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INTRODUCTION |
Mechanosensitivity is a requirement for the survival of an
organism (1, 2). At a cellular level, ion channels often serve as the
unitary element that underlies mechanosensitivity (3, 4).
Mechanosensitive ion channels are of particular relevance in organs
constantly submitted to movement, such as the gastrointestinal tract
and the heart (5, 6). Contractility in both cardiac muscle and
gastrointestinal smooth muscle is initiated by membrane electrical
events as a result of changes in ionic conductances (7, 8). In the
heart, the upstroke of the action potential is mediated via opening of
a tetrodotoxin-insensitive Na+ channel, the
subunit of
which is encoded by SCN5A (9). Mutations in SCN5A can result in
clinically significant cardiac arrhythmias (8, 10). SCN5A is also
expressed in human intestinal circular smooth muscle and the native
Na+ current is mechanosensitive (11-13).
Mechanosensitivity appears to be dependent on the actin cytoskeleton,
since disruption of the actin cytoskeleton by cytochalasin D or
gelsolin abolishes mechanosensitivity (13). This suggests that the
actin cytoskeleton is required to transmit force to the ion channel.
Syntrophins are suggested to be a link between the actin cytoskeleton
and membrane-associated proteins including ion channels, enzymes, and
receptors, since actin filaments are not known to directly interact
with these proteins (14-17). Syntrophins are a multigene family of
homologous proteins (18-22). Five syntrophins,
,
1,
2,
1,
and
2, have been characterized (22, 23). Each syntrophin is encoded
by a separate gene but shares a common domain organization. Each
syntrophin contains two tandem pleckstrin homology domains at the N
terminus, a single PDZ domain, and a highly conserved C terminus
syntrophin-unique region (22, 23). The PDZ domains of syntrophins
,
1, and
2 but not
1 are known to interact with SCN5A and with
SCN4A, a skeletal muscle Na+ channel, via the C terminus
sequence motif (E(S/T)XV) (14, 15, 24). The objective of
this study was to investigate the interaction between SCN5A and
syntrophins in intestinal smooth muscle and the functional consequences
of such an interaction on mechanosensitivity. Our hypothesis was that
syntrophins couple SCN5A with the actin cytoskeleton, providing a
mechanism for mechanical regulation of voltage-dependent
ion channel gating.
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MATERIALS AND METHODS |
Preparation of Single Human Jejunal Circular Smooth Muscle
Cells--
The Mayo Foundation Institutional Review Board approved the
use of human tissue obtained as surgical waste tissue during gastric bypass operations performed for morbid obesity. The method for dissociation of smooth muscle cells from human jejunal circular smooth
muscle strips was as previously described (11, 12). Briefly, the
mucosa, submucosa, and longitudinal muscle of the jejunum were removed
from the specimen by sharp dissection. The circular smooth muscle layer
was then cut into small pieces and incubated with enzyme to release
single smooth muscle cells used for patch clamp studies and single cell
reverse transcription (RT)1-PCR.
Poly(A) RNA Isolation and c-DNA Library
Preparation--
Procedures for RNA isolation and the procedure for
preparation of c-DNA libraries were as described previously (11,
12).
PCR and Single Cell RT-PCR--
All PCR amplifications were
performed using GeneAmp 2400 PCR systems (PerkinElmer Life Sciences)
using standard procedures (12). The protocol for single cell RT-PCR was
as previously outlined (12). Spindle-shaped single smooth muscle cells
were collected directly into PCR tubes containing tRNA and proteinase K. RT was performed using a mixture of random hexamer and oligo(dT) primers following the instructions of the manufacturer (PerkinElmer Life Sciences). The product of the RT reaction was then amplified for
syntrophins using gene-specific primers that were specifically designed
to flank a region that contained introns. All PCR products were
purified and sent to the Mayo Molecular Core Facility for automated DNA sequencing.
Laser Capture Microdissection--
Surgical waste tissue was
fixed in ice-cold acetone according to the protocol described
previously (12). The same number of spots of tissue containing about
1500 smooth muscle cells from the circular muscle layer or the
longitudinal muscle layer were collected using the PIX II cell laser
capture microdissection system (Arcturus Engineering Inc., Santa Clara,
CA) with the 7.5-µm spot size. The caps with collected cells were
then immediately placed into sterile 0.5-ml microcentrifuge tubes
containing 300 µl of RNA STAT-60 reagent (Tel-TEST Inc., Friendswood,
TX) for isolation of total RNA. After washing with 75% ethanol, the
RNA pellet was resuspended in nuclease-free water (Ambion Inc., Austin, TX) and used for the RT-PCR.
Immunohistochemistry--
Pieces of human jejunum (approximately
1 × 1 cm) were prepared for immunohistochemistry as previously
described (12). Briefly, cryostat sections (12 µm thick) were mounted
onto glass slides, air-dried, fixed for 10 min in either cold acetone
or 4% paraformaldehyde, and rinsed in phosphate-buffered saline (PBS).
Sections were incubated with 10% normal donkey serum and 0.3% Triton
X-100 for 1 h to block nonspecific absorption sites and then
incubated overnight at 4 °C with anti-syntrophin
2 rabbit
polyclonal antibody (diluted 1:200 in 5% normal donkey serum; a kind
gift from Dr. Vincenzo Nigro). The specificity of this antibody for
syntrophin
2 has been previously shown by Piluso et al.
(22). After several rinses in PBS, the sections were incubated for
1 h with donkey, anti-rabbit IgG conjugated to CY3 (1:100 dilution
in 2.5% normal donkey serum), rinsed in PBS, and coverslipped in
glycerol-PBS containing an anti-fade reagent.
Yeast Two-hybrid Assay--
Yeast two-hybrid assays were
performed using HybriZAP-2.1 two-hybrid system (Stratagene, La Jolla,
CA). The cDNA fragments encoding the C terminus of SCN5A (amino
acids 1915-2015, CTSCN5A) and the C terminus with last 10 amino acids
truncated (amino acids 1915-2005, CTSCN5A-10) were amplified by PCR
and cloned into EcoRI and SalI restriction sites
of the pBD-GAL4 vector to serve as baits in the yeast two-hybrid
experiments. Two full-length splice variants of syntrophin
2, one
with an intact PDZ domain and the other lacking the PDZ domain, were
inserted into pAD-GAL4 vector (pAD+
2 and pAD+
2-PDZ,
respectively). The nucleotide sequences of the DNA inserts were
confirmed by sequence analysis to verify that inserts did not contain
mutations. The inserts were expressed as fusion proteins with the DNA
binding domain and DNA activating domain of GAL4. Several small scale
yeast transformations were performed using the lithium acetate method
with 40% polyethylene glycol. The plasmids CTSCN5A and CTSCN5A-10 were
cotransformed with the plasmids pAD+
2 or pAD+
2-PDZ into the YRG-2
yeast strain containing HIS3 and lacZ double
reporter genes. After transformation, the yeast was plated on selective
plates lacking tryptophan, leucine, and histidine to show activation of
the GAL4-inducible reporter gene HIS3 through
protein-protein interaction. The colonies that grew on the selective
plates were either due to the leaky expression of HIS3 or to
the specific interaction between proteins resulting in expression of
the HIS3 gene. To distinguish between leaky expression and
specific protein interactions, we used filter lift assays to detect the
expression of a second reporter gene, lacZ. The colonies on
the selective plate were lifted onto a filter and assayed for
-galactosidase activity using
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal)
as a substrate.
GST Pull Downs--
The bacterial expression vector pGEX-5X-1
(Amersham Biosciences) was used to produce a GST fusion protein in
Escherichia coli. The C-terminal fragments of SCN5A with and
without the truncation were fused in frame into pGEX-5X-1 to produce
plasmids pGEX+CT5A and pGEX+CT5A-10. To express GST fusion proteins,
pGEX+CT5A and pGEX+CT5A-10 were transformed into BL21 (DE3)
(Stratagene, La Jolla, CA). Each bacterial culture was induced by
isopropyl-1-thio-
-D-galactopyranoside at 0.1 mM and allowed to express protein at 28 °C for 4 h.
Then the cells were pelleted and resuspended in PBS and 10 mg/ml
lysozyme. After incubation for 30 min on ice, protease inhibitor
mixture (Calbiochem) was added, and the cells were rocked at 4 °C
for 10 min. To destroy the DNA and RNA, the samples were treated with DNase and RNase. The protein extract was then extracted by
centrifugation at 3000 × g for 30 min and incubated
with glutathione beads (Amersham Biosciences).
The complete coded sequences of the splice variants of syntrophin
2
with and without the PDZ domain were linked into the expression vector
pCMV-FLAG, which tags the FLAG epitope at the N terminus, to make
plasmids pCMV-
2 and pCMV-
2-PDZ. After the constructs were
verified by sequencing their inserts, the pCMV-
2 and pCMV-
2-PDZ
constructs were transfected into HEK 293 cells to express the
FLAG-tagged syntrophin
2 proteins. The cells were lysed 24 h
later in lysis buffer, and the supernatants were used for GST pull-down experiments.
100 µl (~200 µg) of HEK 293 cell extracts containing the
FLAG-tagged syntrophin
2 proteins were incubated with 20-25 µl
(~10 µg) of GST + CT5A/GST + CT5A-10 bead-bound fusion proteins
and, as a control, the GST-alone bead-bound fusion protein. After
extensive washing with lysis buffer, the samples were subjected to
SDS-PAGE and transferred to polyvinylidene difluoride membrane. The
membrane was then incubated with anti-FLAG M2 monoclonal antibody
(Sigma). After washing, horseradish peroxidase-conjugated anti-mouse
IgG was used. The immunoreactive bands were visualized by ECL according to the manufacturer's instructions (Amersham Biosciences).
Expression and Purification of Fusion Protein--
The pGEX-5X-1
plasmid constructs for the PDZ domain of syntrophin
2 were generated
by using the same method described above for the C terminus of SCN5A.
The constructs were verified by sequencing, and the plasmids encoding
GST alone and GST plus PDZ were then introduced into BL21 cells for
expression. After induction by isopropyl-1-thio-
-D-galactopyranoside, the cell extracts
were incubated with glutathione-agarose beads for affinity
purification. Following washing, GST and GST plus PDZ were eluted with
10 mM reduced glutathione in 50 mM Tris-HCl (pH
8.0). For patch clamp analysis, the elution was dialyzed in 10 mM HEPES with 50 mM CsCl. The purity of the
proteins was determined by SDS-12% polyacrylamide gel electrophoresis.
The bands of the fusion proteins were of the expected size with high
purity. Protein concentrations were estimated using the Bio-Rad protein
assay kit.
Peptide Synthesis--
A peptide corresponding to the last 10 amino acids (SPDRDRESIV) of the C terminus of the human SCN5A sequence
and a control peptide containing the same amino acids but in random
sequence (PRRSVSDDEI) were synthesized by the Mayo Peptide Synthesis
Facility of the Mayo Proteomics Research Center. The peptides were
purified by reverse phase high performance liquid chromatography using a Vidak C-18 column. Purity was >95% as assessed by amino acid analysis and analytical high performance liquid chromatography. Mass
weight of the peptide was verified by electrospray ionization mass
spectrometry on a Sciex 165B (Concord, Canada).
Plasmid Constructs and Mammalian Cell Transfection--
The
pcDNA3 expression vector (Invitrogen) with human SCN5A (hH1c) was
used in the Na+ channel expression experiments. The
truncation vector containing the full-length SCN5A minus the last 10 amino acids was made by PCR. The cDNAs for syntrophin
2 were
produced by PCR and inserted into the pcDNA3 vector using
EcoRI and SalI. All constructs were verified by
sequencing. LipofectAMINETM 2000 Reagent (Invitrogen) was used to
transfect green fluorescent protein, pEGFP-C1,
(Clontech), sodium channel SCN5A, and syntrophin
2 HEK293 cells (ATCC, Manassas, VA). Transfected cells were
identified by fluorescence microscopy and patch-clamped.
Electrophysiological Recordings--
Whole cell patch clamp
recordings were made using standard patch clamp techniques. Whole cell
recordings were obtained using Kimble KG-12 glass pulled on a P-97
puller (Sutter Instruments, Novato, CA). Electrodes were coated with
R6101 (Dow Corning, Midland, MI) and fire-polished to a final
resistance of 3-5 megaohms. Currents were amplified, digitized, and
processed using a CyberAmp 320 amplifier, a Digidata 1200, and pCLAMP 8 software (Axon Instruments, Foster City, CA). Whole cell records were
sampled at 5 kHz and filtered at 2 kHz with an eight-pole Bessel
filter. 70-75% series resistance compensation (lag of 60 µs) was
applied during each recording. The cell capacitance
(Cm) was 40-100 picofarads in human jejunal
circular smooth muscle cells and 5-30 picofarads in HEK 293 cells. The
access resistance (Ra) was 5-10 megaohms. All
records were obtained at room temperature (21 °C).
For human jejunal circular smooth muscle cell recordings, cells were
held at
100 mV and stepped to
80 through +35 mV at 5-mV intervals
for 50 ms. The interval from the start of one depolarization to the
next was 1 s. SCN5A-overexpressed Na+ currents at a
100-mV holding potential were much larger in HEK 293 than in native
cells. Therefore, for HEK-293 cell records, cells were held at
80 mV
to reduce maximal peak inward current. Transfected cells were then
pulsed to the same voltages as described above. Steady state
inactivation in transfected cells was determined using a pulse protocol
where cells held at
80 mV, stepped to
110 through
60 mV in 5-mV
intervals for 3 s to reach a steady state of inactivation, briefly
stepped to
110 mV for 10 ms (to standardize transients), and finally
stepped to
40 mV. Current was measured at
40 mV. The interval from
the start of one depolarization to the next was 4 s.
The pipette solution contained 145 mM Cs+, 20 mM Cl
, 2 mM EGTA, 5 mM HEPES, and 125 mM methane sulfonate for most
whole cell recordings. In peptide experiments, the terminal 10-amino
acid SCN5A peptide or the control jumbled sequence peptide was added to
this intracellular solution to a concentration of 1 mM. The bath solution contained 149.2 mM Na+, 4.7 mM K+, 159 mM Cl
, 2.5 mM Ca2+, and 5 mM HEPES (normal
Ringers solution) with an osmolarity of 290-300 mosM. All
chemicals other than peptides were obtained from Sigma.
Bath perfusion was used to assess mechanosensitivity as previously
described (25, 26). The bath was perfused at 10 ml/min for 30 s to
create shear stress and activate the mechanosensitive Na+
channel according to a previously established protocol (13).
Data Analysis--
Electrophysiological data were analyzed using
PCLAMP 8 software, custom macros in Excel (Microsoft, Redmond, WA), or
SigmaPlot 2001 for Windows (SPSS Science Marketing, Chicago, IL).
Voltages were adjusted for junction potentials using JPCalcW.
Statistical comparisons were performed by a two-tailed paired
Student's t test, and p < 0.05 was used
for statistical significance. Tau of inactivation values were
determined by fitting a standard two-exponential decay curve between
the points at the peak and at 50 ms. Time-to-peak was measured as the
difference between the time when the pulse started and the time at
maximal peak inward current. Steady state activation and inactivation
curves were fitted with a three-parameter sigmoid (Boltzmann) function.
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RESULTS |
Identification of Syntrophins in Human Jejunal Circular Smooth
Muscle Cells--
To determine which syntrophins are expressed in
intestinal smooth muscle, we used gene-specific primers designed
against the five known syntrophins (
,
1,
2,
1, and
2) to
amplify cDNAs from dissociated human jejunal circular smooth muscle
cell libraries. Products of the expected size were detected for
syntrophin
,
1, and
2 using the appropriate oligonucleotide
primers (Fig. 1A). PCR
amplification for syntrophin
2 produced three bands. Sequence
analysis showed that all three cDNA fragments were different splice
variants of syntrophin
2. Syntrophin
1 was not present in human
jejunal circular smooth muscle (Fig. 1A), consistent with
its limited expression to the brain (22). RT-PCR was also carried out
on aliquots of freshly dissociated smooth muscle cells and cDNA
bands of the expected size for syntrophin
,
1,
2, and
2
obtained (data not shown), again suggesting that these syntrophins were
expressed in intestinal smooth muscle together with the known
expression of syntrophins in cardiac and skeletal muscle (22, 23).

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Fig. 1.
Expression of syntrophins in human intestinal
smooth muscle. A, gene-specific primers designed
against syntrophin , 1, 2, 1, and 2 were used for PCR
amplification from cDNA libraries. Single cDNA bands were
obtained from syntrophin , 1, and 2 primers but not 1.
Three bands were observed on syntrophin 2 amplification. Sequence
analysis showed that all three cDNA fragments were different
transcripts of syntrophin 2. B, to determine anatomical
localization of syntrophins in intestinal muscle layers, ~1500 human
jejunal smooth muscle cells from circular muscle and longitudinal
muscle were collected by laser capture microdissection and syntrophin
message amplified by RT-PCR on multiple aliquots with gene-specific
primers. Bands for syntrophin , 2, and 2 were present in
circular muscle and bands for syntrophin , 1, and 2 in
longitudinal muscle. C, immunolabeling for syntrophin 2.
Human jejunal sections were immunolabeled with an anti-syntrophin 2
antibody. Immunopositive smooth muscle cells were present in the
circular but not longitudinal muscle layer. D, cellular
localization of syntrophins. Primers designed to span introns to
exclude genomic DNA contamination were used for two or three
smooth muscle cell PCR amplification. Products of the expected size for
syntrophin , 2, and 2 were three freshly dissociated human
jejunal circular smooth muscle cells. Product identity was confirmed by
band sequencing.
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To determine the anatomical location of syntrophins within the human
intestinal smooth muscle layers, we used laser capture microdissection
to collect, separately, smooth muscle cells from the jejunal circular
muscle layer and longitudinal muscle layer. Total RNA was extracted
from the harvested cells, and reverse transcription was carried out
using random primers. An aliquot from the reverse transcription was
used for PCR amplification with one specific primer pair for either
,
1,
2, or
2 in each tube. Single bands of the expected
size for syntrophin
and
2 were observed in both circular and
longitudinal smooth muscle (Fig. 1B). Syntrophin
2 was
detected only in circular smooth muscle, and
1 was detected only in
longitudinal smooth muscle (Fig. 1B). The housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase was amplified from the RT
products and used as an internal control.
To further confirm the localization of syntrophin
2 to the circular
muscle layer of the human intestine, we immunolabeled human jejunal
sections with a rabbit polyclonal antibody directed against syntrophin
2 (a kind gift from Dr. Vincenzo Nigro). As shown in Fig.
1C, strong syntrophin
2 immunoreactivity was observed in
the circular smooth muscle layer but not in the longitudinal muscle
layer. Control experiments omitting the primary antibody showed no
immunoreactivity in the circular muscle layer (data not shown). Since
several different cell types reside in the intestinal smooth muscle
layers, single cell RT-PCR was used to localize syntrophins to smooth
muscle cells. Smooth muscle cells were dissociated from strips of
circular smooth muscle and identified according to their spindle shape.
Two or three cells were collected and placed in each tube. Syntrophin
,
2, and
2 were successfully amplified with gene-specific
primer sets designed to span an intron (Fig. 1D) to exclude
genomic sequence. The correct size band was recovered from the agarose
gel using conventional techniques, and results were confirmed by
sequence analysis. There were no products in negative controls (4 µl
of bath solution aspirated just above a smooth muscle cell) (Fig.
1D). These results were duplicated in three additional experiments.
Isolation and Characterization of Syntrophin
2 Splice
Variants--
Similar to the layer-specific expression of syntrophin
2, SCN5A is expressed in human intestinal circular smooth muscle
cells but does not appear to be strongly expressed in longitudinal
cells (12). This cell-specific colocalization between expression of SCN5A and syntrophin
2 led us to focus on syntrophin
2. PCR amplification for syntrophin
2 (Fig. 1A) showed three
bands, suggesting different transcripts of syntrophin
2. To isolate the full-length coding sequence for each transcripts, we designed primers to cover the whole open reading frame. RT-PCR amplification using human jejunal muscle tissue produced a 1.7-kb cDNA fragment, which was subcloned into pCR2.1 plasmid vector using the TOPO-TA protocol (Invitrogen). Sequence analysis showed at least five splice
variants of syntrophin
2 in human jejunal muscle (Fig. 2). Splice variant 1 was identical to the
published sequence of syntrophin
2 with 17 exons (accession number
NM_018968). Splice variant 2 had a 27-bp in-frame deletion in exon 9 that would result in loss of the protein kinase C phosphorylation site.
A similar deletion has been described in human brain tissue (22). In
splice variant 3, exons 3-6 were spliced out, resulting in the nearly complete elimination of the PDZ domain, and a 222-bp in-frame insertion
was present between exons 11 and 12. In another splice variant, variant
4 in Fig. 2, a 256-bp insertion with a stop codon was inserted
between exons 9 and 10, resulting in loss of the pleckstrin homology
and the syntrophin-unique domains and loss of the ATP/GTP-binding site.
The fifth splice variant had a 46-bp insertion with a stop codon
inserted between exons 14 and 15, resulting in loss of the
ATP/GTP-binding site and the syntrophin-unique domains.

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Fig. 2.
Expression of splice variants of
syntrophin 2 in human intestinal circular
muscle. RT-PCR amplification showed at least five splice variants
of syntrophin 2. Splice variant 1 was identical to the published
sequence with 17 exons (accession number NM_018968). Splice variant 2 had a 27-bp deletion in exon 9. Splice variant 3 had exons 3-6 deleted
with a 222-bp insertion ( ) between exons 11 and 12. Splice variant 4 had a 256-bp insertion ( ) with a stop codon between exons 9 and 10. Splice variant 5 had a 46-bp insertion ( ) with a stop codon inserted
between exons 14 and 15.
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Direct Interaction between SCN5A and Syntrophin
2--
To
determine whether syntrophin
2 and SCN5A directly interact in human
intestinal smooth muscle and to determine whether the interaction was
mediated via the PDZ binding domain on syntrophin, we performed yeast
two-hybrid and GST pull-down experiments. The last 100 amino acids of
SCN5A with (CTSCN5A) and without (CTSCN5A-10) the last 10 amino acids
(the last 10 amino acids include the PDZ binding domain) were inserted
into the pBD-GAL4 vector and used as baits (Fig.
3). Splice variant 1 of syntrophin
2
with an intact PDZ domain and splice variant 3 lacking the PDZ domain
were fused into pAD-GAL4 vector and used as prey. In the yeast YRG-2
cells, the reporter gene His was activated only by
the interaction between the splice variant 1 of syntrophin
2 with
PDZ domain and the intact C terminus of SCN5A (Fig. 3b).
Other pairings of syntrophin
2 with SCN5A did not activate
His, indicating that the last 10 amino acids of SCN5A
mediated the binding of SCN5A to the PDZ domain of syntrophin
2. The
specificity of this interaction was confirmed by testing activation of
second reporter gene lacZ (Fig. 3c). In addition, syntrophin
2 and the last 100 amino acids of SCN5A both did not self-activate
when transformed into yeast with empty bait or prey vectors (data not
shown).

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Fig. 3.
Direct interaction between the PDZ domain of
syntrophin 2 and the last 10 amino acids of SCN5A in
vivo. a, schematic diagram of baits and preys used in
the yeast two-hybrid system analysis (CTSCN5A, last 100 aa of SCN5A;
CTSCN5A-10, C terminus lacking the last 10 aa of SCN5A;
Syn- 21, syntrophin- 2 splice variant 1 with an intact
PDZ domain; Syn- 23, syntrophin- 2 splice variant 3 lacking a PDZ domain). b, expression of the reporter gene
HIS3. Each pair of constructs as indicated in A
was co-transfected into the YRG-2 yeast strain. Yeast transformants
were then selected on selective plates and tested for expression of
reporter gene HIS3. Strong expression of HIS3
only occurred when the last 10 aa of SCN5A and the PDZ domain of
syntrophin 2 were both present. c, -galactosidase
activity. Colonies that grew on the selective plates were transferred
onto the filter papers and assayed for -galactosidase activity,
confirming that SCN5A and syntrophin 2 interact and that the
interaction occurs through the C terminus of SCN5A and the PDZ
domain.
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The interaction between the C terminus of SCN5A and the PDZ domain of
syntrophin
2 was verified using GST pull-down assays. Splice
variants 1 and 3 of syntrophin
2 were expressed in HEK 293 cells.
Affinity-purified GST, GST plus CT5A, and GST plus CT5A-10, immobilized
on glutathione-Sepharose beads, were incubated with cell lysates
containing the syntrophin
2 splice variants 1 and 3. Fig.
4 shows that only GST plus CT5A trapped
splice variant 1 of syntrophin
2 (lane 2) and
that there was no interaction between GST plus CT5A with or without the
last 10 amino acids (aa) (lanes 5 and
6) and the splice variant 3 of syntrophin
2, again
suggesting a specific interaction between the PDZ domain of syntrophin
2 and the last 10 aa of SCN5A. GST alone did not bind to either
splice variant of syntrophin
2 (lanes 1 and
4).

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Fig. 4.
Direct interaction between the PDZ domain of
syntrophin 2 and the last 10 amino acids of
the C terminus of SCN5A in vitro. A,
the full-length syntrophin 2 with a PDZ domain (Syn
21) and the syntrophin 2 without a PDZ domain (Syn
23) were transfected into HEK 293 cells. Approximately
200 µg of cell lysate was then incubated with GST, GST plus CT5A
(last 100 aa of the C terminus of SCN5A), or GST plus CT5A-10 (last 100 aa of the C terminus except for the very last 10 aa) beads. After
washing, the proteins bound to the beads were resolved by 10% SDS-PAGE
and identified by Western blots using the anti-FLAG antibody as the
probe. Specific binding was observed only between GST plus CT5A and
syntrophin 21 (with a PDZ domain). B,
lanes 1 and 2 were loaded with 1% of
the cell lysates compared with 10% of the extracts for
lanes 3 and 4.
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The specificity and functionality of the interaction between the C
terminus of SCN5A and the PDZ domain of syntrophin
2 were tested on
the native Na+ current in human intestinal circular smooth
muscle cells. Freshly dissociated cells were patch-clamped, and a 10-aa
peptide (1 mM) corresponding to the last 10 aa of SCN5A was
introduced via the patch pipette into the cells. The peptide completely
abolished the perfusion-induced increase in Na+ current.
The peptide was allowed to diffuse into the cells for 10 min after
breaking in, and the Na+ current was activated by
perfusion. Perfusion did not increase peak inward Na+
current (5 ± 2% increase, n = 8, p > 0.05 compared with preperfusion) (Fig.
5a). In contrast, in control
cells without the peptide, perfusion increased peak inward
Na+ current by 27 ± 3% (data not shown).
Introduction of a control jumbled peptide had no effect on this
perfusion-induced increase in inward Na+ current (27 ± 6% increase in current, n = 6, p < 0.05 compared with preperfusion). Furthermore, introduction of a
GST-bound 98-aa sequence (10 nM) corresponding to the PDZ
domain of syntrophin
2 also blocked the perfusion-induced increase
in inward Na+ current (6 ± 2% increase in current,
n = 6, p > 0.05 compared with
preperfusion) (Fig. 5b). Introduction of GST alone into the human intestinal smooth muscle cells did not block the
perfusion-induced increase in current (19 ± 4% increase in
current, n = 8, p < 0.05 compared with
perfusion without GST). These results suggest that a direct interaction
between the last 10 aa of SCN5A and the PDZ domain of syntrophin
2
is required to maintain mechanosensitivity of the Na+
channel.

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Fig. 5.
SCN5A C terminus peptide and syntrophin
2 PDZ domain peptide block perfusion-induced
increase in peak Na+ current. a, control
Na+ current obtained from a human jejunal circular smooth
muscle cells using the pulse protocol in the inset 10 min after
breaking in with 1 mM C terminus peptide in the pipette
solution and lack of activation of the Na+ current by
perfusion. b, control Na+ current obtained 10 min after breaking in with 1 mM C terminus scrambled
peptide in the pipette solution and activation of the Na+
current by perfusion. c and d, mean
current-voltage relationships for the effects of perfusion in the
presence of the C-terminal peptide and the control scrambled peptide,
respectively. e, mean peak inward Na+ currents.
f, Coomassie Blue-stained recombinant purified GST and GST
plus PDZ shown on 15% SDS-PAGE. g, control Na+
current 10 min after breaking in with 10 nM GST-PDZ peptide
in the pipette solution and lack of activation of the Na+
current by perfusion. h, control Na+ current
obtained 10 min after breaking in with just the GST peptide and
activation of the Na+ current by perfusion. i
and j, mean current-voltage relationships for the effects of
perfusion in the presence of GST-PDZ domain peptide and GST,
respectively. k, mean peak inward Na+
currents.
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Modulation of Gating of SCN5A by Syntrophin
2--
The above
data indicate a specific interaction between syntrophin
2 and SCN5A
and that syntrophin
2 is required for mechanosensitivity of SCN5A.
Syntrophins do not appear to be required for the sarcolemmal localization of sodium channels (27, 28). Therefore, to further delineate the functional consequence of the interaction between the two
proteins, we transfected HEK293 cells with either SCN5A alone or with
SCN5A and syntrophin
2. Co-transfection of syntrophin
2 with
SCN5A shifted in a positive direction the voltage-dependent activation of SCN5A by 8.5 mV. (Fig. 6,
a and b). V1/2 for SCN5A alone
was
43.4 ± 0.3 mV (n = 9) and shifted to
34.9 ± 0.3 mV (n = 9), p < 0.05, when syntrophin
2 was co-transfected with SCN5A. As a result,
maximal peak inward Na+ current shifted from
23 ± 1 to
15 ± 2 mV. At
40 mV, peak inward Na+ current
decreased from
737 ± 94 pA to
281 ± 67 pA when
syntrophin
2 was co-transfected with SCN5A (n = 8, p < 0.05). However, maximal peak inward
Na+ current was unchanged (
1169 ± 174 pA,
n = 7 for SCN5A alone;
1163 ± 278 pA,
n = 8, p > 0.05 for SCN5A with
syntrophin
2). Time to peak current increased at all voltages tested
(Fig. 6c) with a change from 0.87 ± 0.03 to 1.53 ± 0.056 ms at maximal inward Na+ current (
20 mV,
n = 6, p < 0.05). The slope of the
activation curve was also changed (Fig. 6b), with a
k value of 5.1 ± 0.2 mV for SCN5A alone and a
k value of 6.5 ± 0.2 for SCN5A and syntrophin
2.
Inactivation was fit with 2 taus. Co-expression of syntrophin
2
resulted in a slower first tau (fast inactivation) with no change noted
for the second tau (slow decay, Fig. 6c, n = 6, p < 0.05). No effect was noted on the kinetics of
steady-state inactivation (Fig. 6b); therefore, the net
result of the observed changes was a reduction in the overlap of the
activation and inactivation relationships, resulting in a reduced
window current (29) (Fig. 6b) compared with the window
current observed with SCN5A alone. Truncation of the last 10 aa of
SCN5A (n = 6, Fig.
7a) or cotransfection of the
splice variant of syntrophin
2 lacking a PDZ domain (syntrophin
23, n = 6, Fig. 7b) had no
significant effect on Na+ channel gating, suggesting that
the effects of syntrophin
2 on SCN5A current were again mediated by
a specific interaction between the PDZ domain of syntrophin
2 and
the last 10 amino acids of SCN5A.

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Fig. 6.
Effect of co-transfection of syntrophin
2 with SCN5A. a, inward
Na+ current at 40 mV for SCN5A alone and SCN5A plus
syntrophin 2. b, steady state activation and inactivation
curves for SCN5A alone and SCN5A plus syntrophin 2, showing the
right shift in activation and the smaller window current when
syntrophin 2 was co-transfected. c, time to peak
(activation) was slower at all voltages tested when syntrophin
2 was co-transfected with SCN5A. Fast inactivation was also slower
at all voltages tested, whereas slow decay was unchanged (see
"Results" for details).
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Fig. 7.
Effect of truncation of SCN5A and of loss of
the syntrophin 2 PDZ domain on SCN5A
kinetics. a and d, whole cell current traces
at 40 mV for SCN5A, SCN5A without the last 10 aa cotransfected with
syntrophin 2, and SCN5A co-transfected with syntrophin 2 without
the PDZ domain. b and e, steady-state
activation and inactivation curves. c and f,
activation and inactivation kinetics. Both truncation of SCN5A and
absence of the PDZ domain of syntrophin 2 resulted in loss of the
kinetic changes seen when the full-length SCN5A was co-expressed with
syntrophin 2.
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DISCUSSION |
The main finding of this study is that mechanosensitivity of the
human circular smooth muscle and cardiac Na+ channel is
dependent on a specific interaction between the PDZ domain of
syntrophin
2 and the last 10 amino acids of the C terminus of SCN5A,
the
subunit of both the tetrodotoxin-resistant cardiac muscle and
the native intestinal smooth muscle Na+ channel. The
mechanisms that underlie ion channel mechanosensitivity are complex and
vary according to the ion channel studied (3, 30). Potential mechanisms
for ion channel mechanosensitivity include a direct interaction between
the transmembrane portion of the channel and the lipid bilayer,
suggesting that this is an unavoidable consequence of inserting a
channel into the membrane (30-32), activation of mechanosensitive
signaling cascades that subsequently activate ion channels via
phosphorylation or other post-translational modification (3), or force
transmission via protein-protein interactions between the channel and
the cytoskeleton that alter the channel open probability (3, 4, 6, 33, 34). Our data suggest that for SCN5A, the latter mechanism appears to
be a central one for the mechanosensitivity observed upon perturbing the cell's membrane, since mechanosensitivity of the native
Na+ channel was completely lost when the interaction
between the cytoskeleton and the C terminus of SCN5A was disrupted. The
expression of SCN5A and syntrophin
2 in intestinal muscle was
similar, with immunohistochemical and molecular evidence to suggest
that both are expressed in human intestinal circular but not
longitudinal smooth muscle. The co-expression of SCN5A and syntrophin
2 suggests that the interaction may be specific to syntrophin
2
and not be generalizable to all syntrophins with PDZ domains. This is supported by the data from native cells with block of
mechanosensitivity when the specific aa sequence of the PDZ domain of
syntrophin
2 is introduced into the cell. The sequences of PDZ
domains are known to be highly conserved among all five known
syntrophins (22, 23). However, the recently identified syntrophin
1
has a PDZ domain similar to that of other syntrophins and yet does not
bind SCN4A and SCN5A, suggesting different specificity of PDZ domains
of syntrophins (24). Moreover, the Na+ channel-syntrophin
interaction is not necessarily dependent on PDZ domains, since brain
Na+ channels, which lack the consensus motif
E(S/T)XV at their C termini, required to bind PDZ domains,
still copurified with syntrophin (14), suggesting that multiple
interactions occur between syntrophins and Na+ channels and
that the specific interaction between the C terminus of SCN5A and the
PDZ domain of syntrophin
2 may only be only an absolute requirement
for mechanosenstivity.
A link between Na+ channel activity and actin cytoskeleton
has been proposed previously. Treatment of cardiac myocytes with cytochalasin-D to inhibit actin polymerization reduces peak
Na+ current and slows inactivation (35, 36). Disruption of
the cytoskeleton also alters Na+ channel properties in
skeletal muscle, epithelial tissue, and leukemia cells (28, 37-39).
The data presented here suggest that, for SCN5A, the likely link
between SCN5A and the actin cytoskeleton are the syntrophins.
Syntrophins localize associated proteins to the membrane by binding
with dystrophin (40, 41). In genetically modified mice lacking
syntrophin
or the PDZ domain of syntrophin, neuronal nitric-oxide
synthase is absent from the sarcolemma, suggesting dependence on
syntrophin for sarcolemmal localization of neuronal nitric-oxide
synthase (27, 42). Similarly, the membrane localization of water
channel, aquaporin-4, also requires syntrophin (43). Aquaporin-4 is not
localized to the membrane when syntrophin
is absent. However,
syntrophin is not always required for the membrane localization of its
associated proteins. The skeletal muscle sodium channel SCN4A is known
to bind syntrophin
,
1, and
2, but localization to the
membrane does not appear to depend on syntrophins (27, 28). Also, the
absence of syntrophin
does not have an apparent effect on the
distribution or level of expression of the Na+ channel. In
mdx mice, the distribution of Na+ channels remains
identical despite the lack of dystrophin and reduction of sarcolemmal
density of syntrophins (28). These observations suggest that
syntrophins do not play a key role in the sarcolemmal localization of
Na+ channel. Rather, the data presented here indicate that
syntrophins directly alter SCN5A gating behavior. Thus, syntrophin
2
may be an essential Na+ channel-interacting protein
required to recapitulate the full phenotype of the Na+ current.
The changes in activation and inactivation kinetics observed on
cotransfection of syntrophin
2 with SCN5A would have significant effects on the Na+ channel whole cell current. A shift in
the voltage of activation for SCN5A coupled with slowing of activation
will result in a smaller contribution of the peak Na+
current to the action potential. Prolongation of fast inactivation would be expected to increase available Na+ current over a
few milliseconds after the peak, but the reduced window current would
tend to reduce late Na+ current over the window voltage
range (29). No effect on slow decay was seen in our study. However, a
previous study has reported that the last 8 aa of the C terminus of
SCN5A slows inactivation with little effect on activation (44). During
the gastrointestinal slow wave the membrane potential of smooth muscle
is within the window current of SCN5A suggesting that the channel
contributes to the regulation of membrane potential and of
intracellular Na+. Changes in the window current, as seen
with syntrophin
2, may therefore alter membrane potential and
intracellular Na+ homeostasis. Mutations in SCN5A are
associated with changes in gating behavior and clinical disease (10).
Mutations in SCN5A that prolong slow decay result in an enhancement of
inward plateau current and a vulnerability to potentially fatal
heritable arrhythmias like long QT syndrome subtype 3 and rarely sudden
infant death syndrome (45). Mutations that result in a decrease in
Na+ current lead to Brugada's syndrome and Lenegre disease
(10). The changes in SCN5A current kinetics observed on co-transfection of SCN5A in HEK cells with syntrophin
2 may therefore lead to more
than one mechanism for cardiac arrhythmias dependent on the relative
expression of syntrophin
2 variants. It is presently unclear if
syntrophins can directly interact with membrane proteins and the actin
cytoskeleton or if dystrophin is always required. However, considering
that only 65% of long QT syndrome is genotyped presently on the known
long QT-causing genes (46), molecular and functional characteristics of
syntrophin
2 described herein makes this Na+
channel-interacting protein an attractive pathogenomic target for
unexplained, nongenotyped sudden cardiac death.
In summary, mechanosensitivity of the Na+ channel encoded
by SCN5A requires a specific interaction between the C terminus of SCN5A and the PDZ domain of syntrophin
2. Co-expression of SCN5A and
syntrophin
2 results in a smaller Na+ window current,
suggesting the possibility that syntrophins may directly regulate the
cardiac muscle and intestinal smooth muscle Na+ current.
These results also raise the possibility that mutations in the PDZ
domain of syntrophin
2 or differential expression of the identified
splice variants of syntrophin
2 may contribute to clinically
significant arrhythmias.