From the Departments of Physiology and Biophysics, Cell Biology, and Gerentology and Geriatric Medicine, University of Alabama, Birmingham, Alabama 35294
Received for publication, September 28, 2000, and in revised form, December 6, 2000
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ABSTRACT |
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Gene expression, protein expression, and function
of amiloride-sensitive sodium channels were examined in human
lymphocytes from normal individuals and individuals with Liddle's
disease. Using reverse transcriptase polymerase chain reactions,
expression of all three cloned epithelial sodium channel (ENaC)
subunits was detected in lymphocytes. Polyclonal antibodies to bovine
Numerous cell types and tissues express amiloride-sensitive sodium
channels. The most extensively studied are the toad urinary bladder (1, 2), the mammalian renal cortical collecting duct (3, 4),
and the toad renal cell line (A-6) (5, 6). A prominent physiological
function of these cell types is the vectorial transport of
Na+ and water. Epithelial cells of the lung also express
amiloride-sensitive sodium channels. However, the specific function of
these channels is less clearly defined than that of the
salt-reabsorbing epithelia (7). Also, the biophysical and biochemical
characteristics of lung epithelial cell amiloride-sensitive sodium
channels are somewhat different from those of renal cells. These
channels have a different affinity for amiloride, different unitary
conductance, and different cationic selectivities (8-10).
Amiloride-sensitive sodium channels have also been linked to taste
(11). The current carried by these channels can only be partially
inhibited by amiloride (12). The reasons for these differences are not
clear. Possible explanations include multiple channel types derived
from post-translational modifications of
ENaC1 or the expression of
completely different gene products.
Human lymphocytes express a sodium conductance that is activated by
cyclic AMP and inhibited by low concentrations of amiloride (IC50 = ~75 nM) (13). A sodium conductance
with the same properties has been observed in whole cell clamped,
principal cells isolated from the renal cortical collecting duct of the
rat (14). The pharmacological and regulatory properties of the
lymphocyte and renal principal cell channels suggest that they may be
the same protein complex. Also, whole cell clamped lymphocytes isolated from individuals with Liddle's disease have a constitutively activated inward sodium conductance (15). The addition of cyclic AMP to these
whole cell clamped cells has no effect on the sodium conductance. In
contrast, whole cell clamped lymphocytes from unaffected family members
do not exhibit any constitutively active sodium current, even at strong
hyperpolarizing voltage-clamp steps (i.e. Despite these biophysical similarities and the obvious abnormalities in
sodium conductance regulation observed in whole cell clamped
lymphocytes from individuals with Liddle's disease, there has been
some question as to the identity of the protein responsible for the
amiloride-sensitive sodium conductance of lymphocytes. To resolve this
question, we tested the hypothesis that lymphocytes express the ENaC by
four independent methods (electrophysiological, immunocytochemical,
biochemical, and molecular biological). The findings from each method
indicated that ENaC was expressed by human lymphocytes. Also, because
amiloride inhibited all the sodium conductance, the findings ruled out
the possibility that other sodium channels, such as the voltage-gated
sodium channel found in nerve and muscle, were expressed by
lymphocytes. This comprehensive investigation is the only set of data
on ENaC of which we are aware that has utilized molecular biology,
electrophysiology, protein chemistry, and immunofluorescence on a
single cell type. The findings from all of these studies support the
hypothesis that human lymphocyte ENaC has biophysical and regulatory
characteristics that accurately reflect ENaC characteristics expressed
by renal principal cells.
Electrophysiological Methods
Microelectrode pipette solutions contained 100 mM
potassium-gluconate, 30 mM potassium chloride, 10 mM sodium chloride, 20 mM HEPES, 0.5 mM EGTA, 4 mM ATP, and <10 nM free
calcium and were buffered to a pH of 7.2. All electrophysiological
measurements were obtained with a bath solution of serum free RPMI 1640 culture medium, buffered to a pH of 7.4, at room temperature (24 ± 2 °C). Whole cell current records were obtained by formation of
conventional whole cells. Briefly, after formation of high resistance
seals between patch electrodes and lymphocyte plasma membranes (>3
Gohms), a sharp suction pulse was applied to rupture physically the
plasma membrane under the seal, leaving the seal resistance unchanged. The increased cellular capacitance indicated the successful formation of the whole cell configuration. The capacitance was balanced using the
appropriate circuits of the Axopatch 200 patch clamp amplifier (Axon
Instrs.). The voltage clamp protocol was to hold the cells at Single amiloride-sensitive sodium channels were recorded in the
outside-out patch configuration. This configuration was chosen because
amiloride only inhibits the channels from the extracellular face of the
channels. In the outside-out configuration, the outsides of the
channels were exposed to the bath solution. Thus, superfusing the
preparations with 2 µM amiloride-supplemented RPMI was
used as a straightforward method for establishing the identity of the channels. All experiments were performed on Epstein-Barr
virus-transformed non-Liddle's cells (Daudi) or Epstein-Barr
virus-transformed B lymphocytes from the proband for Liddle's disease.
Immunohistochemical Methods
Normal and Liddle's lymphocytes were fixed in a 3:1 mixture of
methanol:acetic acid mixture at The antibody-bound cells were incubated in goat anti-rabbit Oregon
green-conjugated antibodies diluted in blocking buffer for 30 min at
37 °C, washed, and stained with Hoechst stain (20 mg/ml in
phosphate-buffered saline). The cells were washed and mounted in 0.1%
p-phenylene diamine in 9:1 glycerol:phosphate-buffered saline. The mounted cells were examined, and the relative fluorescence intensity was quantitated using fluorescent confocal microscopy (Scanolytics, Fairfax, VA; VayTek Ink., Fairfield, IA).
Molecular Genetic Methods
For total RNA isolation, we used the commercially available
Tri-Reagent total RNA isolation protocol (Molecular Research Center, Inc., Cincinnati, OH). A 250-µl suspension of ~20 × 108 B-lymphocytes (in water) was mixed with 750 µl of
Tri-Reagent to lyse the cells. The lysed cell suspension was allowed to
stand at room temperature for 5 min to dissociate the nucleoprotein complexes. The tube was then spun at 12,000 × g for 10 min at 4 °C. The supernatant was transferred to a fresh tube. To the supernatant was added 200 µl of chloroform, and the contents of the
tube were mixed vigorously for 15 s, stored at room temperature for 10 min, and then centrifuged at 4 °C for 15 min (12,000 × g maximum). The contents separated into three phases. The
colorless aqueous phase contained the RNA. The aqueous phase was
transferred to another tube and mixed with cDNA Synthesis and Gene-specific Amplification
First-strand syntheses were performed using a modification of
the MasterAmp High Fidelity RT-PCR Kit protocol (Epicenter
Technologies, Madison, WI). Briefly, 25 ml of MasterAmp 2× RT-PCR
premix plus 2 µl (40 units/µl) of MMLV-RT Plus 50 ng of total RNA
(prepared as above), and 100 pmol of the gene-specific reverse primer
were combined in a total reaction volume of 50 µl. The mixture was incubated at 37 °C for 1.5 h.
For PCR amplification of specific ENaC subunits ( PCR Parameters
For the Sequencing
PCR products for all subunits were gel purified using the
commercially available QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). Purified products were directly sequenced using the gene-specific forward or reverse primer. Sequencing was performed using the Applied
Biosystems Inc. model 377 sequencer in the University of Alabama
at Birmingham Microbiology Sequencing Core.
Biochemical Methods
Protein Purification--
Lymphocytes isolated from blood
samples collected from patients with Liddle's syndrome (Liddle's) and
from unaffected relatives (normal) were Epstein-Barr virus-transformed
and grown in continuous culture in RPMI 1640 + 10% fetal bovine serum
at 37 °C and 5% CO2 (95% air) in 75-cm2
tissue culture flasks. Cells were collected by centrifugation at
500 × g for 5 min at 4 °C. All subsequent
procedures were carried out at 4 °C. The lymphocytes were washed two
times with phosphate-buffered saline and centrifuged. The cell pellet
was resuspended in Dounce buffer (10 mM Tris-Cl, pH 7.6, 0.5 mM MgCl2, supplemented with protease
inhibitors: 2 µg/ml DNase, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM
phenylmethylsulfonyl fluoride) and incubated 10 min on ice. This
suspension was homogenized for 30 strokes in a Dounce homogenizer.
Tonicity restoration buffer (10 mM Tris-Cl, pH 7.6, 0.5 mM MgCl2, and 600 mM NaCl) was
added to a final NaCl concentration of 150 mM. The
homogenate was then centrifuged at 500 × g for 5 min,
and the pellet was discarded. 0.5 M EDTA, pH 8.0, was added
to the supernatant to achieve a final concentration of 5 mM
and then centrifuged at 100,000 × g for 45 min. The
final pellet was solubilized overnight in a solution of 10 mM NaH2PO4, pH 7.4, 10 mM CHAPS, 10% glycerol, and protease inhibitors (no DNase).
For affinity purification, 5 mg of a polyclonal antibody raised against
a bovine kidney papillary Na+ channel protein complex was
irreversibly bound to protein A immobilized on agarose gel (Pierce). 2 ml of gel were first packed in a plastic column and equilibrated with
50 mM sodium borate, pH 8.2. The gel was resuspended in a
solution of the IgG and incubated at room temperature for 30 min. Free
antibody was washed from the gel with borate buffer. Bound IgG was
cross-linked to the protein A by suspending the gel in a 6.6 mg/ml
solution of dimethylpimelimidate (Pierce) for 1 h at room
temperature. Unreacted imidate groups were blocked by incubating the
column in 0.1 M ethanolamine, pH 8.2, for 10 min at room
temperature. Lastly, the column was washed with 0.1 M
glycine, pH 2.8, and then borate buffer. Before affinity purification,
the column was equilibrated with 10 mM sodium phosphate, pH
7.5.
Solubilized lymphocyte membrane proteins were applied to either column,
allowed to run into the gel bed, and incubated there for 1 h at
room temperature. The column was drained and washed with 10 mM sodium phosphate until the absorbance
(A280) of the wash from the column was the same
as that of phosphate buffer alone. Bound protein was then eluted with
0.1 M glycine-HCl, pH 2.8, in 500-µl fractions that were
neutralized with 1 M Tris, pH 8.0. Absorbance measurements
at A280 were used to determine which fractions
contained antigen.
Iodination--
To visualize the products, immunopurified
proteins were diluted to 100 µl with phosphate-buffered saline prior
to iodination with chloramine T and carrier-free 125I.
Iodinated proteins were separated from unbound label on Sephadex G-25-150 columns, mixed with an equal volume of 2× sample buffer containing 50 mM dithiothreitol (DTT), and electrophoresed
through 8% polyacrylamide gels. Gels were dried and exposed to x-ray film.
Reconstitution--
To study the single channel activity of the
normal and Liddle's protein preparations, they were reconstituted into
proteoliposomes and incorporated into planar lipid bilayers. The
protein-containing fractions eluted from the antibody column were
concentrated to 100 µl and then combined with a mixture of 500 µg
of phosphatidylethanolamine, 300 µg of phosphatidylserine, and 200 µg of phosphatidylcholine. Extracti-Gel D, previously washed with KCl
buffer (400 mM KCl, 5 mM Tris-HCl, pH 7.4, 0.5 mM MgCl2) was added, and the total volume was
brought to 600 µl with KCl buffer. This mixture was incubated first
at room temperature for 45 min and then at 4 °C for 16 h with
agitation. Extracti-Gel D was allowed to settle out of the resulting
proteoliposome suspension by gravity. The protein was aliquoted and
stored at Planar Lipid Bilayers--
Planar lipid bilayers were made from
a phospholipid solution containing a 2:1 mixture of
diphytanoyl-phosphatidyl-ethanolamine/diphytanoyl-phosphatidylserine in
n-octane (final lipid concentration, 25 mg/ml). Bilayers
were bathed with 100 mM NaCl containing 10 mM
MOPS-Tris buffer (pH 7.4). All solutions were filter-sterilized.
Current measurements were performed using a high gain amplifier
circuit. Applied voltage is referenced to the trans-chamber,
which was connected to the current-to-voltage converter, and therefore
was at virtual ground. Acquisition and analysis of single channel
recordings were performed using pCLAMP software and hardware (Axon
Instruments, CA). Data were stored digitally and were filtered at 300 Hz with an 8-pole Bessel filter prior to acquisition at 1 ms/point. All
the analyses were performed for single active channels. The records
shown here are representative of 19 separate experiments. Amiloride
sensitivity of the immunopurified channels in the bilayer was
determined by the addition of the drug to the trans-solution
of the bilayer.
Molecular Biological Analysis of Human B-Lymphocyte RNA--
Using
total RNA prepared from lymphocytes we have synthesized cDNA using
gene-specific primes for the
Sequencing was used as a final confirmation of the subunit segments
amplified from cDNA. Direct sequencing revealed that we indeed
amplified the Normal Human Lymphocyte Amiloride-sensitive Sodium
Conductance--
When human lymphocytes are whole cell clamped using
"normal" ionic gradients, they exhibit a variety of ionic
conductances. However, when the cells are hyperpolarized there is very
little, inward current (Fig. 2, top
left panel). Addition of the membrane permeant active analog of
cyclic AMP (8-CPT-cAMP; final concentration, 40 µM) to
the bath solution significantly (p < 0.05, n = 6) increased the inward conductance at
hyperpolarizing clamp potentials by 25% (Figs. 2, top right
panel, and 3). As shown previously,
the inward current that was activated by cAMP treatment can be
completely inhibited by 2 µM amiloride (13-15).
After formation of the whole cell configuration, slow withdrawal of the
patch electrode often results in resealing of the membrane fracture,
with the outside of the resealed membrane facing the bath solution. The
result of this procedure is an isolated membrane patch in the
outside-out configuration. For the study of ENaC single channels, this
patch orientation is useful because amiloride inhibits these channels
from the outside only. Thus, in the outside-out configuration,
amiloride can be used to identify unambiguously amiloride-sensitive
sodium channels, such as ENaC. When the outside-out configuration was
formed on normal (i.e. non-Liddle's) lymphocytes, no inward
single channel currents were observed when the electrical potential
gradient was directed inward, in six of six consecutive patches. This
finding was consistent with the basal whole cell conductance, which was
very small, even at strong hyperpolarizing potentials. However, single
channels were observed in five of six outside-out patches formed from
cells that were previously treated with 40 µM 8-CPT-cAMP
and had large inward whole cell currents prior to the formation of the
outside-out configuration (Fig. 2, middle left panel). These
channels were completely inhibited by 2 µM amiloride
(Fig. 2, middle right panel). Analysis of the single channel
records yielded a single channel conductance of 10 pS (0.6 pA) of
current at a potential of 60 mV; Fig. 2, bottom left panel).
Also, the time spent in the open configuration was brief ( Amiloride-sensitive Sodium Conductance of Liddle's Disease
Lymphocytes--
The same electrophysiological analysis was performed
on lymphocytes from individuals with Liddle's disease, with somewhat different results. All of the cells examined had a constitutively activated inward conductance at hyperpolarized clamp potentials (Fig.
3, top panel). Unlike non-Liddle's cells, treatment with 8-CPT-cAMP did not significantly alter the whole cell currents. Another
difference between Liddle's lymphocytes and non-Liddle's lymphocytes
was observed after formation of outside-out patches. Single channel
currents were observed in five of six outside-out patches made on
Liddle's lymphocytes, without pretreatment with 8-CPT-cAMP (Fig. 3,
middle left panel). More than 90% of this single channel
activity was inhibited by 2 µM amiloride (Fig. 3,
middle right panel), as determined by amplitude histogram
analysis (Fig. 3, bottom panel). The amplitude histogram
also revealed that the single channel conductance (~10 pS; 0.65 pA at
60 mV) was not different from that measured for amiloride-inhibitable single channels in outside-out patches from non-Liddle's cells.
The basal inward currents from Liddle's cells and the cAMP-activated
inward currents from non-Liddle's cells typically had a "ragged"
appearance. The corresponding single channel records typically showed
bursts of rapid openings and closures from multiple channels within the
patches. The whole cell current characteristics and single channel
characteristics were compared by summing 30 sequential 800-ms segments
of single channel activity at each clamp potential to produce a
"virtual" whole cell current. The parameters underlying this
analysis were that the patch contained eight channels, and that each
cell contained ~716 ± 264 channels within the plasma membrane.
The channel number was estimated from a conventional
[14C]amiloride binding study (assuming 1:1
[14C]amiloride:ENaC binding) shown in Fig.
4. The same [14C]amiloride
binding study was performed on Liddle's lymphocytes. In these cells, a
significantly higher (p = 0.04, n = 10)
number of binding sites were found (2488 + 751). This finding is
consistent with the hypothesis that the Liddle's mutation induces
excessive channel expression.
Using this information, the summed single channel records were
multiplied by 3.3 (800 total/30 segments × 8 channels/patch) to
approximate the current that would be carried by 800 channels. It was
found that the summed single channel currents and the actual whole cell
currents were indistinguishable in both magnitude and morphology,
supporting the hypothesis that the amiloride-inhibitable single
channels were the current carriers for the amiloride-inhibitable inward
whole cell current. Fig. 5 shows the
results of this comparison.
Immunohistochemical Analysis of Human Lymphocyte ENaC--
When
non-Liddle's disease human lymphocytes were treated with antibodies
specific for bovine ENaC, they exhibited considerable specific
immunofluorescence, indicating expression of epitopes that were
recognized by the antibodies (Fig. 6,
left panel). The staining was punctate and intracellular
with some staining at the cell surface. In contrast, Liddle's
lymphocytes, treated identically, were brighter, and more fluorescence
was concentrated at the plasma membrane.
Concurrently, cells of both types were treated for 5 min with 40 µM 8-CPT-cAMP. The fluorescence intensity at the cell
surface of more than 60 cells from each group was measured. It was
found that untreated Liddle's cells were 2.5 times more fluorescent than non-Liddle's cells. Also, treatment with cAMP significantly (p < 0.05) increased the cell surface fluorescence
intensity of both groups of cells. The quantitation of the relative
fluorescence intensity of these cells is shown in Fig.
7. The most straightforward interpretation of these findings is that cells with the Liddle's mutation express many more Na+ channels. This finding
agrees reasonably well with the [14C]amiloride binding
data, which showed a 3.47-fold difference between non-Liddle's and
Liddle's lymphocytes, with Liddle's lymphocytes expressing the higher
number of binding sites. However, these findings do not rule out the
possibility that more epitopes are exposed in Liddle's cells because
the exact number of epitopes is not known and because the channel
stoichiometry is not known. This possibility is somewhat unlikely,
however, because the immunofluorescence and
[14C]amiloride binding results are in reasonable
agreement with respect to the difference in channel expression between
Liddle's and non-Liddle's lymphocytes. One other finding from the
quantitation of the immunofluorescence was that cAMP treatment
significantly increased the relative fluorescence intensity by 25% in
each group. Again, there is no way from these experiments to
distinguish between the possibility that more channels were inserted in
the membrane or more epitopes of nascent channels were exposed by the
treatment. There was no significant increase in the current in
Liddle's cells in response to cAMP treatment. However, the
cell-to-cell variability was sufficiently large enough to preclude a
statistically significant separation of only 25% in Liddle's cells
because of the constitutive activation of the channels and "ragged"
morphology of the whole cell currents.
Biochemical Analysis of Lymphocyte Sodium
Channels--
Immunopurification from normal and Liddle's cells
yielded similar complexes of polypeptides, with molecular masses
of 156, 79.5, and 68.5 kDa. The same banding profile was observed in
the iodinated, reduced immunopruified complexes from normal and
Liddle's lymphocytes using the bovine kidney sodium channel antibody
column (Fig. 8). These immumopurified
polypeptide complexes were then assessed directly for their ability to
function as sodium channels in planar lipid bilayer studies. The 79.5- and 68.5-kDa bands are consistent with
Unreduced immunopurified polypeptide complexes were incorporated into
proteoliposomes and subsequently introduced into planar lipid bilayers
to test the hypothesis that these complexes formed functional ion
channels capable of conducting sodium. It was found that the complexes
from normal lymphocytes and complexes immunopurified from Liddle's
lymphocytes were both able to conduct sodium in planar lipid bilayers.
Consistent with the whole cell findings and with the pathophysiology of
Liddle's disease, the purified channels from Liddle's cells spent a
greater percentage of time in the open state compared with the
immunoprurified channels from non-Liddle's lymphocytes (Fig.
9). Also, amiloride inhibited the single
channel currents recorded from these immunopurified polypeptide complexes (Fig. 9).
One obvious difference between the electrophysiological findings in
intact lymphocytes and purified polypeptide complexes was in the
efficacy of amiloride. Amiloride was approximately 1 order of magnitude
less efficacious in inhibiting Na+ conductance in
incorporated polypeptides than it was in inhibiting whole cell and
single channel conductance in intact lymphocytes. Although the precise
reason for this difference is unknown, it is reasonable to expect that
the three-dimensional structure of the channel complex was somewhat
altered by its removal from its native membrane and subsequent
biochemical manipulation. Therefore, it is possible that the
configuration of the amiloride-binding site was altered, resulting in a
reduction in amiloride binding efficacy.
Another similarity between ENaC and the purified lymphocyte protein
complex was the response to biochemical reduction using DTT. The
addition of DTT to the trans-chamber caused protein
complexes purified from both Liddle's and non-Liddle's lymphocytes to
spend a much higher proportion of their conductive time in lower
conductance states (Fig. 10). In
bilayers, DTT reduced the single channel conductance from 35 to 13 ps,
which is precisely the conductance observed in outside-out patches
(Figs. 2 and 3). This same phenomena was observed when in
vitro translated The hypothesis that the sodium conductance of human lymphocytes is
mediated by a polypeptide complex containing the three cloned elements
of the ENaC was tested by four independent methods. The findings from
each method support the hypothesis that lymphocyte sodium conductance
is accomplished via ENaC exclusively.
Reverse transcriptase polymerase chain reactions using ENaC-specific
primers were amplified each ENaC subunit. The identities of Our inability to demonstrate the presence of Because Electrophysiological examination of lymphocytes revealed a sodium
conductance regulated by cAMP, and the activated current was completely
inhibited by 2 µM amiloride. Obvious abnormalities were
observed in this conductance when normal lymphocyte currents were
compared with those obtained from lymphocytes obtained from individuals
known to have a mutated ENaC Comparative immunofluorescence and [14C]amiloride binding
analysis of normal and Liddle's lymphocytes provided one explanation for the electrophysiological differences between normal and Liddle's lymphocyte, whole cell sodium currents. That Liddle's lymphocytes had
2.5 times more immunofluorescence and 3.5 times more
[14C]amiloride binding suggests that these cells
expressed more channels. Also, the immunofluorescent images indicate
that the majority of the fluorescence increase was located at the
plasma membrane. This finding could account for the larger whole cell
currents and the increased frequency of encountering active channels in outside-out patches. However, a finding of 2.5 times more channels does
not fully explain the 4.5-fold increase in basal whole cell current.
The explanation for this discrepancy was elucidated by examining single
channel kinetics of immunopurified polypeptide complexes incorporated
into planar lipid bilayers.
Removing ion channels from their native plasma membranes and the normal
cellular signaling pathways provides two advantages over studying
channels by patch clamp. First individual channels can be examined,
because channel incorporation into planar lipid bilayers is a rare
event. Typically vesicles are "forced" into the bilayers by
applying a voltage across the bilayer and eliminating the voltage
gradient once a vesicle fusion is observed and then promptly washing
the remaining vesicle from the chamber. This procedure often results in
only a single polypeptide complex being incorporated into the bilayer.
Under these conditions, the intrinsic kinetic properties can be
examined directly. Fig. 10 shows directly that in planar lipid bilayers
stripped of all regulatory elements and cellular connections, normal
channels remain mostly closed interspersed with brief openings. In
contrast, Liddle's channels remain mostly open interspersed with brief
closures. This difference in intrinsic channel kinetics combined with
the increased number of channels observed with immunofluorescence can
fully account for the large basal inward sodium currents typically
observed in whole cell clamped Liddle's lymphocytes.
These studies provide a comprehensive examination of the hypothesis
that human lymphocytes express functional ENaC that is highly
regulated. We know of no other cell type or tissue in which all of
these investigative techniques have been successfully applied to the
study of ENaC. This comprehensive investigation demonstrates directly
the utility of lymphocytes for the investigation of ENaC. The studies
were performed on human cells, thereby eliminating differences between
species. Lymphocytes are available from individuals with suspected ENaC
pathophysiologies such as pseudohyperaldosteronism by obtaining a small
(15 ml) blood sample. Further, lymphocytes are readily transformed,
providing a continuing source of viable cells that express suspected
ENaC polymorphisms. These studies also demonstrated differences in both
electrophysiological characteristics and protein expression between
normal lymphocytes and lymphocytes from individuals with Liddle's
disease. All of the findings were consistent with the pathophysiology
of the hypertensive disorder. Thus, in this well characterized disorder
lymphocytes appear to reflect accurately the cellular physiology of the
principal cells of the renal collecting duct, which produce the
in vivo hypertensive pathophysiology.
-ENaC bound to the plasma membrane of normal and Liddle's
lymphocytes. A quantitative analysis of fluorescence-tagged ENaC
antibodies indicated a 2.5-fold greater surface binding of the
antibodies to Liddle's lymphocytes compared with normal lymphocytes.
The relative binding intensity increased significantly (25%;
p < 0.001) for both normal and Liddle's cells after
treatment with 40 µM 8-CPT-cAMP. Amiloride-sensitive
whole cell currents were recorded under basal and cAMP-treated
conditions for both cell types. Liddle's cells had a 4.5-fold larger
inward sodium conductance compared with normal cells. A specific 25%
increase in the inward sodium current was observed in normal cells in
response to cAMP treatment. Outside-out patches from both cell types
under both treatment conditions revealed no obvious differences in the
single channel conductance. The Popen was
4.2 ± 3.9% for patches from non-Liddle's cells, and
27.7 ± 5.4% in patches from Liddle's lymphocytes.
Biochemical purification of a protein complex, using the same
antibodies used for the immunohistochemistry, yielded a functional
sodium channel complex that was inhibited by amiloride when
reconstituted into lipid vesicles and incorporated into planar lipid
bilayers. These four independent methodologies yielded findings
consistent with the hypotheses that human lymphocytes express
functional, regulatable ENaC and that the mutation responsible for
Liddle's disease induces excessive channel expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
140 to
160
mV). The apparent constitutive activation of the Na+
conductance could account for the hypertensive pathophysiology associated with Liddle's disease.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
60 mV
for 200 ms and clamp to membrane potentials ranging from
160 to + 40 mV for 800 ms in sequential 20 mV increments, returning to the holding
potential for 200 ms between each clamp step.
20 °C, for 30 min. After fixation, the cells were treated with a 3% formaldehyde solution, subsequently washed twice, and resuspended in phosphate-buffered saline. The fixed
cells were treated with 1% bovine serum albumin in phosphate-buffered saline to eliminate nonspecific protein binding and subsequently incubated in the same solution, supplemented with anti-bENaC
(amiloride-sensitive sodium channels purified from bovine kidney)
antibodies or nonimmune rabbit IgG as a negative antibody control for
30 min at 37 °C. The cells were then rinsed in phosphate-buffered saline.
,
, and
), we
used 10 µl of the reversed transcribed product for each specific gene
as template in a 25-µl reaction volume containing 100 pmol of each
specific subunit forward and reverse primer for 50 cycles. Primers were
designed based upon GenBankTM sequences (
-subunit,
SCNN1A, accession number Z92981;
-subunit, SCNN1B, accession number
U16023; and
-subunit, SCNN1G, accession number L36592). The specific
PCR primers and expected product sizes were:
PCR primers: forward,
5'-GAA CAA CTC CAA CCT CTG GAT GTC-3', and reverse, 5'-TCT TGG TGC AGT
CGC CAT AAT C-3', with an expected product size with cDNA of 257 bp;
PCR primers: forward, 5'-TGC TGT GCC TCA TCG AGT TTG-3', and
reverse, 5'-TGC AGA CGC AGG GAG TCA TAG TTG-3', with an expected
product size with cDNA of 277 bp; and
PCR primers: forward,
5'-TCA AGA AGA ATC TGC CCG TGA C-3', and reverse, 5'-GGA AGT GGA CTT
TGA TGG AAA CTG-3', with an expected product size with cDNA of 237 bp. The gene-specific reverse primers were used for cDNA synthesis. As a control for our gene expression assay, primers for the
-subunit were designed to span introns between exons 5 and 6 and between exons 6 and 7. Thus, the expected size for the
-subunit using genomic DNA
was 1023 bp. PCR products derived from
-subunit cDNA using the
same primer set were expected to be 257 bp. Primers for the
-subunit
were within a single exon. Given the partial and equivocal sequence
data for the
-sequence in GenBankTM, primer design was
guided by published intron/exon boundaries (16). Primers known to
reside in introns were used to test for the presence of genomic DNA.
-subunit, PCR reaction was performed in a final
reaction volume of 25 µl using 7.5 µl of double-distilled
H2O, 2.5 µl of 10× PCR buffer (final concentration, 1×
(Promega); final concentration, 1.5 mM MgCl2),
200 µM each dNTP (2.0 µl), 100 pmol each of the
-forward (100 pmol) and
-reverse (100 primers), 5 units
Taq DNA polymerase (Promega), and 10 µl of template
cDNA. Cycle conditions were 94 °C for 5 min for 1 cycle followed
by 94 °C for 1 min, 60 C for 1 min, and 72 °C for 1 min for 50 cycles. The PCR reaction mix for the
-subunit and
-subunit were
the same except for the appropriate gene-specific primers. The PCR amplification conditions for
-subunit and
-subunit were also the
same as for the
-subunit except the annealing temperature for
was 63 °C. A second round of PCR amplification was performed using 2 µl of the 50-µl reaction product as template for a total of 30 cycles.
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-,
-, and
-subunits of the human
amiloride-sensitive epithelial sodium channel. Fig. 1 shows the PCR products obtained using
gene-specific cDNA prepared from B-lymphocytes as template. To test
the efficacy of our cDNA preparation method and to confirm that our
PCR products were indeed derived from cDNA and not genomic DNA, we
designed primers flanking two introns in the
-subunit. When the same
PCR primers were used for amplification of a segment of the
-subunit
using genomic DNA and cDNA, two bands of different but expected
sizes were obtained: 1023 bp for the genomic DNA and 257 bp for the
-subunit cDNA. Based upon the intron/exon composition and/or the
lack of a clear and complete sequence for the
- and
-subunits, a
similar strategy for these units was not productive. Particularly
problematic was designing primers that yield a product size amenable to
direct sequencing. However, when one of the primers known to occur
within an intron was used in an attempt to amplify these subunits, no amplification was obtained with our cDNAs as would be expected. PCR
products for the
- and
-ENaC subunits obtained with our cDNA
were of expected size and are shown in Fig. 1.
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Fig. 1.
RT-PCR products from lymphocyte mRNA
using primers specific for each of the cloned ENaC subunits. The
predicted sized for each product is: , 257 bps;
, 277 bps. Direct
sequencing of the products confirmed that the
and
products were
ENaC.
- and
-subunits. Our cDNA sequencing results for the
-subunit were problematic. This problem persisted even though the same primer sets were used to amplify genomic DNA and the
product was successfully sequenced. The reverse primer of the same
primer set was used for the cDNA synthesis and yielded the expected
size band. Attempts at resolution have included efforts to sequence in
both forward and reverse directions and amplification with the original
primer set and then using nested primers to reduce the likelihood of
any nonspecificity. Finally, we have considered the occurrence of a
polynucleotide tract causing the Taq DNA polymerase drop-off
as well as the presence of secondary structure. The addition of
Me2SO was not useful. Additionally, alternative primer sets
have been designed; however, there remains a persistent lack of
sequence data. This was a consistent negative observation, irrespective
of whether the primer set is the one described or alternative ones
tested. This finding would suggest that there is little or no
expression of the
-subunit in B-lymphocytes.
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Fig. 2.
Normal cultured lymphocytes were whole cell
clamped, and then treated with 40 µM 8-CPT-cAMP. The treatment
activated inward currents. After activation, outside-out patches were
formed, and single channel currents were recorded. These were
completely inhibited by amiloride (2 µM). The
middle panels show a recording from the same outside-out
patch before (left) and after (right) superfusion
with amiloride. Amplitude histogram analysis showed that each patch
contained a minimum of five channels. Open dwell time analysis showed
that the channels had relatively fast open-to-closed transitions
( < 10 ms).
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Fig. 3.
Liddle's disease lymphocytes were whole cell
clamped, constitutively activated inward currents were recorded, and
outside-out patches were formed. Constitutively activated single
channels were observed in five of six outside-out patches. The
constitutively active channels were inhibited >90% by 2 µM amiloride. Amplitude histogram analysis showed that
the number of openings was reduced but the single channel conductance
remained unchanged by amiloride.
< 10 ms; Fig. 2, bottom right panel), with relatively long
closures between multiple channel bursts of opening.
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Fig. 4.
The number of [14C]amiloride
binding sites was determined for non-Liddle's (Daudi) and Liddle's
human lymphocytes. These studies utilized saturating
concentrations of [14C]amiloride (500 and 1000 nM) on both cell types. Cells were treated with 40 µM 8-CPT-cAMP prior to use in the binding assay.
[14C]Amiloride was competed away with 100 µM amiloride. Assuming 1:1 binding, these findings
predict that non-Liddle's lymphocytes express 716 ± 264 sites/cell and Liddle's lymphocytes express 2488 ± 751 sites/cell after treatment with cAMP.
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Fig. 5.
Upper panel, cell-attached patch single
channel openings. The inward currents (downward deflections) were
carried by Na+ because the main cation in the pipette
solution was Na+. Thirty 1-s records were recorded at each
of 11 clamp potentials ( 160 mV to +40 mV in 20 mV increments).
Lower panel, thirty sequential digital 1-s records for each
potential was synchronized and summed to produce the virtual whole cell
current shown in the lower panel. The morphology of these
virtual whole cell currents is indistinguishable from actual whole cell
currents.
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Fig. 6.
Confocal images of lymphocytes fixed and
treated with polyclonal antibodies generated against the
-subunit of bovine ENaC. Both non-Liddle's
and Liddle's lymphocytes exhibited antibody binding, with the
Liddle's lymphocytes exhibiting considerably more
immunoreactivity.
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Fig. 7.
The fluorescence intensity of four groups of
cells treated with anti-bENaC antibodies and fluorescence-labeled
secondary antibodies was quantitated relative to a single,
non-Liddle's cell. The findings were that cells from individuals
known to have the original Liddle's mutation had ~2.5 times the
fluorescent intensity as cells from unaffected members of the same
family. Also, treatment with 40 µM 8-CPT-caMP for 5 min
prior to fixation significantly increased the relative fluorescence of
each group by 25%.
-,
-, or
-hENaC (either
glycosylated or unglycosylated), which typically run between 65 and 100 kDa. The larger band may correspond to a multimeric combination of
these subunits. Others have purified bands of similar size from A6
epithelial cells and have speculated that this band was an xENaC
multimer (19).
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Fig. 8.
Left panel, iodinated polypeptide
complex from immunopurified from normal lymphocytes and run under
reducing conditions produced this multiband set of polypeptides.
Right panel, the immunopurified polypeptide complex from
Liddle's lymphocytes produced a set of reduced polypeptides similar to
that found in normal lymphocytes.
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Fig. 9.
Single channel recordings showing sodium
conduction via polypeptide complexes immunopurified from human
lymphocytes using an antibody directed directed against bovine
ENaC. Amiloride inhibited these single channels. However, the
efficacy of amiloride was considerably less than was observed in whole
cell clamps and outside-out patches. The single channel currents shown
here were recorded with 50 mV of + potential applied cis to
trans.
rENaC was subjected to DTT reduction in planar
lipid bilayer preparations (17), and sodium channels was purified from
bovine kidney using the same immunopurification procedure as was used
on lymphocytes (18).
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Fig. 10.
The effects of DTT on immunopurified ENaC
from human lymphocytes. These effects are similar to those seen on
reconstituted ENaC. The single channel currents shown here were
recorded with 100 mV of + potential applied cis to
trans. Before DTT treatment the conductance was 35 pS and
was similar to the conductance measured at other potentials and the
single channel conductance measured in ENaC purified from bovine renal
tissue. After DTT the conductance was reduced to 13 pS.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ENaC and
ENaC products were subsequently confirmed by direct sequence
analysis of the cDNA. However, whereas the RT-PCR reactions using
ENaC-specific primers yielded products of the correct size, subsequent sequence analysis failed to confirm the identity of these
products. Thus, it remains unresolved as to whether or not
ENaC
plays a role in the function or regulation of the amiloride-sensitive sodium channels expressed by lymphocytes.
-hENaC by sequence
analysis of lymphocyte RT-PCR products raises the possibility that
lymphocytes express amiloride-sensitive sodium channels composed of
only the
-hENaC and
-hENaC subunits. In oocytes it has been shown
that sodium channels are formed by
-
,
-
, and
-
-
expression combinations (20-22). Thus, it is possible that
lymphocytes express an
-
combination. However, as shown in Fig.
2, normal unstimulated lymphocytes have virtually no
amiloride-sensitive sodium current. This is not consistent with
reported constitutive activity of
-
channels (20). Also, the
channels expressed by human lymphocytes appear to be highly regulated.
They are activated by stimulation of
1-adrenergic
receptors (23). They are activated by (14, 15, 24), and they are
activated by aldosterone.2
Further, lymphocyte amiloride-sensitive, whole cell sodium currents are
electrophysiologically indistinguishable from the amiloride-sensitive, whole cell sodium currents found in rat renal principal cells (14).
-
,
-
, and
-
-
expression combinations all
produce amiloride-sensitive sodium currents in oocytes, it is possible that any or all of these combinations are expressed by lymphocytes and
principal cells. However, the most likely possibility is that principal
cells express
-
-
channels. This is because mutations in the
-subunit are known to produce Liddle's disease (25), and a
truncation mutation of the
-subunit has been linked to familial low
renin hypertension in one family (26). Oocyte studies indicate that
maturation and assembly of subunits into channels is a "slow
inefficient process" (21). Thus, there may be considerable variability of the mRNA levels, and that could be responsible for
our inability to demonstrate
-hENaC directly. Because of the
similarity in current morphology, magnitude, response to agonists, and
pharmacological profile of amiloride and its analogs (13) between the
amiloride-sensitive whole cell sodium currents in lymphocytes and
principal cells, it seems likely that both cell types express similar
channels. Because the differences induced by the prototypical Liddle's
mutation produce an excess of channel expression in both oocytes and
lymphocytes, the current evidence supports the hypothesis that both
cell types respond similarly to mutation of
-hENaC. Thus, despite
our inability to directly sequence
-hENaC from lymphocyte mRNA,
the other pieces of information currently available are consistent with
the hypothesis that lymphocytes and principal cells express the same
type of amiloride-sensitive sodium channels.
-subunit, (i.e. lymphocytes from individuals with Liddle's disease). Liddle's lymphocytes had a
constitutively activated sodium conductance, whereas non-Liddle's lymphocytes did not. This finding alone indicates that minimally, the
-ENaC subunit plays a role in lymphocyte sodium conductance, because
the only difference between Liddle's and non-Liddle's lymphocytes is
the mutation in the
-ENaC subunit. Two other electrophysiological findings further our understanding of lymphocyte sodium conductance. There was no difference in the single channel conductance. Thus, mutation of the
-ENaC subunit does not appear to affect the
conductive pore of the channels. However, the kinetic activity of the
channels expressed by normal cells appeared to be considerably less
frequent than the channels expressed by Liddle's cells. The basal
whole cell currents in Liddle's cells are significantly larger than the basal currents observed in normal non-Liddle's lymphocytes. The
difference could result from different numbers of channels (suggested
by the greater plasma membrane immunofluorescence and greater number of
[14C]amiloride binding sites) or different frequencies of
opening and closing. Because single channel openings were rare events in membrane patches from non-Liddle's cells, there is no basis for
comparison of basal single channel kinetics between non-Liddle's and
Liddle's lymphocytes. To resolve this problem additional techniques were applied to lymphocytes, protein biochemistry, and immunofluorescence.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK 37207 (to D. J. B.) and DK52789 (to J. K. B.).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.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed. Tel.: 205-934-6214; E-mail: bubien@uab.edu.
§ Present address: Dept. of Biol. Chem., Johns Hopkins School of Medicine, Baltimore, MD 21205.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M008886200
2 Z.-H. Zhou and J. K. Bubien, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: ENaC, epithelial sodium channel; hENaC, human ENaC; PCR, polymerase chain reaction; RT, reverse transcriptase; bp, base pair(s); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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