + channel type III m R N A
Up to 20 ng of Na + channel transcript $34 were
injected into each oocyte. Four to 6 days later, oocytes
had expressed inward currents which we measured with
two-microelectrode voltage clamp and cell-attached
patch clamp. Identification of these currents as arising
from voltage-dependent Na + channels rests on the
evidence that (a) the current was highly sensitive to
tetrodotoxin ( T r X ) ; (b) the reversal potential was 43
mV, a value expected for Na + current, after inactivation
had been removed by TsIV-5, and a-scorpion toxin; (c)
and the inward current peaked at -5 mV, at which point
CI- currents would be outward 5 (E o = -24 mV).
Fig. 3A shows a family of macroscopic type III Na +
currents in oocyte under two-microelectrode voltage
clamp. The most striking difference to Na + currents
endogenous to the oocyte 3° or those expressed after
injection of total brain RNA 3"11 (Fig. 3B), total muscle
RNA 34, total heart RNA 36, type II Na + channel
m R N A 35, type I I A N a + channel m R N A 3, and to native
Na + currents in neuronal cells 19 is a 10-fold or greater
prolongation in the decay of the current. In addition,
time to peak current is prolonged. Since the microelectrode voltage clamp does not accurately clamp the large
and convoluted oocyte membrane 25, we examined
whether the large difference in macroscopic current
108
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ATCGGGCTG•AGCTCTTCATGGGCAACCTGAGGAATAAATGCTTGCAGTGGCCC•CGAGCGATTCAGCTTTTGAAA•CAACACTACTTCCTACTTCAATGGCA•AATGGATTCAAATGGG
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2521
CTGGCAAATGTGGAGGGGCTGTCTGTGCTTCGGTCCTTCAGACTGCTCCGAGTCTTCAAGTTGGCAAAG•CCTGGCCCACA•TGAACATGCTCATTAAGATCATCGGCAACTCGGTGGGC
L
A
N
V
g
G
L
S
V
L
R
S
F
R
L
L
R
V
F
K
L
A
K
S
W
P
T
L
N
M
L
I
K
I
I
G
2641
A
TAAGGAGT GGAGGAACCGGAGG
K
E
W
R
N
R
R
GTGCTCTCCCCACCAGTCTCTC
TT G A G T A T C C G T G G C T C C C T G T T T
TCCC CAAGACGCAATAGCAA~CGAGCATTTT
C
S
P
H
Q
S
L
L
S
I
R
G
S
L
F
S
P
R
R
N
S
K
T
S
I
GCGAAGGACGTGGGGTCTGAGAATGACTTT
A
K
D
V
G
S
E
N
D
Q
G C A C TG G G C A A C C T G A C C C T G G TG CT G G C C A T C A T C G T C T T C A T T T
A
L
G
N
L
T
L
V
L
A
I
I
V
F
I
TT G C C G TG GT C G G C A T G C A G C T G T T T G G A A A G A G C T A C A A G G A G T G
F
A
V
V
G
M
Q
L
F
G
K
S
Y
K
E
C
TAATGGAG CTAGGC
L
M
E
L
G
N
S
V
G
TG TC TG C A A G A T C A A T G T G G A C T G C A A G
V
C
K
I
N
V
D
C
K
2761
CTGCCGCGCTGGCACATGAACGACTTCTTCCACTCCTTCCTGATCGTGTTCCGAGTGCTGTGTGGGGAGTGGATAGAGACCATGTGGGACTGCATGGAGGTCGCGGGCCAGACCATGTGC
L
P
R
W
H
M
N
D
F
F
H
S
F
L
I
V
F
R
V
L
C
G
E
W
I
E
T
M
W
D
C
M
E
V
A
G
Q
T
M
C
2881
CTTATTGTGTTCATGTTGGTCATGGTGATTGGGAACCTTGTGGTTCTGAACCTCTTTCTGGCCTTATTGTTGAGTTCCTTTAGTTCAGATAACCTTGCTGCTACTGACGATGATAACGAA
L
I
V
F
M
L
V
M
V
I
G
N
L
V
V
L
N
L
F
L
A
L
L
L
S
S
F
S
S
D
N
L
A
A
T
D
D
D
N
E
3001
3121
ATGAACAACCTC CAGATC GCGGTG GGAAGGATGCAAAAGGGAAT
M
N
N
L
Q
I
A
V
G
R
M
Q
K
G
GAAG GCAACA~TAGACAG
E
G
N
K
I
D
S
I
T G A T TT T G T G A A A A A T A A G A T A C G G G A G T G C T T C
D
F
V
K
N
K
I
R
E
C
CT GC A T G T C C A A T A A C A C G G G C A T C G A ~ T A A G C A A A G A G C T
C
M
S
N
N
T
G
I
E
I
S
K
E
L
F
C G A A A A G C G T TT TT C A G A A A G C C G A A A G T G A T A G A A A T CCAA
R
K
A
F
F
R
K
P
K
V
I
E
I Q
T A A C T A C C T T A A A G A C G G T A A T G G A A C C A C C A G C GG CG TG GG A A C C G G A A G C A G T G T G
N
Y
L
K
D
G
N
G
T
T
S
G
V
G
T
G
S
S
V
3241
GASdkAATACGTAAT CGATGJ~ZkAATGACTACATGT CATT CATA/L~CAAT CC CAGC CT CACC GT GACT GTGCCAAT TG C TGT GGGAGAGTCTGACT TT G A A / ~ T T T A A A T A C G G A A G A G T T C
E
K
Y
V
I
D
E
N
D
Y
M
S
F
I
N
N
P
S
L
T
V
T
V
P
I
A
V
G
E
S
D
F
E
N
L
N
T
E
E
F
3361
AG C A G T G A G T C A G A A T TG G A A G A A A G T A A G GA G A A A T T A A A T G C A A C C A G C T CT TC T G A A G G A A G C A C A G
S
S
E
S
E
L
E
E
S
K
E
K
L
N
A
T
S
S
S
E
G
S
T
TT G A T G T F GC TC CA CC CC GA G A A G G T G A A C A A G C A G A A A T T G A A C C TG AG
V
D
V
A
P
?
R
E
G
E
Q
A
E
I
E
P
K
Fig. 2. Nucleotide sequence of rat brain Na' channel type II!. The D N A sequence corresponding to the R N A sequence transcribed off $34
is shown in capital letters and the derived amino acid sequence in the single letter code. Position I is the start site of T7 R N A polymerase,
and the transcribed polylinker is shown in small letters. Differences in the published type lI1 sequence are shown on top of the nucleotide
109
decay was present in cell-attached patch recordings where
isopotentiality is established. Fig. 3C shows the average
current obtained by summing 100 records from a patch
containing two type III Na + channels superimposed on
3481
GAGGACCT TAAGCCAGAAGCTTGTTT
E
D
L
K
P
E
A
C
the average current obtained by summing 4 records from
a large patch containing more than 40 Na + channels
expressed after injection of total brain RNA. The slow
decay of the type III Na ÷ current was clearly present in
TACT GAAGGGTGCATTAAAAAATTCCCCTTCTGTCAAGTAAGTACAGAAGAAGGTAAAGGAAAAATAT
F
T
E
G
C
I
K
K
F
P
F
C
Q
V
S
T
E
E
G
K
G
GGTGGAAT CTTAGGAAGACATGCTAC
K
I
W
W
N
L
R
K
T
C
CATTGTGTTCATGATTCTCCTCAGTAGTGGCGCTTTGGCCTTTGAGGATATATACATTGAGCAACGAAAGACGATCAAGACCAT
I
V
F
M
I
L
L
S
S
G
A
L
A
F
E
D
I
Y
I
E
Q
R
K
T
I
K
T
M
Y
A
3601
3721
3841
3961
4081
4201
4321
4441
4561
AGCATTGTGGAGCACAACTGGTTTGAGACGTT
S
I V
E
H
N
W
F
E
T
GAGTATGCAGACAAGGTCTT
E
Y
A
D
K
V
4801
4921
5041
5161
5281
5401
G
A
A
V
TTCTATCACT
F
Y
H
F
GTGTTAACACGACAACAGGCAACATGTTTGAAATAAAAGAAGTGAACAATTT
C
V
N
T
T
T
G
N
M
F
E
I
K
E
V
N
CTATGGATTTCAAACCTATT
Y
G
F
Q
T
Y
CATCAAATCCCTAC
I
K
S
L
ATGAGGGTGGTTGTGAATGCTCTTGTTGGTGCAATTCCCTCCATCATGAATGTGTTATTGGTGTGT
M
R
V
V
V
N
A
L
V
G
A
I
P
S
I
M
N
V
L
L
N
C
AACCTGTACATGTACCTGTACTTTGTCATCTTCATCATCTTCGGCTCGTTCTTCACTCTAAATCTATT
N
L
Y
M
Y
L
Y
F
V
I
F
I
I
F
G
S
F
F
T
L
CAAGACATCTTTATGACAGAAGAACAGAAGAAATAC
Q
D
I
F
M
T
E
E
Q
K
K
Y
N
TCACCAATGCCTGGTGCTGGTTGGACTT
F
T
N
A
W
C
W
L
D
GGACACTGAGAGCTCTGAGGCCGC
R
T
L
R
A
L
R
P
CAGTGACTGTCAGGCTCTTGGCAAGCAAGCCC
S
D
C
Q
A
L
G
K
Q
A
L
CCTGATCGTT
L
I V
TCCGAGCC TTATCCCGCTTTGAAGGC
L
R
A
L
S
R
F
E
G
F
V
D
S
GGGACGTCAAACTGCAGCCCATATATGAAGAA
R
D
V
K
L
Q
P
I
Y
E
CATCGGTGTCATCATAGACAACTTCAACCAGCAGAAGAAGAAGTTT
I
G
V
I
I
D
N
F
N
Q
Q
K
K
K
TACAATGCAATGAAGAAGCTCGGCTCAAAGAAAC
Y
N
A
M
K
K
L
G
S
K
K
CTCAGAAGCCCATCCCTCGGCC
P
Q
K
P
I
P
R
P
K
GGTGGAAGAATGTGAAAGTCAACTTT
R
W
K
N
V
K
V
N
F
F
E
GGAGGT
G
G
TGCAAACAAATTTCAAGGGATGGTCTTT
A
N
K
F
Q
G
M
V
F
GATTTTGTAACCAGACAAGTGTTTGACATCAGCATCATGATCCTCATCTGCCTCAACATGGTGACCATGATGGTGGAAACGGATGACCAGAGCAAATACATGACC C T G G T T T T G T C C C G A
F
V
T
R
Q
V
F
D
I
S
I
M
I
L
I
ATCAACCTAGTGTTCATTGTCCTCTTCACTGGGGAGTTTCTGCTGAAGCTCATC
I
N
L
V
F
I
V
L
F
T
G
E
F
L
L
K
G
ATTGTAGGAATGTTTCTCGCAGAGCT
TATAGAGAAGTATTTCGTGTCCCC
I
V
G
M
F
L
A
E
L
I
E
K
Y
F
V
S
ATCCGCACTCTGCTCTTTGCTTTGATGATGTCCCTTCCTGCGCT
I
R
T
L
L
F
A
L
M
M
S
L
P
AAAAAAGAGGCTGGAATTGATGACATGTTCAACTTT
K
K
E
A
G
I
D
D
M
F
N
F
A
L
C
L
L
I
P
N
M
V
T
M
M
V
E
T
D
D
Q
S
TCCCTCAGATACTACTACTTCACGATAGGGTGGAACAT
S
L
R
Y
Y
Y
F
T
I
G
W
N
I
TACCCTGTTC CGAGTCATCCGCCT GGCCAGGATTGGAC
T
L
F
R
V
I
R
L
A
R
I
G
GTTCAACATCGGCCTCCTGC
F
N
I
G
L
L
K
Y
M
T
L
G
V
CATC CTGGAGAACTTCAGCGTCGC
I
L
E
N
F
S
V
A
CACCGAAGAAAGTGCAGAGCCC
CTGAGTGAGGACGACT
T
E
E
S
A
E
P
L
S
E
D
D
S
R
F
TGCC TATGTT
A
Y
V
TGGGACGGACTGCT GGCC CCCATCCTCAAC
W
D
G
L
L
A
P
I
L
N
AGCGCACCTCCCGACTGTGACCCCGATGCAATTCACCCTGGAAGCTCGGTGAAGGGGGACTGTGGGAACCCATCCGTGGGGATTTTCTTTTTTGTCAGCTACATCATCATATCCTT•CTG
S
A
P
P
D
C
D
P
D
A
I
H
P
G
S
S
V
K
G
D
C
G
N
P
S
V
G
I
F
F
F
V
S
Y
I
I
GTGGTGGTGAACATGTACATCGCTGT
V
V
V
N
M
Y
I
A
L
GAATCCTACGCCTGAT
CAAAGG CGCCAAGGGG
R
I
L
R
L
I
K
G
A
K
G
TTTTCCTGGTCATGTT
CATCTACGCCATCTTTGGGATGTCCAACTT
L
F
L
V
M
F
I
Y
A
I
F
G
M
S
N
GAGACT TTTGGCAACAGCATGATCTGCTTGTTCCAAATCACCACCTCTGCCGGC
E
T
F
G
N
S
M
I
C
L
F
Q
I
T
T
S
A
V
C T T T G A C T T T G T G G T G GT G A T T C T C T C G
F
D
F
V
V
V
I
L
S
I
S
F
L
TTGAGATGTTCTAC GAGGTCTGGGAGAAGTTC
F
E
M
F
Y
E
V
W
E
K
F
GACCCTGACGCCACTCAGTTCATAGAGTTCTGCAAG•TTTCTGACTTTGCAGCTGCCCTGGATCCTCCCCTCCTCATCGCAAAGC•AAACAAAGTC•AGCT•ATTGCCATGGACCTGCCC
P
D
A
T
Q
F
I
E
F
C
K
L
S
D
AT GGTGAGTGGAGACCGCATCCACTGCCTGGACATCTTGTTTGC
M
V
S
G
D
R
I
H
C
L
D
I
L
F
A
F
A
A
A
L
D
P
P
L
L
I
A
K
P
N
K
V
TTTTACA~GCGGGTCCTGGGCGAGAGTGGAGAGATGGACGCTCTTCGAATC
F
T
K
R
V
L
G
E
S
G
E
M
D
A
L
R
I
Q
L
I
A
M
D
L
P
CAGATGGAAGATCGCTTCATGGCT
Q
M
E
D
R
F
M
A
5641
TCCAACCCCTCCAAGGTCTCTTATGAGCCCATTACCACCACCCTGAAACGGAAACAAGAGGAGGTGTCTGCTGCTATCATTCAGCGTAATTATAGATGTTATCTTTTAAAGCAACGGTTA
S
N
P
S
K
V
S
Y
E
P
I
T
T
T
L
K
R
K
Q
E
E
V
S
A
A
I
I
Q
R
N
Y
R
C
Y
L
L
5761
AAAAACATATCGAG TAAATACGACAAAGAGACAATCAAGG
K
N
I
S
S
K
Y
D
K
E
T
I
K
5881
F
GC TG
L
CT C A T C T T C T G G C T G A T T T T T A G C A T C A T G G G T G T G A A T C T G T T T G C T G G A A A G
L
I
F
W
L
I
F
S
I
M
G
V
N
L
F
A
G
GACAACGTTGGGGCTGGCTACCTGGCATTGCTGCAAGTGGCCACATTCAAAGGCTGGATGGACATCATGTATGCAGcTGTTGATTCGC
D
N
V
G
A
G
Y
L
A
L
L
Q
V
A
T
F
K
G
W
M
D
I M
Y
A
A
D
5521
CACGTACATCTTCATCCTGGAGATGCTCCTCAAATGGGTGGC
T
Y
I
F
I
L
E
M
L
L
K
W
V
GATGTTTCTTTGGTTAGCCTGGTAGCCAATGCTCTTGGTTACTCAGAACTTGGTGC
D
V
S
L
V
S
L
V
A
N
A
L
G
Y
S
E
L
D
4681
F
F
K
Q
R
L
G A A G G A TT G A C T T G C C T A T A A A A G G A G A T A T G G T
T A T T G A C A A A T T G A A T GG G A A T TC C A C C C C A G A A A A G A C G G A T G GG
G
R
I
D
L
P
I
K
G
D
M
V
I
D
K
L
N
G
N
S
T
P
E
K
T
D
G
A
A G T T CC T C C A C A A C CT CT C C TC CT T C C T A T G A C A G T GT A A C A A A A C C A G A T A A G G A A A A G TT T G A G A A A G A C A A A C C A G A A A A A G A A A T C A A A G G G A A A G A G GT CC G A G A G A A T C A A A A G
S
S
S
T
T
S
P
P
S
Y
D
S
V
T
K
P
D
K
E
K
F
E
K
D
K
P
E
K
E
I
K
G
K
E
V
R
E
N
Q
K
6001
TAAAAAGAGACAAAGAAATGTCTTTGTAATCAATTGTTTACAGC
end
CTCTGAAGGTAAAGTATCCGTGTCAACT
G G A C TC T A A G G A G A G G T C C A T G C C A A A C T G A C T G T T T C A A C A A A T A C T
6121
CAAGGTCAGTGCCTATAC
6241
TTGTGCAAGT
6361
CCCCACTTCATAGTCTGTTCATAATACTATGTCACTATTTTTGTAAATGAAGTTTACGTTAAGGGAAAATATATATATAAGAATCCCATGTTGCTAAGTCCACAAGTTTCTCCAGTAATC
6481
A T A A A A A A A T A T T T TG CC T G A G A G A T G A A A T T A T TG CT C A A
CAGACAGT GACCTCTGTCACTGCCACTCTGTGAGACAGGGTATCAACATTGACAAGAGGTTGCTGC
T
GACCCGTCATCACGCCCC
CAAACT CCATTAGTACAACGCTCCTGTCAT
TT C C A T T A C C A G C T G A C A C T
CTATTT TTAACATTCACATTTGCCATATTTTTACAAAATCTGTC
GCTGAGGAGAACTCCA
CCAGTGTATC TTCCTGGT
sequence, and the corresponding amino acid changes are indicated below. The position and length of the first two inserts (in clone 5) are shown
by (O); the 96 nucleotide insert (in clone 5) is shown in small letters (pos. 1924-2019).
110
the
cell-attached
patch.
Time
to
peak
current
was
A
p r o l o n g e d in t y p e III N a ÷ c h a n n e l s (3.1 ms c o m p a r e d
¢
-~
20 rnV
~ 200~-
with 0.9 ms in the c h a n n e l s e x p r e s s e d by total brain
RNA).
~
A t t h e single c h a n n e l level, t h e s e effects w e r e
5
0
nA
s e e n to be the result of p r o l o n g e d bursts and d e l a y e d
",.~_.
° ,oo~
o
~o~
latency to first o p e n i n g of the type III N a ÷ channels. A n
2°
0
20 mV
a c c o u n t of the single t y p e III N a ÷ c h a n n e l p r o p e r t i e s will
/~jJ
be r e p o r t e d separately.
T h e level of e x p r e s s i o n of t y p e III Na ÷ currents r a n g e d
b ,r-'
B
up to 1500 n A p e r o o c y t e , which did not readily allow
No5
m a c r o s c o p i c c u r r e n t r e c o r d i n g s f r o m large c e l l - a t t a c h e d
patches. W e instead a n a l y z e d p e a k c u r r e n t - v o l t a g e ( l - V )
relationship,
rates of c u r r e n t
ings. Since the rise t i m e of the voltage step and r e s o l u t i o n
of the first 5 - 1 0 ms of m e m b r a n e current are limited by
the large c a p a c i t a n c e of the o o c y t e , the p e a k c u r r e n t m a y
not be m e a s u r e d accurately. W e instead e x t r a p o l a t e d the
initial a m p l i t u d e s of the currents f r o m the decay phase to
c o n s t r u c t the p e a k I - V
relationship and the steady-state
A
t y p e III
~
brain RNA
~00msec
O
Z
decay, and steady-state
inactivation and i n a c t i v a t i o n gating in w h o l e cell record-
C
B
D
~IO
,,
L - 8~0
-60
-40
,4
-20
O
20 rn¢
Fig. 4. Macroscopic type III Na ~ current kinetics. A: currents
evoked by voltage steps to -20, -10, and -5 mV from the family
shown in Fig. 3A are superimposed on sums of two exponentials
functions of the form l(t) = A o + A~ exp - t / r 1 + A 2 exp - t / r e. For
the traces shown, the values of A 0, AI and A2 in nA are (-20 mV):
-7.5, -82,-74; (-10 mV): -1.6,-394, -98; and (-5 mV): -1,-470,
-81; and the values of rl and r 2 in ms are (-20 mV) 39.4, 168, (-10
mV) 24.9, 87 and (-5 mV) 20.0, 69. For B and D, the amplitude of
the currents was measured as the sum of A0, A 1 and A:. In B-D,
data points are the mean of 2-6 oocytes. Error bars are S.E.M. and
are usually smaller than the symbols. B: current-voltage relationship. The finding of peak current amplitude at approximately -5 to
0 mV was confirmed in more than 50 other oocytes. C: time
constants of decay as a logarithmic function of test potential in the
same oocyte. D: steady-state inactivation as a function of 5 s (filled
triangles) and 50 s (open triangles) prepulse potential, and normalized conductance from holding potential -100 mV as a function of
test potential (open circles). The smooth lines are Boltzmann
functions of the form l(v) = l / { + [ e x p ( V m - V u 2 ) / k ] } . After 5 s
prepulses, Vv2 = -36.1 mV and k = 7.8 mV; after 50 s prepulses,
the values are -51.0 and 6.7 mV. For activation, Vv2 = -10.7 mV
and k = -4.7 mV.
br@in RNA
500
1 O0
msec
nA
~1type III
activation and inactivation relationships. This t e c h n i q u e
has the a d v a n t a g e of analyzing t h e c u r r e n t trace well after
the capacity c u r r e n t has s u b s i d e d and the m e m b r a n e
voltage is m a x i m a l l y c o n t r o l l e d . It is l i m i t e d t h e o r e t i c a l l y
Fig. 3. Na ÷ currents expressed by type llI Na ÷ channel mRNA and
by total rat brain. RNA. All the electrophysiological recordings were
made at room temperature in 145 mM [Na+]o . A: family of currents
evoked by 400 ms depolarizing voltage steps from a holding
potential of-90 to -40 mV and in 5 mV increments thereafter in an
oocyte injected with 20 ng of $34 mRNA. To reduce the capacity
current, the holding potential was changed to -60 mV for 50 ms
prior to the test pulse. Depolarization for this short period at this
potential does not produce significant activation or inactivation of
type III Na ÷ current. Calibration bars are 50 nA and 100 ms. B:
currents expressed after injection of 150 ng of total rat brain RNA.
Depolarizing steps to -30 through -10 mV in 5 mV increments are
shown. Conditions are identical to A. Calibration bars are 500 nA
and 100 ms. C: expressed Na ÷ currents through cell-attached
patches. The recording of total rat brain RNA Na ÷ currents is the
average of four 20 ms depolarizing steps, and the type III Na ÷
current is the average of a hundred 100 ms idealized traces from a
patch containing two channels. The holding potentials are -90 and
-100 mV, respectively, and the test potential is -20 mV in both. The
peak currents have been scaled - - the peak Na ÷ current from total
brain RNA is 42.8 pA, and from type III Na ÷ channel RNA 0.38
pA. Calibration bar = 20 ms.
by the a s s u m p t i o n that the p r o c e s s e s of activation and
inactivation are i n d e p e n d e n t , u n c o u p l e d processes. This
a s s u m p t i o n was used by S t t i h m e r and c o w o r k e r s 35 in
studying e x p r e s s e d type II N a ÷ currents.
Fig. 4 A shows the c u r r e n t s p r o d u c e d by 3-step d e p o larizations and the fit to these c u r r e n t s with b i e x p o n e n t i a l
decay functions. T h e d e p e n d e n c e of the two rates of
decay on the test p o t e n t i a l is s h o w n in Fig. 4C. B o t h fast
and slow d e c a y
depolarizations.
rates
The I-I/relationship
increased
with
increasing
test
(Fig. 4B) shows p e a k c u r r e n t at
- 5 to 0 m V and agrees well with the r e p o r t of Suzuki et
al. 37. T h e steady-state a c t i v a t i o n c u r v e (Fig. 4 D ) was
c o n s t r u c t e d by p l o t t i n g n o r m a l i z e d c o n d u c t a n c e versus
test p o t e n t i a l and was fit by a B o l t z m a n n f u n c t i o n with
m i d p o i n t - 1 0 . 7 m V and slope factor - 4 . 6 6 mV. B e c a u s e
the decay of the m a c r o s c o p i c c u r r e n t was p r o l o n g e d , we
111
A
-4-0 mV
+1
.
0
~
control
~
=
C
.
__J
--40
mV
toxin
g
Z
0.1
1
10
!}
100
['q-X ] nM
!1
-40
- - controq
- -~ toxin
-Ct
and ~
toxins
' -s;
~
1
/
c~ a n d
fl t o x i n s
Ii
~\ //
~-~m~
~/
;
mV
//
'
5;~v
Fig. 5. Effect of toxins on type III Na ÷ currents. A: dose-response
relationship to TTX. Data points are the mean of 3 experiments,
and the S.D. is smaller than the symbols. The smooth line is the
theoretical fit for a single binding site with a IC50 = 1.8 nM. B-E
show the response to a(TslV-5) and fl(Ts-y) toxins. The I-V curves
were obtained by changing the membrane potential from -100 to 50
mV in a continuous ramp lasting 1 s. Individual step depolarizations
to -40 and -5 mV in control (C), a-toxin alone (5 ~M) (D), and
combined a- and fl-toxins (5/~M) (E) are shown, a-toxin increased
peak current without altering the I-V relationship, and fl-toxin had
the additive effect of shifting the threshold for current activation to
more negative potentials. Calibration bars = 50 nA and 100 ms.
used prepulses of 5 and 50 s in analyzing the voltage
dependence of steady-state inactivation (Fig. 4D). After
a prepulse to varying poten{ials, the residual Na ÷ current
was measured at a test potential of 0 mV (Fig. 4D). The
two smooth curves are Boltzmann functions with midpoints -36.1 and -51.0 mV and slope factors 7.79 and
6.73 mV for 5 and 50 s prepulses, respectively. The
prepulse duration did not significantly affect the kinetics
of current decay at the test pulse to 0 mV.
Effects of toxins on type III Na ÷ currents
Fig. 5 shows the effect of Na ÷ channel-specific toxins.
T T X blocked the Na ÷ currents reversibly with an IC5o of
1.8 nM (Fig. 5A). Fig. 5 B - E shows the effect of toxins
from the scorpion Tityus serrulatus. The current-voltage
relationships in Fig. 5B were obtained by changing the
m e m b r a n e potential from -100 to 50 mV in a continuous
ramp lasting 1 s. This technique appears acceptable for
this analysis because of the very slow inactivation of type
III Na ÷ currents, especially after the addition of ascorpion toxin. The peaks of the ramp I - V (Fig. 5B) and
the peak current I - V (Fig. 4B) coincide at - 5 to - 1 0 mV,
confirming the reliability of the ramp I - V for these slowly
inactivating currents.
TslV-5, an a-toxin 19'31, increased current without
altering the I - V relationship (Fig. 5B). Step depolarizations to - 4 0 mV did not evoke current either before or
after this toxin was added to a final bath concentration of
5/~M (Fig. 5D); the current at test potential - 5 mV was
increased after toxin treatment compared to control, and
a large non-inactivating component was present. This
effect is similar to that in neuroblastoma cells 19 and in
oocytes injected with high molecular weight R N A from
brain 21. Ts-y, a fl-toxin from the same species, was added
to the same oocyte after the a-toxin effect had stabilized.
The ramp I - V showed activation at more negative
potentials and a decrease in the peak current (Fig. 5B).
This was confirmed in step depolarizations to - 4 0 and - 5
mV (Fig. 5E). Ts-y alone shifted the peak of the I - V by
-25 mV (not shown, n = 3). These toxin effects are
similar to those seen in neuroblastoma cells 38 and heart
cells 39.
DISCUSSION
Slow inactivation of macroscopic type III Na + currents
We isolated several overlapping c D N A clones of the
rat brain Na ÷ channel type III and used them to construct
a full-length channel gene. R N A transcript from construct $34 leads to surface expression of Na ÷ channels
when injected into Xenopus oocytes.
Oocyte-expressed Na ÷ currents encoded by total brain
R N A 3'21 (Fig. 3B) decay at r o o m temperature with a
single exponential time course with a time constant of less
than 10 ms. Stiihmer and colleagues 35 likewise found
oocyte-expressed type II Na ÷ currents to decay with time
constants less than 10 ms at all test potentials even at
15 °C. Currents encoded by high molecular weight brain
R N A 2x and type I I A N a ÷ channel m R N A 3 decay more
slowly, but still with time constants less than 10 ms at
room temperature. The decay of the expressed type III
Na ÷ current is markedly slower and occurs in two phases.
Although Suzuki and coworkers 37 do not quantify the
decay time constant in their report of expressed type III
Na ÷ currents, their published figures appear similar to
ours, showing the time until 50% decay of peak currents
to be 10-20 ms.
The level of expression in our experiments was 5- to
10-fold lower than that reported by Suzuki and
coworkers 37, despite our injecting up to 4 times as much
m R N A (20 ng compared with 5 ng per oocyte). This
difference may reflect geographical variation in oocyte
expression or differences in the 5" untranslated ends. It
is unlikely to affect our analysis of macroscopic inactivation, as (a) our results were the same for 200 and 1500
n A currents, (b) as noted above, the rate of current decay
appears similar in the figures of Suzuki and colleagues 37,
and (c) reducing current amplitude with T r X did not
affect inactivation rate (Fig. 5A).
We found further evidence of very slow inactivation of
type III Na ÷ currents in two-pulse protocols. The amount
112
of type III Na + current remaining at a test potential of 0
m V depended greatly on prepulse duration. In experiments using 50 s prepulses, the midpoint of the fitted
Boltzmann function (-51.0 mV) was 15 mV more
negative than for the 5 s prepulse experiments (-36.1
mV). The first value is similar to that found in type II and
III Na + currents using 2 s prepulses at room
temperature 37 and in Na + currents expressed by total
lasted 50 s. This is due to the large shift in activation
gating to more positive potentials in our experiments. We
found the midpoint of the normalized conductance curve
to be -10.7 mV compared with -40.5 m V for type II Na +
currents at 15 °C35. Some of the difference may lie in the
inaccuracy of the microelectrode clamp compared with
cell-attached patch recordings 25 or in the temperature
difference.
brain R N A using 50 ms prepulses at room temperature 2~.
This midpoint value, however, is about 15 or more mV
more positive than the value reported for type II Na +
currents using 32 ms prepulses at 15 °C35, and in native
Na + currents u n d e r various experimental conditions in
heart cells 22, neuroblastoma cells 19, and rat peripheral
nerve 27. It is noteworthy that Stfihmer and colleagues 35
found a small slowly or non-inactivating component of
current in their two-pulse experiments, which may
correlate with our slowly inactivating current. Slowly
inactivating TTX-sensitive components of neuronal Na +
currents have been only rarely reported w'16.
Scorpion toxin effects
The site of action of scorpion toxins has been unclear.
Data from radiation inactivation experiments pointed to
the large a-subunit as the site of toxin binding 1'4.
However, photoactivatable derivatives of a- or fl-scorpion toxins predominantly label one of the small subunits
of the Na + channel 17'33. Krafte and co-workers 21 have
shown that a-scorpion toxin prolongs Na + currents
expressed by oocytes injected with high-molecular weight
m R N A from rat brain. O u r results show that a cloned ct
The expected result of very slowly decaying neuronal
Na + currents would be prolonged neuronal action potentials. Such, however, have not been reported. The
action potential of a n e u r o n containing type II1 Na +
channels need not be prolonged if (a) neurons modulate
the channel in such a way as to hasten inactivation or (b)
other, faster inactivating Na + channels were more nu-
subunit expressed in oocytes contains both functional aand fl-scorpion toxin binding sites. The intermediate
effect of the mixture of a- and fl-scorpion toxin is in
keeping with the concept of different toxin binding sites v.
We cannot rule out the possibility that the oocyte
provides its own fl-subunit-like component. A definite
answer to this problem requires the cloning and expression of the gene(s) encoding the fl-subunits.
merous in the m e m b r a n e . Long-term modulation of
n e u r o n a l function might be achieved by varying the level
of expression of fast and slowly inactivating Na + channels.
Despite the shift of inactivation gating to more positive
potentials, the overlap of type III Na + current activation
and inactivation curves after 5 s inactivating prepulses is
small. The overlap is further reduced when the prepulse
Acknowledgements. We thank Dr. M.E. Herrero-Zabaleta and
Stacey Gouzene for excellent help in the initial phases of this work,
Georges Frech for constructing one of the cDNA libararies, and Dr.
Lourival D. Possani, Universidad National Autonoma de Mexico,
for scorpion toxins. This work was supported by grants from the
American Heart Association, Texas Affiliate (R.H.J. and G.E.K.),
the Sealy Memorial Endowment (J.R.M.), and National Institutes
of Health Grants KL01858 (J.R.M.) and HL36930 and HL37044
(A.M.B.).
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