From the Department of Neurology and the
§ Paralyzed Veterans of America/Eastern Paralyzed
Veteran Association Neuroscience Research Center, Yale
University School of Medicine, New Haven, Connecticut 06510 and the
¶ Rehabilitation Research Center, Veterans Affairs Connecticut
Healthcare System, West Haven, Connecticut 06516
Received for publication, July 15, 2002, and in revised form, October 17, 2002
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ABSTRACT |
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We have previously shown that fibroblast growth
factor homologous factor 1B (FHF1B), a cytosolic member of the
fibroblast growth factor family, associates with the sensory
neuron-specific channel Nav1.9 but not with the other
sodium channels present in adult rat dorsal root ganglia neurons. We
show in this study that FHF1B binds to the C terminus of the cardiac
voltage-gated sodium channel Nav1.5 and modulates the
properties of the channel. The N-terminal 41 amino acid residues of
FHF1B are essential for binding to Nav1.5, and the
conserved acidic rich domain (amino acids 1773-1832) in the C
terminus of Nav1.5 is sufficient for association with this
factor. Binding of the growth factor to recombinant wild type human
Nav1.5 in human embryonic kidney 293 cells produces a
significant hyperpolarizing shift in the voltage dependence of channel
inactivation. An aspartic acid to glycine substitution at position 1790 of the channel, which underlies one of the LQT-3 phenotypes of cardiac
arrythmias, abolishes the interaction of the Nav1.5 channel
with FHF1B. This is the first report showing that interaction with a
growth factor can modulate properties of a voltage-gated sodium channel.
Voltage-gated Na+ channels, which are essential for
the generation of action potentials and cell excitability, are
complexes of a pore-forming Different classes of proteins have been shown to interact with several
sodium channels and influence their surface expression and/or the
properties of their Na+ currents. Auxiliary Channel partners can modulate the Na+ current either as a
direct effect of binding or by inducing post-translational
modifications, for example, phosphorylation. Peak Na+
current amplitude in cultured hippocampal neurons is reduced upon
activation of dopamine (D1) receptors via
cAMP-dependent protein kinase that is anchored to
the Fibroblast growth factor homologous factor 1B (FHF1B, also known as
FGF12B)2 belongs to the FGF
family of growth factors (18). FHF1-4 (FGF11-14) form a subfamily
whose members lack canonical signal peptides and are not secreted into
the medium of transfected cells (18-21). Recently, FHF1 and FHF2 have
been shown to bind to the scaffold protein Islet of
Brain 2 (IB2) and recruit the mitogen-activated protein kinase p38 Plasmids--
Yeast expression vector pDBleu (Invitrogen) and
pPC86 (Invitrogen) are fusion vectors for the linkage of proteins to
the Gal4 DNA-binding domain and to the VP16 transactivation domain,
respectively. Fragments encoding the C termini of all known vertebrate
voltage-gated sodium channels were amplified by PCR and cloned in-frame
into the SalI/NotI sites of pDBleu
(pDB-rNav1.1C, pDB-rNav1.2C,
pDB-rNav1.3C, pDB-rNav1.4C,
pDB-rNav1.5C, pDB-rNav1.6C,
pDB-hNav1.7C, pDB-rNav1.8C, and
pDB-rNav1.9C) respectively. The coordinates of the C
termini fragments are: a.a. 1790-2009 of rNav1.1
(GenBankTM accession number NM_030875); a.a. 1780-2005 of
rNav1.2 (GenBankTM accession number NM_01264);
a.a. 1726-1977 of rNav1.3 (GenBankTM
accession number NM_013119); a.a. 1595-1840 of rNav1.4
(GenBankTM accession number Y17153); a.a. 1778-2019 of
rNav1.5 (GenBankTM accession number AF353637);
a.a. 1766-1976 of rNav1.6 (GenBankTM accession
number L39018); a.a. 1753-1977 of hNav1.7
(GenBankTM accession number X82835); a.a. 1728-1957) of
rNav1.8a (GenBankTM accession number X92184);
and a.a. 1588-1765 of rNav1.9 (GenBankTM
accession number AF059030). Fragments encoding subregions of the C
terminus of rNav1.5a were also amplified and subcloned into
the SalI/NotI sites of pDBleu: CR1 plus CR2 (a.a.
1773-1885; pDB-Nav1.5CR1-2), CR1 (a.a. 1773-1832;
pDB-Nav1.5CR1), CR2 (a.a. 1832-1885;
pDB-Nav1.5CR2), and UR (a.a. 1885-2016;
pDB-Nav1.5UR). Fragments encoding point mutations (E1784K,
D1790G, Y1795H, and Y1795C) and the multiple residue substitution
(E1784K/D1790G/Y1795H) of the C terminus of Nav1.5 channel
were also cloned into the pDBleu vector: E1784K
(pDB-Nav1.5CK), D1790G (pDB-Nav1.5CG), Y1795H (pDB-Nav1.5CH), Y1795C (pDB-Nav1.5CC), and KGH
(pDB-Nav1.5CKGH). The full coding sequence of rat FHF1B
(GenBankTM accession number AF348446) was cloned in-frame
into the SalI/NotI sites of pPC86 vector to
generate plasmid pPC86-FHF1B and pPC86-FHF1B, respectively.
The bacterial expression vector pGEX-3X (Invitrogen) was used to
produce recombinant proteins in Escherichia coli. A fragment encoding the C terminus of Nav1.5 was subcloned in-frame
into the BamHI/EcoRI sites of pGEX-3X to produce
the plasmids pGEX-Nav1.5C.
For expression of GFP fusion proteins in mammalian cells, the complete
coding sequence of FHF1B and a series of N-terminally truncated
segments were subcloned in-frame into the
BamHI/EcoRI sites of pEGFP-N1 vector
(Clontech) to produce plasmid pFHF1B-GFP, pFHF1B
A construct of full-length human Nav1.5 was a generous gift
from Dr. Mike O'Leary. The complete open reading frame and flanking untranslated sequences were cloned into the
HindIII/XbaI sites of the vector pcDNAI. The
mutant D1790G was generated using site-directed mutagenesis. The
forward mutagenic primer
(5'-AGCCCCTGAGTGAGGACGGCTTCGATATGTTCTATG-3') and the reverse mutagenic primer (reverse complement sequence of
the forward primer) were used in the QuikChange mutagenesis system
(Stratagene); the single nucleotide substitution G, underlined and in
bold type, causes the D1790G LQT-3 mutation. The sequence of the entire
insert was verified to eliminate the possibility of additional
unintended substitutions.
All of the constructs were verified by sequencing their inserts.
Sequencing was carried out at the Howard Hughes Medical Institute/Keck Biotechnology Resource Laboratory at Yale University. Sequence analysis
was done using Lasergene (DNASTAR) and BLAST (National Library of
Medicine) software.
Yeast Two-hybrid Assay for Protein-Protein
Interactions--
Three independent colonies were analyzed for the
interaction in yeast between two proteins, of which one was fused to
the Gal4 DNA binding domain and the other was fused to the VP16
transactivation domain. The procedures of Vojtek et al. (24)
and Hollenberg et al. (25) were followed for (i) growing and
transforming the yeast strain MAV203 with the selected plasmids and
(ii) testing by the In Vitro Binding Assay--
For expression of GST fusion
proteins, the plasmid pGEX-Nav1.5C was transformed into
E. coli DH5 (Invitrogen). The fusion proteins were
affinity-purified on glutathione-agarose beads as previously described
(26). To examine the binding of FHF1B and its derivatives to the C
terminus of rNav1.5 in vitro,
glutathione-Sepharose beads (50 µl) preincubated with purified GST
(0.5 µg), serving as control, or GST-Nav1.5C (0.5 µg)
were incubated with extract (500 µg of protein) of HEK293 cells
transfected with an expression plasmid encoding either full-length
FHF1B-GFP or its derivatives (i.e. FHF1B Co-immunoprecipitation--
HEK293 cells were grown in 100-mm
dishes in Dulbecco's modified Eagle's medium containing
heat-inactivated 10% fetal bovine serum. After reaching 50%
confluence, the cultures were co-transfected with the mammalian
expression plasmids pCMV-Nav1.5 and pEGFP-N1 or pFHF1B-GFP.
The cultures were harvested 48 h later, and the cell extracts were
prepared according to Liu et al. (27). After 1 h of
incubation with anti-GFP (25 µg/ml) antibodies
(Clontech), aliquots (200 µl) from this mixture
were incubated with 30 µl of protein A-agarose (Invitrogen) at
4 °C overnight. After washing five times with immunoprecipitation
buffer (28), the bound proteins were released by boiling in 20 µl of
2× SDS loading buffer for 3 min (26). The released proteins were
examined by Western blotting with sodium channel pan-specific
antibodies (Upstate), and the signal was detected using the ECL
chemiluminescent system (Amersham Pharmacia Biotech).
Expression of Recombinant Na+ Channels--
HEK 293 cells were grown under culture conditions and co-transfected with 1 µg of wild type human Nav1.5 or its D1790G mutant cDNAs, together with 0.9 µg of FHF1B-GFP and pEGFP-N1 cDNAs,
using a calcium-phosphate protocol. Transfected cells were identified by green fluorescence from recombinant FHF1B-GFP and EGFP proteins.
Electrophysiology--
Nav1.5 sodium currents were
measured by whole cell patch-clamp protocols (29) with Axopatch 200B
amplifiers (Axon Instruments) using the following solutions: internal,
140 mmol/liter CsF, 1 mmol/liter EGTA, 10 mmol/liter NaCl, and 10 mmol/liter HEPES, pH 7.3, adjusted to 310 mOsmol/liter with glucose;
external, 140 mmol/liter NaCl, 3 mmol/liter KCl, 1 mmol/liter
MgCl2, 1 mmol/liter CaCl2, and 20 mmol/liter
HEPES, pH 7.3, adjusted to 320 mOsmol/liter with glucose. The pipette
potential was zeroed before seal formation; the voltages were not
corrected for liquid junction potential. Capacity transients were
cancelled, and series resistance was compensated (90%) as necessary.
Leakage current was digitally subtracted on-line using hyperpolarizing
control pulses, applied before the test pulse, of one-sixth test pulse
amplitude ( Experimental Protocols and Data Analysis--
Steady-state
activation curves were constructed with the membrane potential held at
The voltage-dependent steady-state inactivation
relationship was investigated with a standard two-pulse protocol. For
maximum recovery from slow inactivation of Na+ channels, a
test potential to FHF1B Associates with Cardiac Channel Nav1.5 and
Sensory Neuron-specific Channel Nav1.9 in Yeast Two-hybrid
Assays--
We reported previously that FHF1B binds directly to the C
terminus of sensory neuron-specific, voltage-gated TTX-R sodium channel
Nav1.9 (23). The C-terminal polypeptides of sodium channels vary in length from 177 amino acids for Nav1.9 to 251 amino
acids for Nav1.3a (brain type IIIA); alignment of these
polypeptides (not shown) shows that the similarity to the C-terminal
polypeptide of Nav1.9 ranges from 50% for
Nav1.1a to 56.7% for Nav1.5. Despite the 50%
similarity of the C termini of the sodium channels, FHF1B did not bind
to the C termini of Nav1.7 and Nav1.8, which
are expressed, as is Nav1.9, in adult rat dorsal root
ganglia neurons (23). FHF1B is expressed in tissues where other sodium
channels are expressed; therefore, we tested whether the C-terminal
polypeptides of other sodium channels bind FHF1B in yeast two-hybrid
assays. As shown in Fig. 1, FHF1B binds
to the C-terminal polypeptide of Nav1.5 and
Nav1.9 but not to other sodium channels in this assay.
FHF1B Associates with Nav1.5 Both in Vitro and in
Vivo--
To test whether FHF1B binds to the C-terminal polypeptide of
Nav1.5 in vitro, we used the pull-down assay. We
expressed the FHF1B-GFP fusion protein in HEK293 cells and the
C-terminal polypeptide of Nav1.5 as a GST fusion protein
(GST-Nav1.5C) in bacteria. Affinity-purified GST and
GST-Nav1.5C immobilized on glutathione-Sepharose beads were
incubated with lysates of HEK293 cells expressing either GFP or
FHF1B-GFP. Purified GST or GST-Nav1.5C did not pull down GFP protein in this assay (Fig.
2A, lanes 1-3).
Purified GST did not pull down FHF1B-GFP fusion protein (Fig.
2A, lane 5), whereas GST-Nav1.5C
efficiently pulled down the FHF1B-GFP fusion protein (Fig.
2A, lane 6).
To examine whether FHF1B also binds to full-length Nav1.5
in vivo, we performed a co-immunoprecipitation assay (Fig.
2B). In this assay, HEK293 cells were transfected with
full-length human Nav1.5 together with GFP (lanes
1 and 3) or Nav1.5 together with
full-length FHF1B-GFP (lane 2 and 4). Comparable
levels of Nav1.5 were detected in total lysates from both
conditions using a pan-specific sodium channel antibody (lanes
1 and 2). Lysates from sister cultures were incubated
with anti-GFP antibodies (lane 3 and 4), and the
immunoprecipitated complexes were counter-tested with the sodium
channel pan-specific antibodies. A single immunoreactive band of
Nav1.5 expected size is detected in the sample brought down
by anti-GFP antibodies from the cell lysates expressing full-length Nav1.5 together with FHF1B-GFP (lane 4) but not
from the lysates expressing full-length Nav1.5 with GFP
(lane 3). Thus, FHF1B specifically binds to full-length
Nav1.5 in vivo.
The N-terminal Segment (a.a. 1-41) of FHF1B Is Required for
Association with the C-terminal Polypeptide of
Nav1.5--
The segment of FHF1B that is critical for the
binding of the C terminus of Nav1.5 was mapped by a
pull-down assay using a series of N-terminal deletions of FHF1B. FHF1B
derivatives were linked to GFP (Fig.
3A), and the constructs were
used to transfect HEK 293 cells. Pull-down assays were performed using
immobilized GST (Fig. 3B, lanes 2, 5,
8, 11, 14, and 17) and
GST-Nav1.5C (Fig. 3B, lanes 3,
6, 9, 12, 15, and
18), and cell extracts were prepared from transfected HEK293
cells. The cell lysates show the presence of the respective GFP-FHF1B
derivative (Fig. 3B, lanes 1, 4,
7, 10, and 13) or GFP alone (Fig.
3B, lane 16). Immunoblotting using anti-GFP
antibodies showed that deletion of the N-terminal tetrapeptide of FHF1B
significantly reduced association with GST-Nav1.5C compared with the full-length factor (compare lanes 3 and
6). Deletion of N-terminal 41 amino acid residues totally
disrupted the interaction of FHF1B and Nav1.5C (lane
9). FHF1B lacking the N-terminal 77 or 143 residues (lane
11 and 15), similar to GFP alone (lane 18), did not show any interaction with GST-Nav1.5C. As a negative control, GST did not pull down either of the FHF1B derivative polypeptides (see
Fig. 6B, lanes 2, 5, 8,
11, 14, and 17). These data show that
the N-terminal segment (a.a. 1-41) contains molecular determinants for
the interaction of FHF1B with the C-terminal polypeptide of Nav1.5.
Conserved Segments of the C-terminal Polypeptide of
Nav1.5 Are Important for FHF1B Binding--
Alignment of
the C-terminal polypeptides of the three TTX-R Na+ channels
(Fig. 4) and all mammalian sodium
channels (not shown) identified two conserved regions (CR1 and CR2) of
60 and 54 amino acids each and a terminal unique region of 132 amino
acids for Nav1.5. We subcloned fragments encoding the
individual conserved and unique regions (Fig.
5A) to identify the
Nav1.5 segment, which is necessary for FHF1B binding. We
carried out yeast two-hybrid assays using CR1, CR2, CR1+CR2, and the
unique region linked to GAL4DBD and full-length FHF1B fused toVP16AD.
As shown in Fig. 5, removal of the unique region (a.a. 1885-2016) and
CR2 (a.a. 1832-1885) from the C terminus did not disrupt the
interaction with FHF1B. CR1 (a.a. 1773-1832) alone is necessary and
sufficient to bind FHF1B.
LQT-3 Point Mutation in Nav1.5 Abolishes Interaction
with FHF1B--
CR1 of Nav1.5 C terminus (also referred to
as the acidic rich domain) includes a number of negatively charged
residues (Fig. 4), which is important in the control of the channel
inactivation (30, 31). Several inherited mutations that are associated with the cardiac arrhythmias Long QT syndrome and Brugada disease have
been reported in this domain. These include E1784K (32), D1790G
(33-35), and Y1795H and Y1795C (36). To determine whether these
mutations affect the interaction of Nav1.5 channel with FHF1B, we first introduced these point mutations into the C terminus (a.a. 1773-1832) of Nav1.5 and cloned them into a yeast
expression plasmid (Fig. 6A).
Y2H assays revealed that the mutations E1784K, Y1795H, or Y1795C did
not affect FHF1B binding (Fig. 6B). However, the D1790G
mutant totally abolished the interaction with FHF1B (Fig.
6B). As might be expected, the triple mutant KGH (E1784K, D1790G, and Y1795H combined) did not show any interaction (Fig. 6B).
The effect of the D1790G mutation on the interaction of FHF1B and
full-length Nav1.5 in vivo was tested by an
immunoprecipitation assay (Fig. 6C). In this assay, extracts
of cells expressing the mutant D1790G (lanes 1 and
2) or wild type human Nav1.5 (lanes 3 and 4) together with full-length FHF1B-GFP were first
incubated with anti-GFP antibodies (lane 2 and
4), and the immunoprecipitated complexes were counter-tested
with sodium channel pan-specific antibodies. Both wild type
Nav1.5 and the D1790G mutant channel were expressed
efficiently as comparable levels of the channels were detected in the
cell lysates (lanes 1 and 3). Full-length wild
type Nav1.5 (Fig. 6C, lane 4) but not
the D1790G mutant (lane 2) co-immunoprecipitates with
FHF1B-GFP. Thus, FHF1B specifically binds to full-length
Nav1.5 in vivo, and the LQT-3 mutation D1790G abolishes this interaction.
Voltage-dependent Sodium Currents in HEK293 Cells
Transfected with Nav1.5 or
Nav1.5+FHF1B--
The observations that FHF1B binds to the
C-terminal polypeptide of Nav1.5 and that anti-GFP antibody
precipitates the full-length channel together with FHF1B-GFP, prompted
us to investigate whether this binding modulates Nav1.5
currents. For this purpose, Nav1.5 channels were
co-expressed together with FHF1B-GFP in HEK 293 cells and were compared
with Nav1.5 channels that were co-expressed with GFP alone.
Voltage-gated Na+ currents with fast activation and fast
inactivation kinetics were evoked by depolarizing pulses in cells
transiently transfected with Nav1.5 plus GFP or
co-transfected with Nav1.5 plus FHF1B-GFP (Fig.
7, A and B).
Functional association of the FHF1B protein with Nav1.5
channel did not significantly change the peak current densities. The
peak Na+ current density (elicited by voltage pulses from
Nav1.5 Channel Activation Is Not Modulated by
FHF1B--
Fig. 7C shows the current-voltage (I-V)
relationship of the voltage-dependent activation of
Nav1.5 with or without FHF1B. Sodium currents were
activated at around FHF1B Modulates Nav1.5 Channel Inactivation--
The
effects of FHF1B on voltage-dependent inactivation were
examined by measuring the amplitude of peak currents evoked by a
two-pulse protocol as detailed under "Experimental Procedures." The
average V1/2 of the voltage-dependent inactivation of Nav1.5 was FHF1B Does Not Modulate D1790G Mutant Channel--
Because LQT-3
point mutation D1790G abolishes the interaction between
Nav1.5 channel and FHF1B in yeast two-hybrid and
biochemical assays (Fig. 6), we confirmed a lack of interaction with
the mutant channel by electrophysiology. Voltage-gated Na+
currents were evoked by depolarizing pulses in HEK 293 cells transiently co-transfected with D7190G plus GFP or co-transfected with
D1790G and FHF1B-GFP (Fig. 8,
A and B). As seen in wild type Nav1.5
currents, co-expression of FHF1B protein with the D1790G mutant channel
did not significantly enhance the peak current density. The peak
Na+ current densities (elicited by voltage pulses from
The Na+ currents produced by the D1790G mutant channel
showed faster inactivation kinetics (Fig. 8A) compared with
wild type Nav1.5 channels, as reported earlier (35). The
inactivation kinetics are 3-fold faster than the wild type
Nav1.5 currents at
The D1790G mutation causes a marked negative shift, The results of this study show, for the first time, that
interaction with a growth factor can modulate the properties of a voltage-gated sodium channel. Our findings show that a member of the
FGF family (FGF12; also termed FHF1B) binds to two members of the
voltage-gated sodium channel family (Nav1.5 and
Nav1.9). The association of FHF1B with Nav1.5
(this study) and with Nav1.9 (23) were verified by both
in vitro and in vivo assays. We show that the
N-terminal 41 amino acids of FHF1B and the 60-amino acid CR1 (acidic
rich domain, a.a. 1773-1832) in the proximal region of the C terminus
of Nav1.5 are important for the interaction of these two
proteins. Our results further demonstrate that binding of FHF1B to
Nav1.5 results in a significant alteration in the voltage
dependence of inactivation. The LQT-3 channel mutant D1790G abolishes
the binding of Nav1.5 to FHF1B. Our findings thus
demonstrate the binding of FHF1B to the C terminus of
Nav1.5 and provide the first evidence for the modulation of
a sodium channel by a growth factor.
FHF1B binds to the proximal segment of the C terminus of
Nav1.5 and modulates the voltage dependence of inactivation
of the channel with no effects on channel activation (this study).
Nav1.5 is primarily expressed in heart muscle (38, 39) but
is also expressed in embryonic dorsal root ganglia neurons (40) and in
limbic regions of rat brain (41). FHF1B also binds to the analogous
region of the sensory neuron-specific TTX-R sodium channel Nav1.9 (23). The effect of FHF1B interaction with
Nav1.9 has yet to be determined.
Nav1.8 is a sensory neuron-specific sodium channel that
produces a TTX-R current with different kinetic properties compared with Nav1.5 and Nav1.9 (42-44). Although
Nav1.5 and Nav1.8 share ~80% amino acid
identity in the CR1+CR2 region, Nav1.8 failed to interact
with FHF1B (Fig. 1 and Ref. 23). In contrast, Nav1.5 and
Nav1.9 share ~70% amino acid identity in the CR1+CR2
segment, and both bind FHF1B. Alignment of the C-terminal polypeptide
of the three TTX-R channels (Fig. 4) identifies multiple residues in
the CR1+CR2 region that are identical in Nav1.5 and
Nav1.9 but that are different from Nav1.8. It
is possible that one or more of these residues may block or destabilize
the interaction of FHF1B with the C terminus of Nav1.8.
FHF1B lacks a signal peptide, similar to FGF2, and is not secreted into
the medium by transfected HEK 293 cells (18-21). FGF2 is exported to
the extracellular space via an endoplasmic reticulum/Golgi-independent mechanism that may involve the An early study speculated that the interaction between FHF1B and
Nav1.9 might modulate the channel properties either
directly or indirectly by recruiting other proteins to the channel
complex (23). FHF1 has been shown to interact with the scaffold protein IB2 and recruit mitogen-activated protein kinase p38 Sodium channels Nav1.1 and Nav1.5 and the
auxiliary subunits The conserved acidic-rich domain in the C terminus of
Nav1.5 is important in the control of inactivation (30,
31). Our findings that FHF1B binds to this domain and that it causes a significant shift in the voltage dependence of channel inactivation to
more negative potentials support this concept. Our results also show
that the D1790G mutation abolishes the interaction with FHF1B. If the
FHF1B-mediated hyperpolarizing shift of the channel inactivation is
caused by the recruitment of a kinase to the channel complex, the
D1790G hyperpolarizing shift of channel inactivation might be produced
by an alternate mechanism. Irrespective of this, the present results
provide, for the first time, evidence that FHF1B, a member of the
fibroblast growth factor family, can bind to and functionally modulate
the cardiac sodium channel.
Because FHF1B binding to the Nav1.5 mimics the effects of
D1790G mutation, it is possible that the level of expression of FHF1B
can affect heart function. Accordingly, the low level of FHF1B
expression in adult heart (23) may be functionally important. Whether
overexpression of FHF1B in the heart under pathological conditions or
by transgenic techniques produces physiological changes similar to
those of the Long QT syndrome needs to be further explored.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit and auxiliary
-subunits (1).
Ten distinct pore-forming
-subunits of sodium channels have been identified in vertebrates (2). Dysfunction or dysregulation of sodium
channels can cause a number of disorders, including life-threatening
cardiac arrhythmias (3, 4) and neuropathic pain (5). Thus, factors that
modulate sodium channel function or subcellular localization are of
interest from both a pathophysiological and a therapeutic standpoint.
-subunits
affect the assembly, trafficking, and electrophysiological activities
of
-subunits (6) and have recently been shown to link sodium
channels to extracellular matrix proteins via their association with
neurofascin (7). The neuronal adhesion molecule contactin/F3 increases
the membrane insertion of sodium channels by direct (8) or indirect (9)
association with the
-subunit of the channels. The association with
contactin/F3 may also mediate the interaction of the channel with the
extracellular protein tenascin (8). Annexin II light chain (p11) binds
to the N terminus of sensory neuron-specific sodium channel
Nav1.8 and enhances its functional expression (10). The C
terminus of the skeletal muscle and cardiac muscle
-subunits,
Nav1.4 and Nav1.5, respectively, contain a
PDZ-binding motif that links these channels to syntrophin (11).
-subunit by the channel partner AKAP15 (12, 13). Receptor
protein-tyrosine phosphatase
interacts with brain sodium channels,
phosphorylates tyrosine residue(s) of the channels, and causes a
depolarizing shift in the voltage dependence of inactivation of
recombinant Nav1.2 (14). The Ca2+-binding
protein calmodulin interacts with the C terminus of Nav1.2 (15), Nav1.5, Nav1.4 (16, 17), and
Nav1.61 and
modulates the Na+ currents in an isoform-specific manner.
to the signaling complex (22). Previously, we
reported the interaction of FHF1B with a sensory neuron-specific, tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel
Nav1.9 (23). The functional significance of this
interaction has yet to be determined. FHF1B did not interact with the C
termini of the sodium channels Nav1.7 and
Nav1.8, which, similar to Nav1.9, are expressed
in the sensory neurons of the dorsal root ganglia (23). In this study,
we demonstrate that FHF1B associates with another TTX-R sodium channel,
the cardiac channel Nav1.5. We show that FHF1B binds to
Nav1.5 both in vitro and in vivo and
that the co-expression of FHF1B with wild type human Nav1.5
causes a hyperpolarizing shift in steady-state inactivation of this channel.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N4-GFP, pFHF1B
N41-GFP, pFHF1B
N76-GFP, and
pFHF1B
N142-GFP.
-galactosidase assay and by growth phenotypes on
(SD leu/trp/his/ura/3AT+) plates and on plates containing
5-fluoroorotic acid (SD leu/trp/5FOA+).
N4-GFP,
FHF1B
N41-GFP, FHF1B
N76-GFP, and FHF1B
N142-GFP), as
indicated, in 150 µl of buffer AM (10 mM Tris, pH 7.9, 10% glycerol, 1 mM MgCl2) supplemented with
100 mM KCl and 0.5 mg/ml bovine serum albumin (26). The
bound proteins were denatured in sample buffer and separated by 12%
SDS-PAGE, and GFP fusion proteins were detected by immunoblotting with
anti-GFP antibodies (Clontech).
P/6 procedure). Whole cell currents were
elicited from a holding potential of
130 mV and filtered at 5 kHz and
acquired at 50 kHz using Clampex 8.1 software (Axon Instruments). For
current density measurements, the membrane currents were normalized to
membrane capacitance, which was calculated as the integral of the
transient current in response to a 5-ms hyperpolarizing pulse from
120 mV (holding potential) to
130 mV. All of the experiments were
performed at room temperature (21-25 °C).
130 mV and a series of 100 ms test pulses that ranged from
100 to
+60 mV in increments of 5 mV. The peak value of
INa (INa,peak) at each
membrane potential (Vm) was plotted. The
relationship of peak INa versus
Vm was fitted with the following equation
where Vrev is the reversal potential of
INa, and GNa is the
voltage-dependent sodium current conductance.
GNa was fitted using the following Boltzmann
distribution equation
(Eq. 1)
where GNa,max is the maximum conductance,
V1/2 is the membrane potential at half-maximal
conductance, and k is the slope factor.
(Eq. 2)
20 mV was preceded by a 500-ms preconditioning
pulse ranging from
130 to
10 mV in wild type and
150 to
10 mV
in mutants. The normalized curves
(INa/INa,max) were fitted
using the following Boltzmann distribution equation
where INa,max is the peak Na+
current at
(Eq. 3)
20 mV measured from the most negative preconditioning
pulse potential (
130 mV for wild type or
150 mV for mutant),
Vm is the preconditioning pulse potential,
V1/2 is the membrane potential of half-maximum
INa, and k is the slope factor. The
data are represented as the means ± S.E.; a value of
p < 0.05 was considered statistically significant.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
FHF1B associates with Nav1.5 and
Nav1.9. The -galactosidase assay was used to test
the interaction between FHF1B and the C termini of the voltage-gated
sodium channels (rNav1.1, rNav1.2,
rNav1.3, rNav1.4, rNav1.5,
mNav1.6, hNav1.7, rNav1.8, and
rNav1.9). The known interaction between c-Jun and c-Fos
serves as a positive control, whereas the lack of interaction of
retinoblastoma (Rb) and laminin serves as a negative
control. Three independent yeast transformants for each pair of
plasmids were transferred onto the nitrocellulose membrane, and the
-galactosidase activity was determined.
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Fig. 2.
FHF1B associates with Nav1.5 both
in vitro and in vivo.
A, binding of FHF1B to Nav1.5C in
vitro (GST pull-down assay). Purified GST (lanes 2 and
5) or GST-Nav1.5C fusion protein (lanes
3 and 6) immobilized on glutathione-Sepharose beads
were incubated with extracts prepared from HEK293 cells transfected
with a plasmid encoding either GFP alone (lanes 1-3) or
FHF1B-GFP fusion protein (lanes 4-6). HEK293 proteins that
were trapped by the interaction with GST or GST-Nav1.5C
(lanes 1 and 4, respectively) were examined by
immunoblotting with anti-GFP antibodies. The positions of GFP and
FHF1B-GFP are indicated. B, FHF1B binds to
Nav1.5 in vivo (co-immunoprecipitation assay).
Equal amounts of protein extracts prepared from HEK293 cells
transfected with Nav1.5 expression plasmid with either
pEGFP-N1 (lanes 1 and 3) or pFHF1B-GFP
(lanes 2 and 4) were incubated with anti-GFP
polyclonal antibodies (lanes 3 and 4). The
immunoprecipitated protein complex and cell extracts (lanes
1 and 2), which provides a positive control) were
examined by immunoblotting with a sodium channel pan-specific antibody.
The position of Nav1.5 is indicated.
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Fig. 3.
Identification of rFHF1B segments binding to
Nav1.5. A, schematic diagram of FHF1B-GFP
constructs used to map the binding of FHF1B to Nav1.5. The
numbers refer to the amino acid residues in FHF1B. Each
segment (i.e. a.a. 1-4, red; 5-41,
white; 42-76, magenta; 77-142, blue;
143-181, yellow; GFP, green) is encoded by a
single exon. The binding or lack of binding of the various FHF1B
segments to Nav1.5, as revealed in B, are
indicated by plus or minus signs,
respectively. B, 0.5 µg of GST (lanes 2,
5, 8, 11, 14, and
17) or 0.5 µg of GST-Nav1.5C (lanes
3, 6, 9, 12, 15, and
18) immobilized on glutathione-Sepharose beads were
incubated with cell extracts prepared from HEK293 cells transfected
with either full-length FHF1B-GFP (lanes 1-3),
FHF1B N4-GFP (lanes 4-6), FHF1B
N41-GFP (lanes
7-9), FHF1B
N76-GFP (lanes 10-12), FHF1B
N142-GFP
(lanes 13-15), or GFP alone (lanes 16-18). The
bound proteins and respective cell extracts (lanes 1,
4, 7, 10, 13, and
16) were examined by immunoblotting with anti-GFP
antibodies. An unknown protein X is indicated. For additional details,
see "Experimental Procedures."
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Fig. 4.
Amino acid alignment of the C-terminal
polypeptides of the tetrodotoxin-resistant channels Nav1.5,
Nav1.8, and Nav1.9. The C-terminal
polypeptide amino acid sequences of the channels are aligned using the
Clustal W program. CR1 and CR2 are underlined. Residues in
CR1 and CR2 that are conserved in Nav1.5 and
Nav1.9 but that are different in Nav1.8 are
shown in bold type. The shaded sequences
represent the predicted six helical segments in the C terminus of
Nav1.5 (31). The position of the cardiac arrythmia mutants
E1784K (32), D1790G (33-35), and Y1795H and Y1795C (36) are shown. An
asterisk represents identical amino acid residues in the
three channels; a colon represents conservative
substitutions; a dot represents a semi-conservative change;
and a space represents a missing residue.
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Fig. 5.
CR1 (a.a. 1773-1832) of the C-terminal
polypeptide of Nav1.5 is sufficient for FHF1B binding.
A, schematic diagrams of DBD-Nav1.5C fusion
proteins used to map the sites of FHF1B binding on the C-terminal
polypeptide of Nav1.5. The numbers refer to
amino acid residues in the C terminus of Nav1.5. CR1
(green), CR2 (brown), and UR (magenta)
stand for conserved region 1, conserved region 2, and unique region,
respectively. The binding or lack of binding of FHF1B to the various
DBD-Nav1.5C segments, as revealed in B, are
indicated by plus and minus signs, respectively.
B, binding of FHF1B by the C terminus of Nav1.5
and its derivatives. The -galactosidase assay was used to test the
interaction between FHF1B and derivatives of the C terminus of
Nav1.5. Three independent yeast transformants for each pair
of plasmids were transferred onto the nitrocellulose membrane, and the
-galactosidase activity was determined as previously
described.
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Fig. 6.
The LQT-3 point mutation D1790G in the C
terminus of Nav1.5 abolishes the interaction between FHF1B
and Nav1.5. A, schematic diagrams of wild
type (Nav1.5C), naturally occurring point mutants (E1784K,
D1790G, Y1795H, and Y1795C) and combination mutant (KGH). The numbers
refer to amino acid residues in the C terminus of Nav1.5.
The binding or lack of binding of FHF1B to the wild type and various
point mutants, as revealed in B, are indicated by
plus and minus signs, respectively. B,
binding of FHF1B to the wild type and various mutants of the C terminus
of Nav1.5. The -galactosidase assay was used to test the
interaction between FHF1B and mutants of the C terminus of
Nav1.5. Three independent yeast transformants for each pair
of plasmids were transferred onto the nitrocellulose membrane, and the
-galactosidase activity was determined. C, D1790G mutant
abolishes the binding of FHF1B to Nav1.5 in vivo
(co-immunoprecipitation assays). Extracts prepared from HEK293 cells
transfected with pFHF1B-GFP with either wild type Nav1.5
(lanes 3 and 4) or D1790G mutant (lanes
1 and 2) expression plasmids were incubated with
anti-GFP polyclonal antibodies (lanes 2 and 4).
The immunoprecipitated protein complex and respective cell extract
(lanes 1 and 3, which provides a positive
control) were examined by immunoblotting with a sodium channel
pan-specific antibody. IP, immunoprecipitation;
CE, cell extract.
130 to
30 mV) was 0.47 ± 0.11 nA/pF (n = 24)
for Nav1.5 plus GFP and 0.28 ± 0.03 nA/pF for
Nav1.5+FHF1B-GFP (n = 22, p = 0.18), respectively.
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Fig. 7.
Modulation of Nav1.5 channel
inactivation by FHF1B. A, current traces recorded from HEK
293 cells co-transfected with Nav1.5 and GFP. B,
current traces recorded from HEK 293 cells co-transfected with
FHF1B-GFP. C, normalized current-voltage relationships for
Nav1.5 (n = 11) and
Nav1.5+FHF1B-GFP (n = 19) are shown.
D, voltage-dependent activation (normalized
conductance, circles) and inactivation (fraction available,
squares) were determined as described under "Experimental
Procedures." The lines connecting the symbols are
Boltzmann fits to the data.
60 mV and reached maximal amplitude at
30 mV
for both Nav1.5 (n = 11) and
Nav1.5+FHF1B (n = 19). The general shape of
the I-V relationship did not change. The midpoint of the normalized I-V
curve (from
60 to
30 mV) for Nav1.5+FHF1B showed a
slight shift in the positive direction (Fig. 7D, open
circles) but is not statistically significant (p = 0.21). The average V1/2 and k (slope) values for the fitted functions were
50.41 ± 1.32 mV and
5.6 ± 0.47 mV/e-fold potential change, respectively, for
Nav1.5 and
46.24 ± 1.13 mV and
5.8 ± 0.44 mV/e-fold potential change, respectively, for
Nav1.5+FHF1B.
76.40 ± 1.2 mV with a
k value of 8.4 ± 0.5 mV/e-fold potential
(n = 16; Fig. 7D, open squares).
In contrast, co-expression of FHF1B with Nav1.5 caused a 9 mV hyperpolarizing shift of the V1/2 (
85.66 ± 1.4 mV; p < 0.001; Fig. 7D, closed
squares) but no change in the slope (k value of
9.1 ± 0.2 mV/e-fold potential change (n = 21)).
Thus, the functional association of FHF1B with Nav1.5
causes a significant hyperpolarizing shift in the
voltage-dependent inactivation of the channel.
130 to
30 mV) were 0.17 ± 0.06 nA/pF (n = 17)
for D1790G and 0.17 ± 0.09 nA/pF for D1790G+FHF1B
(n = 18, p > 0.05), respectively.
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Fig. 8.
FHF1B does not modulate the D1790G mutant
channel. A, current traces recorded from HEK 293 cells
co-transfected with D1790G and GFP. B, current traces
recorded from HEK 293 cells co-transfected with FHF1B-GFP.
C, normalized current-voltage relationships for D1790G
(n = 8) and D1790G+FHF1B (n = 11) are
shown. D, voltage-dependent activation
(normalized conductance, circles) and inactivation (fraction
available, squares) were determined as described under
"Experimental Procedures." The lines connecting the
symbols represent Boltzmann fits to the data.
30 mV, where maximal current amplitude
is measured (data not shown). Co-expression of FHF1B did not alter the
kinetics of inactivation of D1790G currents (Fig. 8B). The
I-V relationships of D1790G and D1790G+FHF1B were not significantly
different (Fig. 8C). Voltage-dependent activation of D1790G and D1790G+FHF1B currents are shown in Fig. 8D (circles). The average V1/2
and k values for the fitted functions were
40.27 ± 3.15 mV and
8.68 ± 0.59 mV/e-fold potential change,
respectively, for D1790G (n = 8) and
41.92 ± 3.46 mV and
10.56 ± 1.27 mV/e-fold potential change,
respectively, for D1790G+FHF1B (n = 11). Compared with the wild type Nav1.5 channels, the D1790G mutant channels
show a depolarized shift of ~10 mV in voltage-dependent
activation (p < 0.05) in agreement with published
results (35). Co-expression of FHF1B and D1790G did not modulate the
voltage-dependent activation of the mutant channels.
22 mV, in the
voltage dependence of inactivation (Fig. 8D, open
squares; p < 0.001), in agreement with published
results (35, 37). The average V1/2 and k
values for the fitted functions were
98.74 ± 2.82 mV and
10.82 ± 1.06 mV/e-fold potential change, respectively, for D1790G
mutant (n = 16). Co-expression of FHF1B with D1790G
channels failed to affect voltage-dependent inactivation of
the channel. The average V1/2 and k
values for the fitted functions were
98.43 ± 2.91 mV and 10.33 ± 1.7 mV/e-fold potential change, respectively, for
D1790G+FHF1B (n = 16). The lack of modulation of D1790G
channels by co-expression with FHF1B is consistent with the biochemical
data showing that this mutation abolishes the binding of the proteins.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of
Na+/K+-ATPase (45, 46). We previously posited
that Nav1.9, in an analogous fashion to the
-subunit of
Na+/K+-ATPase, might participate in the export
of FHF1B to the cell surface in native neurons (23). Nav1.9
produces only a very small current when expressed in HEK 293 cells (47)
perhaps due, among other possibilities, to a low channel density at the
cell surface. Thus, the lack of FHF1B in the medium of HEK 293 cells co-transfected with Nav1.9 and FHF1B was not surprising.
Therefore, because recombinant Nav1.5 produces a robust
current in HEK 293 cells and is modulated by FHF1B, indicating a
functional interaction in the cell, we tested the FHF1B export
hypothesis in the present study. Using anti-GFP antibodies, FHF1B-GFP
was precipitated from HEK 293 cells co-transfected with
Nav1.5 and FHF1B-GFP but was undetectable in the medium
(data not shown), suggesting that the Nav1.5 channel does
not enhance the export of FHF1B to the medium.
to the complex (22). The present study clearly shows that co-expression of FHF1B
produces a significant hyperpolarizing shift of the voltage dependence
of inactivation of Nav1.5. This shift is similar to that
attributed to the phosphorylation of Ser1505 in the
cytoplasmic linker joining domains 3 and 4 by protein kinase C (48,
49). Other residues, for example Ser526 and
Ser529 in the cytoplasmic loop linking domains 1 and 2 (L1)
of Nav1.5, have been reported to be phosphorylated by
cAMP-dependent protein kinase (50). Nav1.2
sodium channels are phosphorylated at serine residues in L1 by
cAMP-dependent protein kinase anchored to the channel via
the protein AKAP15 (13). Thus, it is possible that the binding of FHF1B
to CR1 of the Nav1.5 C terminus might recruit a kinase to
the channel complex to phosphorylate either Ser1505 or
Ser526 and Ser529 or other residues and
influence the inactivation of the channel. The identity of the kinase
that might be recruited to Nav1.5 and Nav1.9
remains to be determined.
1,
2, and
3 are present in cardiac myocytes
(39, 51, 52). The association of Nav1.5 with
1 or
3
may produce a depolarizing shift in voltage dependence of inactivation
(33, 51, 52) and in recovery from inactivation (51). Several inherited
mutations linked to Long QT syndrome and Brugada syndrome have been
reported in the acidic-rich domain in the C terminus of the cardiac
Nav1.5 channel, which overlaps with CR1, i.e.
E1784K (32), D1790G (33-35), and Y1795H and Y1795C (36). An et
al. (33) postulated that the D1790G mutation prevents the binding
of
1 to Nav1.5 and causes a significant shift in the
opposite direction. Similarly, FHF1B may interfere with the binding of
subunits to Nav1.5. Co-expression of FHF1B and
1
subunit, however, did not affect the modulation of Nav1.5
by FHF1B (data not shown), suggesting that the two proteins do not
compete for the same binding site or that FHf1B binds to Nav1.5 with higher affinity than the
-subunits.
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ACKNOWLEDGEMENTS |
---|
We thank Lynda Tyrrell and Bart Toftness for assistance and Dr. Mike O'Leary for the generous gift of Nav1.5 plasmid.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Rehabilitation Research and Development Service and Medical Research Services, by Department of Veterans Affairs, and the National Multiple Sclerosis Society Grant RG-1912, and by gifts from the Paralyzed Veterans of America and Eastern Paralyzed Veterans Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Spinal Cord Research Fellowship from the
Eastern Paralyzed Veteran Association.
** To whom correspondence should be addressed: Paralyzed Veterans of America/Eastern Paralyzed Veteran Association Neuroscience Research Center, Yale University School of Medicine, 127A, Bldg. 34, 950 Campbell Ave., West Haven, CT 06516. Tel.: 203-937-3802; Fax: 203-937-3801; E-mail: sulayman.dib-hajj@yale.edu.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M207074200
1 R. I. Herzog, T. R. Cummins, C. Liu, and S. G. Wax, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: FHF, fibroblast growth factor homologous factor; FGF, fibroblast growth factor; TTX-R, tetrodotoxin-resistant; a.a., amino acid(s); GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase.
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REFERENCES |
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