Introduction cardiac cells [2,3,6,10,11,20,22,23,34,42,46,48,49,54,60]. Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol887.Issue2.Dec2000:
Brain Research 887 2000 335–343 www.elsevier.com locate bres
Research report
The cardiac sodium channel mRNA is expressed in the developing and adult rat and human brain
a , b
b c
Laurel M. Donahue , Penelope W. Coates , Vaughan H. Lee , Denise C. Ippensen ,
c c
Steven E. Arze , Shirley E. Poduslo
a
Cascade Biologics , Inc., 4475 SW Scholls Ferry Road, Portland, OR 97225, USA
b
Department of Cell Biology and Biochemistry , Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
c
Division of Neurology , Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
Accepted 26 September 2000
Abstract
Expression of the rat RH-I SkM2 and human hH1 SCN5A tetrodotoxin-resistant TTX-R, voltage-sensitive sodium channels is thought to be specific to cardiac tissue. We detected RH-I SkM2 mRNA in newborn rat brain using both RNase protection assay analysis
and in situ hybridization and in adult rat brain using RNase protection assay analysis. This expression was observed primarily in developing limbic structures of the cerebrum and diencephalon, and in the medulla of the brain stem. Using RT-PCR analysis, we
detected hH1 SCN5A mRNA in both fetal and adult human brain. Interestingly, mutations in the human cardiac sodium channel are known to lead to cardiac abnormalities, which result in arrhythmias and frequently in sudden cardiac death. If these mutant channels were
also expressed in limbic regions of the brain, alterations in channel function could have drastic effects on the brain’s signaling ability, possibly promoting seizure activity.
2000 Elsevier Science B.V. All rights reserved.
Theme : Excitable membranes and synaptic transmission
Topic : Sodium channels
Keywords : Cardiac sodium channel; RH-1 SkM2; hH1 SCN5A; Limbic; mRNA expression; Long QT3 syndrome
1. Introduction cardiac cells [2,3,6,10,11,20,22,23,34,42,46,48,49,54,60].
In addition, they are also expressed in glial cells Voltage gated sodium channels sodium channels are
[3,6,10,24,48,51] and
other peripheral
tissues essential for the electrical excitability of neurons and other
[3,8,20,23,24,26,28,35,36,42]. Although the sodium chan- excitable cells. Sodium channels comprise a multigene
nel isoforms were originally defined based on the tissue or family with two subfamilies determined by sequence
cell type from which they were isolated rat brain I, II, and homology [10]. Within each family, the channel subtypes
III, the cardiac channel, and the glial channel, they are are distinguished by their single channel conductance and
actually expressed in a variety of tissues. In fact, many sensitivity to pharmacological agents [22,34,60]. Sodium
tissues express
multiple channel
isoforms [5,7–
channels are expressed in a number of different cell types, 10,17,18,21,22,24,29,39,47,53,56,57]. For example, the rat
including excitable cells, such as central nervous system brain I and II sodium channels, as well as the glial sodium
CNS neurons, peripheral nervous system PNS sensory channel, are expressed in rat spinal sensory neurons [9].
neurons, neuroendocrine cells, skeletal muscle cells, and Sodium channels vary in their sensitivity to tetrodotoxin
TTX, a guanidinium neurotoxin that interacts exclusively with sodium channels [22,28,60]. Sodium channels are
broadly divided into three classes based on their sensitivity to TTX [18,62]. Those demonstrating the highest TTX
Corresponding author. Tel.: 11-503-292-9521; fax: 11-503-292-
sensitivity are designated TTX-sensitive TTX-S channels
0566. E-mail address
: lauriedcascadebio.com L.M. Donahue.
and are blocked with low concentrations of TTX 1–5
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 3 0 3 3 - X
336 L
nM; those with intermediate sensitivity, TTX-resistant 2. Materials and methods
TTX-R channels, require higher concentrations of TTX 0.2–1 mM for inactivation; and those with the lowest
2.1. RNA isolation and quantification sensitivity, TTX-insensitive TTX-I channels, are blocked
only with very high concentrations of TTX 30–100 mM. Total RNA from adult rat heart, and adult and postnatal
To date, there are five cloned sodium channels that are not day 0 P0 Sprague–Dawley rat brains was isolated as
1
TTX-S: the rat RH-I SkM2 and the human HH1 SCN5A described previously [19]. Poly A
RNA from HeLa cardiac sodium channel genes, whose gene products are
cells human cervical epithelial carcinoma cells; American TTX-R [60], the rat and the human NaN SNS2 sensory
Type Culture Collection, Rockville, MD was also isolated neuron sodium channel gene products which are TTX-I
as described previously [17]. Total RNA from adult human [13], and the rat SNS PN3 sensory neuron sodium channel
heart, and adult and fetal human brain was obtained from gene product, which is TTX-I [2,46].
Clontech Laboratories, Inc. Palo Alto, CA. The con- One of the most extensively studied sodium channels is
centration of each RNA was estimated by optical density the cardiac sodium channel. It has been cloned from rat
measurements and the relative amounts of RNAs used in RH-I SkM2 and human HH1 SCN5A cardiac cDNA
the RNase protection assays were normalized to cyto- libraries [25,33,45,55]. Electrophysiological studies of rat
plasmic actin mRNA levels. and human cardiac sodium channels expressed in Xenopus
oocytes demonstrated that RH-I SkM2 and HH1 SCN5A are the functional TTX-R sodium channels expressed by
2.2. RNase protection assay cardiac cells [22,32]. RH-I SkM2 is expressed in neonatal
and adult rat heart: neonatal heart expresses solely RH-I RNase protection assays were performed as previously
SkM2, whereas adult heart expresses both RH-I SkM2 and described [19]; RPA II kit, Ambion, Austin, TX. In
a TTX-S NaCh current [33,45,49]. RH-I SkM2 expression assays for RH-1 SkM2 expression, 50 mg of total RNA
is also present in neonatal skeletal muscle and in de- from P0 and adult rat brain, 5 mg of total RNA from adult
nervated adult muscle, but is absent in innervated adult rat heart, and 5 mg of total RNA from yeast were
4 32
muscle [33,45]. The expression of HH1 SCN5A has only hybridized to 5310 cpm of the
P-labeled RH-1 SkM2 been examined in adult tissues and while generally re-
antisense RNA probe. The 414 nucleotide nt gene-spe- ported to be specific to cardiac tissue [25], a few reports
cific probe for RH-1 SkM2 was generated by BamHI demonstrate its localization in adult rat brain [18,30,59,61].
linearization of the plasmid SK- 15-2-1 and transcription To date, the sodium channel genes that have been cloned
with T7 polymerase. The Sk- 15-2-1 plasmid [33]; gift from rat brain are TTX-S I, II, III, and NaCh6
from Dr. Roland Kallen, University of Pennsylvania, [4,16,27,38,50,53]. Although earlier studies failed to
Philadelphia, PA contained nucleotides 6718–7076 from distinguish between the TTX-R and TTX-I sodium channel
the 39 untranslated region of the mRNA. The expected size currents, several studies suggest that both kinds of current
of the protected fragment was 358 nt. RPAs were also exist in the CNS [14,31,43,52,58,60]. However, the genes
performed using a cytoplasmic actin antisense RNA probe responsible for the TTX-R and the TTX-I currents in the
to control for RNA load. In this case, 5 mg of each of the
4 32
CNS have not yet been identified. Because we demon- RNAs were hybridized to 1310 cpm of the
P-labeled strated expression of the rat RH-I SkM2 cardiac channel
actin antisense RNA probe. The template for cytoplasmic mRNA in the neuronal cell lines, RT4-B8 and RT4-E5
actin pTRI-b-Actin-125-Rat was obtained from Ambion [17] and others detected its expression in B104 neuro-
Austin, TX; a 218 nt antisense RNA probe was syn- blastoma cells [29], we hypothesized that expression of
thesized using SP6 polymerase. The predicted size of the this gene might be responsible for the TTX-R current in
protected fragment was 126 nt. Both the RH-1 SkM2 and the nervous system. In fact, using RNase protection assays
actin antisense RNA probes were synthesized using the of total brain RNA, we and others have observed RH-I
Riboprobe System kit Promega Corporation, Madison, SkM2 mRNA expression in neonatal and adult rat brain
WI. RPAs were visualized both by autoradiography and [18,59,61].
by using a Molecular Dynamics PhosphoImager 445SI In this study, we demonstrate that RH-I SkM2 and
Sunnyvale, CA. Band intensities were quantified using HH1 SCN5A mRNAs were expressed in both developing
ImageQuaNT software Molecular Dynamics. and adult rat and human brain by RNase protection assay
and RT-PCR analyses. In situ hybridization of P0 rat brain further demonstrated that RH-I SkM2 mRNA expression
2.3. In situ hybridization was localized to limbic and certain autonomic structures of
the newborn rat brain. Interestingly, the localized expres- In situ hybridizations were performed as previously
sion we observed may implicate the cardiac sodium described [37,44]. Briefly, four P0 rat brains were fixed in
channel in sudden unexplained death in epilepsy. 4 paraformaldehyde, embedded in paraffin, and cut in 5
L .M. Donahue et al. Brain Research 887 2000 335 –343
337
mm serial coronal sections. Sections were de-paraffinized, expression of the RH-I SkM2 cardiac sodium channel
dehydrated through a series of ethanol washes, washed in gene in P0 and adult rat brain Fig. 1. When qualitatively
PBS, subjected to Proteinase K 20 mg ml digestion, and normalized to levels of cytoplasmic actin mRNA expres-
pre-hybridized for 2–4 h at room temperature [32,38]. sion Fig. 1B, RH-I SkM2 mRNA was detected at
Sections were hybridized at 508C for 16–20 h with 2 ng of roughly equal levels in both P0 and adult rat brain in three
35
either the S-labeled RH-1 SkM2 sense or antisense RNA
separate experiments representative example shown in probe from the plasmid SK- 15-2-1 [33]; specific activity
Fig. 1A. As expected, RH-I SkM2 mRNA was detected of the probes ranged from 12 to 23 mCi mg. The sense
in the rat heart RNA positive control, but not in the probe was generated by linearizing the SK- 15-2-1 plas-
negative control, yeast RNA. mid with SalI followed by transcription with T3 poly-
merase. The antisense probe was generated by linearizing the plasmid with SacI followed by transcription with T7
polymerase. After hybridization, the sections were washed for 30 min in 50 formamide, 13 SSC 0.15 M NaCl
0.015 M Na citrate, 10 mM DTT at 508C, followed by a 30-min wash in 0.53 SSC at room temperature. The
sections were then treated for 30 min with 20 mg ml RNase A at room temperature, washed for 2 h in 0.13
SSC at 758C, and dehydrated through a series of ethanol washes. Sections were dipped in NTB2 emulsion Eastman
Kodak Company, Rochester, NY, exposed for 3 weeks at 48C, developed, and counterstained with Harris modified
hematoxylin [37,44]. The sections were examined under both brightfield and darkfield optics on an Olympus BX-60
microscope. Identification of rat P0 brain regions was based on Paxinos et al. [41].
2.4. RT-PCR amplification The GeneAmp RNA PCR Core Kit Perkin Elmer
Cetus, Norwalk, CT was used to reverse transcribe and amplify 500 ng of DNase-treated human heart and brain
total RNAs, as well as 200–500 ng of DNase-treated HeLa
1
poly A RNA. Here, 100 pmol of each primer were used
in 50 ml reactions. The primers used were from the domain I-II linker region of hH1 SCN5A [55] and were used to
amplify a 410 base pair bp fragment. The forward primer was 59-CAGGACTTCTATGAAGCCACG-39, which ex-
tends from nucleotides 1698 to 1718, and the reverse primer
was 59-AAGCCATCTACACACGGAGC-39,
which extends from nucleotides 2089 to 2108 [55]. Ampli- fication conditions were 958C for 5 min, 30 cycles of 948C
for 1 min, 628C for 1.5 min, and 728C for 1 min, followed by an incubation at 728C for 5 min. The PCR products
were separated by electrophoresis on 2 agarose gels.
Fig. 1. RNase protection assays of RH-I SkM2 and cytoplasmic actin. Lane designations: M, markers; P, probe; Y, yeast; Hrt, adult rat heart; Ad,
adult; P0, postnatal day 0; Wb, whole brain. A RH-I SkM2 RPA. Fifty