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Enhanced DNA-Binding Activity of a Stat3-Related Protein in Cells Transformed by the Src Oncoprotein

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Cytokines and growth factors induce tyrosine phosphorylation of signal transducers and activators of transcription (STATs) that directly activate gene expression. Cells stably transformed by the Src oncogene tyrosine kinase were examined for STAT protein activation. Assays of electrophoretic mobility, DNA-binding specificity, and antigenicity indicated that Stat3 or a closely related STAT family member was constitutively activated by the Src oncoprotein. Induction of this DNA-binding activity was accompanied by tyrosine phosphorylation of Stat3 and correlated with Src transformation. These findings demonstrate that Src can activate STAT signaling pathways and raise the possibility that Stat3 contributes to oncogenesis by Src.
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Journal of the American Society of Nephrology 2543
A Novel Variant of the p-Subunit of the Amiloride-Sensitive
Sodium Channel in African Americans1
Yan Ru Su,2Mark P. Rutkowski,2 Charles A. Klanke, Xiaomin Wu, Yong Cul, Paymund Y.K. Pun,
Vicki Carter, Max Reif, and Anil G. Menon3
YR. Su, CA. Klanke. X. Wu. V. Cui. AG. Menon,
Department of Molecular Genetics, Biochemistry and
Microbiology. University of Cincinnati Medical Center,
Cincinnati. OH
M.P. Rutkowski, V. Carter, M. Reif, Department of
Internal Medicine, Division of Nephrology and Hyper-
tension, University of Cincinnati Medical Center. Cm-
cinnati, OH
R.Y.K. Pun, Department of Molecular and Cellular
Physiology. University of Cincinnati Medical Center.
Cincinnati. OH
(J. Am. Soc. Nephrol. 1996; 7:2543-2549)
ABSTRACT
The amiloride-sensitive sodium channel is responsible
for the rate-limiting step of sodium reabsorption in the
distal renal tubule, and thus may play a key role in the
maintenance of sodium balance and blood pres-
sure. In this study, a genetic variant that results in a
change ofthreonine to methionine at amino acid 594
(T594 M) in the carboxy-terminus of the p-subunit of
the amiloride-sensitive sodium channel has been
identified. This variant was present in 6.1 % of African-
American subjects (N =231) but was not seen in
Caucasians (N =192). Whole cell voltage clamp of
B-lymphocytes from individuals with the T594 M van-
ant showed similar basal membrane slope conduc-
tance, compared with the wild-type but increased
response to cAMP analog.
Key Words: Blacks. blood pressure, ion channels. patch
clamp techniques. sodium balance
The regulation of sodium balance in mammals is a
process that involves a number of sodium trans-
porters. many of which are expressed in the kidney.
Exact sodium balance is ultimately determined by the
distal nephron. Because the amiboride-sensitive so-
dium channel is a key regulator of salt reabsorption in
the nephron. we booked for mutations in the gene
encoding the 3-subunit of this channel in African
1Received June 4, 1996. Accepted July 23. 1996.
2Both authors contributed equally to this work.
3Correspondence to Dr. AG. Menon, Department of Molecular Genetics, Bio-
chemistry and Microbiology University of Cincinnati Medical Center, 231 Be-
thesda Avenue, Cincinnati OH 45267O524.
104&6673/07 12-2543103.00/0
Journal of the American Society of Nephrology
Copyright © 1996 by the American Society of Nephrology
Americans-a group known to have a higher preva-
lence of both hypertension and salt-sensitivity when
compared with Caucasians ( 1-5). The channel Is corn-
posed of at beast three subunits: a, 3, and y (6.7). The
human genes encoding all three subunits have been
identified and cloned (8-1 1). Mutations In the car-
boxy-terminus of the f3 and y subunits of this channel
have been proposed to be the underlying cause of
Llddbe’s syndrome. a rare autosomab dominant form of
hypertension ( 1 2, 1 3). The f3-subunit of the amiboride-
sensitive sodium channel has been shown to be ex-
pressed in the renal collecting duct (14) and in B-
lymphocytes ( 15. 16). and the electrophysioboglcal
characteristics of the channel in these two tissues
have been shown to be indistinguishable ( 1 7). Whole
cell patch clamp studies in B-lymphocytes from mdi-
viduals with Liddle’s syndrome ( 1 8) and expression of
Liddle’s syndrome mutant channels in Xenopus oo-
cytes ( 1 9) have shown increased channel activity.
More recently, this increased sodium conductance
has been correlated with an increased number of
channels on the oocyte membrane (20).
We report a novel variant that results in a substitu-
tion ofrnethionine for threonine at Position 594. In the
carboxy-terminus of the 3-subunit. This variant was
detected in African Americans but not in Caucasians.
Whole cell voltage clamping in B-lymphocytes show
that the variant was associated with enhanced so-
dium conductance in response to a cAMP analog.
METHODS
Subjects were recruited from the Hypertension Clinics of
the University of Cincinnati and the Veterans Administration
Hospital, as well as from the community at large. The study
was approved by the Institutional Review Board of the Uni-
versity of Cincinnati.
African Americans
All subjects underwent a brief clinical evaluation and
provided a blood sample for measurement ofplasma electro-
lytes and DNA isolation. Subjects were seated for 5 mm. after
which blood pressure recordings were done in triplicate by
using a mercury manometer with the appropriate cuff size
based on the upper mid-arm circumference. Korotkoff Phase
V was used to define the diastolic pressure.
Treated hypertensive subjects were included if they re-
ported that the onset ofhypertenslon was before the age of 60
and that they had been under continuous treatment with
antihypertensive medication for the previous 6 months. In
hypertensive subjects not on antihypertensive treatment,
average diastolic blood pressure was confirmed to be 90
mm Hg on a second visit a week later. All hypertensive
subjects met the following criteria: no reported history of
secondary hypertension: serum potassium level 3.5 mEq/L
(unless they were being treated with thiazide or loop diuret-
Amiloride-Sensitive Sodium Channel Variant
254.4 Volume 7 -Number 12 -1996
Ics); serum creatinine concentration 1 .6 mg/dL for men
and s 1 .4 mg/dL for women; alcohol intake 1 .5 ounces of
ethanol per day: and no use of steroids or estrogen before the
diagnosis of hypertension. In subjects with non-insulin-
dependent diabetes mellitus, the diagnosis of hypertension
had to precede the diagnosis of diabetes mellitus by at beast
5 yr.
Normotensive subjects were included if systolic pressure
was <150 and diastolic pressure was <90 mm Hg. based on
the average of three readings on a single visit while not being
treated with antlhypertensive medications.
Caucasians
To determine allele frequency of the T594 M variant In
Caucasians. two populations were combined: ( 1) 1 20 unre-
lated grandparents from the Centre d’Etude du Polymor-
phisme Humain (CEPH) pedigree for whom hypertension
status is unknown (2 1 ), and (2) 72 unrelated Caucasians
from the Cincinnati area (30 normotensive, 32 hypertensIve.
and ten of indeterminate blood pressure status).
DNA Analysis
DNA was extracted from peripheral blood by using Pure-
gene DNA isolation kit (Gentra Systems Inc. Minneapolis.
MN). Single-strand conformatlonal polymorphism analyses
were performed by amplifying DNA fragments of the final
exon of human amlborlde-sensitive sodium channel 3-sub-
unit (-ENaC) from genomic DNA. Oligonucleotide primers
ENaC 1746 and f3ENaC 1940 were used in polymerase chain
reactions. as previously described ( 1 2). Products were frac-
tionated on 0.6X MDE (Mutation Detection Enhancement)
gels (AT Biochem. Malvern. PA) and 0.8X TBE (Tris Borate
EDTA) as electrophoresis buffer at room temperature for 12
to 1 4 h. After autoradiography. the variant band was cut
from the gel. and the DNA was eluted and reamplified before
being sequenced with an ABI automated DNA sequencer
(ABI. Foster City, CA). In all cases, both strands of DNA were
sequenced.
Aldosterone Profiling
Eight subjects with the T594 M variant. of which four were
hypertensive. were randomly chosen for aldosterone profiling
and ebectrophysiobogical evaluations. Eight African-Amen-
can subjects without the T594 M variant (ofwhom three were
hypertensive) were chosen as control subjects such that age,
sex. and body mass index were matched between the two
groups. This aldosterone profiling protocol has been used to
classify Individuals in Liddbe’s syndrome kindreds (22). All
subjects collected urine overnight. At approximately 8 am.
the next day. blood was collected after the subject sat upright
for 30 mm. All subjects continued to follow their usual diet
and medication treatment. Serum and urine aldosterone
levels were measured by RIA (Coat-A-Count: Diagnostic
Product Corporation. Los Angeles. CA). Normal ranges for the
upright position are 5 to 30 ng/dL for serum aldosterone and
2 to 14 g/24 h for urine abdosterone. Although no normal
range has been established for overnight 1 2-h collections,
12-h aldosterone excretion rates have been shown to come-
late with 24-h collection excretion rate in adults (23). Plasma
renin samples were determined by RIA (New England Nu-
clear. Boston, MA). The normal range for upright plasma
renin with this assay Is 0.3 to 3.0 ng/mL per h.
Electrophysiological Studies
The electrophyslologlcal measurements on lymphocytes
were conducted In a single-blind manner (presence or ab-
sence of the variant was not revealed to experimenter). The
bath medium was RPMI 1640 contaIning low Ca2 (0.54 mM)
supplemented with 10 mM N-hydroxyethylpiperazmne-N’-2-
ethanesulfonic acid to buffer the pH. The perforated-patch
method was used (24,25). The pipette was tip-filled with
recording medium containing (in mM): KC1, 70; K2S04. 30;
NaC1, 12; EGTA, 0.5: MgCl2. 1: HEPES, 20; glucose, 10 (pH
was adjusted to 7.2 and osmolarity to 300 mosmol). The
pipette was then back-filled with nystatin-containing record-
ing solution (200 to 300 tg/mL) prepared from a fresh stock
consisting of 50 mg/mL of nystatin sonicated in dimethyb
sulfoxide. Resistances of the pipettes were between 12 to 15
Mohm.
The slope conductance was measured from the linear
portion of the ramp current elicited by a voltage ramp from
-150 to +50 mV over the duration of 1 s, using the software
pCbamp (Version 5.5. Axon Instruments, Foster City, CA). At
beast four ramps at 0.2 Hz were averaged for each measure-
ment. After the control ramps were obtained (two to three
measurements), 300 j.tM 8-CPT-cAMP (8-CPT 8-(4-chboro-
phenylthio) adenosine 3.5-cyclic monophosphate). a mem-
brane-permeabbe analog of cAMP, was supemfused onto the
cell from a blunt-tipped pipette (5 to 10 .tm) placed nearby,
and two to three more measurements were performed. Cur-
rent records were filtered at 5 kHz. digitized, and stored for
analysis.
Statistical Analysis
Results are expressed as mean ±SD or 95% confidence
Intervals. Plasma renin bevels, abdosterone bevels, sodium
and potassium excretion rates, and ratio of urine aldosterone
to potassium were log-transformed before analysis of van-
ance. To correlate the aldostemone profile results with the
electrophysioboglc data. the ratio of the 8-CPT-cAMP-stimu-
lated to basal conductance was averaged for each subject
and used as the dependent variable in an analysis of variance
(Statistical Analysis System; SAS Institute, Cary, NC).
RESULTS
Identification of a Missense Mutation at Codon
594 of the Amiloride-Sensitive Sodium Channel
f3 Subunit
Single-strand conformationab polymorphism analy-
sis of the 3’ end of the gene showed a variant band in
some individuals of African-American descent. Elu-
tion of this band and subsequent sequence analysis
revealed a single nucleotide substitution (C-T) In the
coding strand at amino acid position 594. Sequencing
the opposite strand confirmed the initial observation.
The normal allele was sequenced and found to be
identical to that reported by McDonald et at. (10).
These results are shown in Figure 1 .All individuals
with this variant were heterozygous at this locus.
The frequencies of the T594 M variant are shown in
Table 1 .The clinical characteristics of the African
Americans with and without the T594 M variant are
shown in Table 2. There were no statistically signifi-
cant differences between African Americans with and
without the T594 M.
AWT AGCCTGACACGGCCC
*
VAR AGCCTGACATGGCCC
a 2 12.2, p =0.002 for the entire table.
bf.0.12. P=0.72 versus hypertensive subjects.
C 2 =]2.0. P=0.0005 versus African-American subjects (first two rows
combined).
BWT GGGCCGTGTCAGGCT
*
VAR G G GCC A I G T C A GG C I
Su et al
Journal of the American Society of Nephrology 2545
Figure 1.Sequence comparison of both strands of DNA from
wild-type and variant. Panel A shows a comparison of
wild-type sequence with sequence of sense strand. The C-IT
transition is highlighted with an asterisk. Panel B shows
comparison of wild-type sequence with anti-sense strand.
The complementary G-A transition is highlighted with an
asterisk.
Amiloride-Sensitive Sodium Conductance in
Lymphocytes With and Without the T594 M
Variant
Membrane slope conductance of isolated B-bympho-
cytes from individuals without the T594 M variant
(wild-type) and B-lymphocytes harboring the T594 M
variant were measured. Slope conductances before
and in the presence of 8-CPT-cAMP from the two
groups were then compared. The basal slope conduc-
tances for the two groups were similar: 0.43 ± 0.24 nS
(N =15) forwild-type cells and 0.45 ±0.21 nS(N =12)
for T594 M variant cells (P =0.79). In the presence of
8-CVT-cAMP, slope conductance of responsive cells
TABLE 1.T594M variant in populations of unrelated
subjects
Group NT594M/
NTested Percentagea
African-American Hypertensive
Subjects
African-American Normotensive
Subjects
Caucasian Subjects
7/126
7/105
0/192
5.6
67b
OC
was increased to 1 .20 ±0.72 nS for the wild-type cells
(paired t test, P<0.05) and 1 .99 ±1 .22 nS for the
variant cells (paired t test, P<0.05). One-way analysis
of variance shows that the 8-CPT-cAMP-stimulated
slope conductance Increase In the T594 M variant is
significantly higher than that for the wild-type (P <
0.05). Representative traces of the recordings ob-
tamed are shown in Figure 2. Because the amiboride-
sensitive sodium channel Is most active at very nega-
tive membrane potentials (more negative than -120
rnV), the presence of channel activities obscured the
measurements of the slope conductance at the nega-
tive potentials (see Figure 2). Thus, the slope conduc-
tance that we measured during 8CPT-cAMP applica-
tion is likely to be an underestimation of the actual
changes that had occurred.
The change in slope conductance in response to
8-CPT-cAMP measured for the variant could result
from a higher number of amiboride-sensitive sodium
channels (also an increase in probability of channel
opening. unitary conductance. etc.) present in the
variant lymphocytes or from a change in responsive-
ness of the channel to 8-CPT-cAMP. To compare the
responsiveness of the amiboride-sensitive sodium
channel with 8-CPT-cAMP in the wild-type and the
variant, and to rule out the possibility that the re-
sponse is rebated to the basal conductance, we exam-
med the ratio of the 8-CPT-cAMP response to basal
slope conductance in the same cell. The response ratio
was 2.97 ±1.13 (N =15) for the wild-type. This ratio
is in good agreement with the ratio we derived (3.12)
from the data reported for normal B-lymphoid cells by
Oh et at.. ( 15). For the variant, the response ratio was
4.72 ±1.94 (N =12), which is significantly higher
when compared with the wild type (P =0.007). These
results indicate that the T594 M variant is more
responsive to 8-CPT-cAMP.
When amiloride (2 jIM) was coapplied with 8CPT-
cAMP, the slope conductance were 0.36 ± 0.24 nS
(N =10: control subjects without arniboride 0.39 ±
0.23 nS) and 0.57 ±0.30 nS (N =7; control subjects
without amiboride, 0.55 ± 0.26) for wild-type and T594
M variant, respectively (Figure 2). The result showed
that the enhancement in slope conductance by 8-CPT-
cAMP occurs via an arniboride-sensitive conductance.
Amiloride-Sensitive Sodium Channel Variant
2546 Volume 7 -Number 12 -1996
TABLE 2. Clinical characteristics of African-American subjects#{176}
Group Genotype N (mit) Age (yr) Age Dx
(yr) BMI
(kg/m2) SBP
(mm Hg) DBP
(mm Hg) Rx
(%)
Hypertensive Subjects Wild-type 1 19 (47/72) 52 ±11 39 ±12 32.6 ±7.9 144 ±22 92 ±14 87
Hypertensive Subjects T594M 7 (5/2) 62 ±15 44 ±16 28.4 ±3.8 149 ±18 91 ±6 87
Normotensive Subjects Wild-type 98 (30/68) 57 ±17 n/a 28.1 ±6.9 124 ±14 77 ±7 0
Normotensive Subjects T594M 7 (3/4) 55 ±14 n/a 27.2 ±4.2 125 ±10 77 ±8 0
0Values shown are mean SD. N. number of subjects; m/f, Nmale/Nfemaie; Age Dx, age of diagnosis of hypertension; SBP. systolic blood pressure;
DBP, diastolic blood pressure; Rx, subjects on antihypertensive medications; BMI. body mass index.
Aldosterone Profiles of Subjects With and
Without T594 M
The aldosterone profiles of the 16 subjects whose
lymphocytes were studied electrophyslobogically are
shown In Table 3. Although the serum aldosterone
and urine abdosterone bevels were statistically higher
In the subjects with the T594 M variant. there was no
statistical difference when the urine aldosterone bevel
was normalized for potassium excretion. The levels of
circulating aldosterone in individuals with the T594 M
variant were of particular Interest because earlier
studies in mammalian nephrons show that aldoste-
rone increases the Na+ conductance in the apical
membrane through the activation of amiboride-sensi-
tive sodium channel (26,27). To assess whether the
higher abdosterone bevels observed in Individuals with
the T594 M variant could account for the higher
8-CPT-cAMP-stimubated slope conductance in the
lymphocytes from these individuals. an analysis of
variance was performed using the ratio of 8-CPT-
cAMP-stimulated- to-basal slope conductance as the
dependent variable. The covariates urine sodium ex-
cretlon, urine potassium excretion, and abdosterone
excretion did not abolish the relationship between
presence of the variant and higher 8-CPT-cAMP-stlm-
ubated slope conductance (P =0.00 1).
DISCUSSION
We have Identified a missense mutation T594 M in
the carboxyb terminus of the 13-subunit of the amibo-
ride-sensitive sodium channel that is present in nor-
motensive and hypertensive African Americans (6.1%)
but not in Caucasians. Ebectrophysbobogicab studies of
the amiboride-sensitive sodium conductance In bym-
phocytes revealed that this variant is associated with
an enhanced 8-CPT-cAMP-stlmulated response when
compared with wild-type (4.72- versus 2.97-fold. re-
spectiveby).
There Is evidence that mutations in the carboxy-
terminus of the 3 subunit of the amiloride-sensitive
sodium channel can bead to abnormal channel behav-
ior, causing sodium retention and hypertension. Mu-
tatlons causing a premature truncation of the last 43
to 75 amino acids of the carboxy-termmnus of the
amiloride-sensitive sodium channel a-subunIt have
been Identified in Liddle’s syndrome. an autosomal
dominant form of hypertension (12). The T594 M
variant that we have identified is located within this
region (47 amino acids from the carboxy-termmnus).
Our electrophysioboglc and clinical data for the T594
M variant differ from that reported for Liddbe’s syn-
drome. The amiboride-sensitive sodium channels of
B-bymphoid cells from Liddle’s patients did not re-
spond to 8-CPT-cAMP ( 1 8), whereas the response to
8-CPT-cAMP in the T594 M variant was enhanced
when compared with the wild-type. This lack of re-
sponse in Liddle’s syndrome has been attributed to
increased inward baseline sodium current. Recently,
it has been reported that Liddle’s syndrome trunca-
tion mutations in the carboxy terminus of the a-sub-
unit result in an increased number of channels ex-
pressed in Xenopus laevis oocytes ( 1 9). Analysis of the
carboxy-terminus revealed a conserved eight-amino-
acid motif(PPPXYXXL) that when mutated, resulted in
an increased number of sodium channels expressed
on the cell surface (20). Although we cannot rube out
whether the enhanced responses induced by 8-CPT
cAMP in the T594 M variant result from an increase in
the channel density on the cell surface, the increase in
response ratio suggests that the enhanced slope con-
ductance is likely a result of a change in an Intrinsic
property of the channel. Thus It Is possible that the
physiological robe played by the T594 M variant (which
is not contained within the PPPXYXXL motif) may be
different from variants observed in Liddle’s syndrome.
The back of suppression of aldosterone in individuals
with the T594 M variant and Its presence In normo-
tensive individuals (five of seven of whom were over
the age of 50) also contrasts with Liddbe’s syndrome
patients, in whom abdosterone secretion is suppressed
secondary to sodium retention and in whom hyperten-
sion usually develops during the second decade of life
(22).
Other investigators have suggested that cAMP can
either activate or inhibit the activity of the amiboride-
sensitive sodium channel. Bubien et at. .have shown
in lymphocytes that cAMP usually activates the amilo-
ride-sensitive sodium conductance but inhibits chan-
neb activity after exposure to pertussis toxin (28).
Stutts et at. .reported that coexpressing the cystic
fibrosis transmembrane regulator with the rat renal
amlboride-sensitive sodium channel suppressed
cAMP-stimulated channel activity (29). Our studies
indicate that lymphocytes expressing the T594 variant
Wild-type
-1 50 mV
Variant 1
-1 50 mV
B
Variant 2
+5OmV
2OpA[
100 msec
mV
Variant 3
-1 50 mV
-1 50 mV
+5’
+5OmV
B
Su et al
Journal of the American Society of Nephrology 2547
Figure 2. Current-Voltage (l-V) relation measured in lymphocytes from Individuals withoutthe variant (left panel, wild-type) and
from individuals harboring the variant (right panel) under perforated-patch recording. I-V relation was elicited and the slope
conductance obtained as described in the method section. Trace A in both panels shows the I-V relation under control conditions.
Trace B In each panel shows the I-V relation in the presence of 8-CPT-cAMP. Trace C in both panels shows the I-V relation in the
presence of both of 8-CPT-cAMP and amilonide (2 1.tM). Variants are representative fracings from three different individuals.
sodium channel not only show an enhanced response
to cAMP but also show a greater susceptibility to
opening at negative potentials on some occasions.
possibly because of the boss of a suppressive effect.
The threonine residue at position 594 in the a-subunit
may be Involved in the regulation of channel activity,
as It Is a potential target for phosphorybation by
protein kinase C (PKC) (10). It has been shown, for
example. that activation of PKC by addition of phorbol
1 2, 1 3-dibutyrate or mezerein abolished amiboride-
sensitive sodium channel activity (30). The substitu-
tion of methionine for threonine at this site may
therefore cause the channel to become resistant to the
negative-regulatory effect of PKC, and could explain
the enhanced response to 8-CPT-cAMP seen in the
T594 variant. It is possible that the mechanism of
regulation of channel activity involves at least two
distinct sites-a positive modulatory site that in-
creases channel activity and a negative modulatory
site that decreases channel activity. The sites may be
on the sodium channel itself or on its associated
regulatory complex. Regulatory sites other than the
cAMP and PKC sites, such as G protein binding sites,
may also exist (28).
An important and yet unresolved question Is
whether this variant, apparently specific to African
Americans. plays a role In the higher prevalence of
salt-sensitive hypertension seen in this population.
Although there was no significant difference In the
prevalence of the T594 M variant between the hyper-
Amiloride-Sensitive Sodium Channel Variant
2548 Volume 7 -Number 12 -1996
TABLE 3. Aldosterone profile of a subset of African-American subjects with and without T594M vaniant#{176}
Parameter Wild-Type (N =8) T594M Variant (N =8)
Serum Potassium (mEq/L) 4.2 (3.9 to 4.6) 4.5 (4.3 to 4.7)
Plasma Renin Activity (ng/mL per h) 0.45 (0. 19 to 1.04) 0.37 (0. 17 to 0.78)
Serum Aldosterone (ng/dL) 6.9 (4.4 to 10.8) 13.3 (9.9 to 17.9)b
Urine Sodium (mEq/ 12 h) 70 (55 to 89) 62 (40 to 62)
Urine Potassium (mEq/12 h) 21 (14 to 32) 27 (17 to 41)
Urine Aldosterone (g/12 h) 2.0 (1.2 to 3.2) 4.4 (2.8 to o.7)b
Aldosterone:Potassium Ratioc 94 ( to 135) 172 (92 to 325)
aincludes four hypertensive and four normotensive subjects in the T594M group. and three hypertensive and five normotensive subjects in the
wild-type group. All values reported except potassium are the anti-log of the mean log-transformed value (95% confidence intervals).
bp 0.03 by analysis of variance; all others are not significantly different.
CNanograms of urine aldosterone per milliequivalent of urine potassium.
tensive and normotensive populations that we stud-
led, this does not preclude it from being one of the
contributing factors to the trait. The phenotypic con-
tributlon of the T594 M variant may not be evident in
normotensive subjects who harbor the variant with-
out the additive effects ofother genetic or environmen-
tab factors.
It Is Important to point out some of the limitations in
the interpretation of our observations. First, our elec-
trophysiobogical studies were whole cell patch clamp-
Ing measurements and not single channel recordings.
Second, although it has been shown by reverse tran-
scrlption- pobymerase chain reaction that mRNA en-
coding the 3- subunit is present in lymphocytes from
human (15) and rat (16), the alpha subunit of this
channel is probably a different isoform both in rat and
human lymphocytes from the alpha subunit ex-
pressed in the renal collecting duct (15, 16).
Nonetheless, It has been shown that amiboride-
sensitive sodium channel activity is increased in lym-
phocytes from individuals with Liddle’s syndrome
(similar to what has been observed in the oocyte
expression system of Liddle’s mutations). Therefore,
although it is possible that the difference in whole cell
ebectrophyslobogicab activity conferred by the n-sub-
unit variant T594 M in B-lymphocytes would also be
present in the sodium channel expressed in the renal
collecting tubule. this has not been proven in our
study. If this variant does predispose to salt-sensitive
hypertension, it would have to alter the function of the
amilorlde-sensltive sodium channel in the renal col-
becting duct.
Whether the T594 M variant confers any selective
advantage to individuals who harbor the variant gene
remains unknown. It is possible that the increased
ability to reabsorb sodium conferred by the variant
channel may be an advantage In certain conditions of
salt deprivation, or, alternatively. increased protection
against volume depletion in conditions of water scar-
city.
The dIscovery of the T594 M genetic variant in the
13-subunit of the amiloride-sensitive sodium channel,
a key regulator of renal sodium handling, and the
altered electrophysiobogicab properties associated with
it may be one of the first steps in establishing the
amiboride-sensitive sodium channel as a genetic com-
ponent of essential hypertension in African Amen-
cans.
ACKNOWLEDGMENTS
This work was supported in part by grants from the National Insti-
tutes of Health Program of Excellence and Markey Foundation to
A.G.M. .from Dialysis Centers Inc. to M.P.R and MR. and a Cardlo-
vascular Center Research Grant from the University ofClnclnnati. We
thank Drs. Peter Gartslde. Kenneth Blumenthal. John Cupoletti,
Jerry Lingrel. Robert Luke. and Gary Shull for discussions and
helpful comments on the manuscilpt. and acknowledge the excellent
technical assistance ofTerri Lewis and Kristen Braig.
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We studied c-fos gene expression in rat fibroblasts by microinjection of regulatory DNA sequences, such as the serum response element (SRE) present in c-fos promotor, in order to compete directly with such sequences for binding of putative regulatory factors. We show that an additional fos intragenic regulatory element (FIRE) is located at the end of exon 1. When coinjected with an SRE oligonucleotide, it induced c-fos expression in quiescent cells, whereas injection of SRE sequence alone failed to do so. Moreover, injection in quiescent cells of an SRE oligonucleotide together with a p-fos-lacZ construct containing the c-fos SRE as well as an in-frame insertion of FIRE resulted in a block to beta-galactosidase expression that can be relieved by coinjection of the FIRE sequence.
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pp60c-src is phosphorylated in vivo at tyrosine 527, a residue not present in pp60v-src (its transforming homolog), and not at tyrosine 416, its site of in vitro autophosphorylation. To test the hypothesis that tyrosine phosphorylation regulates pp60c-src biological activity, we constructed and studied pp60c-src mutants in which Tyr 527 and Tyr 416 were separately or coordinately altered to phenylalanine. Tyr----Phe 527 mutation strongly activated pp60c-src transforming and kinase activities, whereas the additional introduction of a Tyr----Phe 416 mutation suppressed these activities. Tyr----Phe 416 mutation of normal pp60c-src eliminated its partial transforming activity, which suggests that transient or otherwise restricted phosphorylation of Tyr 416 is important for pp60c-src function even though stable phosphorylation is not observed in vivo.
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Thirteen clones of hybrid cells which synthesize antibodies directed against the Rous sarcoma virus (RSV) transforming protein, pp60src, were isolated. Mouse myeloma cells were fused with spleen cells from mice that had been immunized with purified pp60src from bacterial recombinants which direct the synthesis of the RSV src gene. The hybridomas which survived the selection medium were screened by immunoprecipitation of pp60src from 32P-labeled lysates of RSV-transformed cells. Monoclonal antibodies produced by subclones derived from 13 hybridomas recognized pp60src encoded by the Schmidt-Ruppin and Prague strains of RSV and the cellular homolog of pp60src. Antibody from clone 261 had a high affinity for the viral yes gene product, and antibodies from clones 443 and 463 recognized the transforming proteins encoded by viruses containing the related transforming genes fps and ros. Several other clones had a low affinity for the viral yes, fps, and ros gene products which could be detected by in vitro phosphorylation of the transforming proteins after immunoprecipitation with the monoclonal antibody. All of the monoclonal antibodies allowed phosphorylation of pp60src and casein in an immune complex-bound reaction.
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Binding of interferons IFN-alpha and IFN-gamma to their cell surface receptors promptly induces tyrosine phosphorylation of latent cytoplasmic transcriptional activators (or Stat proteins, for signal transducers and activators of transcription). Interferon-alpha activates both Stat91 (M(r) 91,000; ref. 1) and Stat113 (M(r) 113,000; ref. 2) whereas IFN-gamma activates only Stat91 (refs 3, 4). The activated proteins then move into the nucleus and directly activate genes induced by IFN-alpha and IFN-gamma. Somatic cell genetics experiments have demonstrated a requirement for tyrosine kinase-2 (Tyk2) in the IFN-alpha response pathway and for Jak2 (ref. 6), a kinase with similar sequence, in the IFN-gamma response pathway. Here we investigate the tyrosine phosphorylation events on Stat and Jak proteins after treatment of cells with IFNs alpha and gamma and with epidermal growth factor (EGF). Stat91 is phosphorylated on Tyr701 after cells are treated with IFN-alpha and EGF, as it was after treatment with IFN-gamma (ref. 8). We find that Jak1 also becomes phosphorylated on tyrosine after cells are treated with these same three ligands, although each ligand is shown to activate at least one other different kinase. Jak1 may therefore be the enzyme that phosphorylates Tyr 701 in Stat91.