J C E M O N L I N E

A d v a n c e s i n G e n e t i c s — E n d o c r i n e R e s e a r c h

Variations in the Potassium Channel Genes KCNK3

and KCNK9 in Relation to Blood Pressure and
Aldosterone Production: An Exploratory Study

Jeesun Jung, Paula Q. Barrett, George J. Eckert, Howard J. Edenberg,

Xiaoling Xuei, Wanzhu Tu, and J. Howard Pratt
Departments of Medical and Molecular Genetics (J.J.), Biostatistics (G.J.E., W.T.), Biochemistry and
Molecular Biology (H.J.E., X.X.) and Medicine (J.H.P.), Indiana University School of Medicine, and the
Veterans Affairs Medical Center (J.H.P.), Indianapolis, Indiana 46202; and Department of Pharmacology
(P.Q.B.), University of Virginia School of Medicine, Charlottesville, Virginia 22908

Context: Two potassium (K) channel genes, Kcnk3 and Kcnk9, when deleted in mice, produced a
model of hyperaldosteronism and hypertension.

Objective: Our objective was to explore genetic variation [single-nucleotide polymorphisms (SNP)]
in KCNK3 and KCNK9 in relation to blood pressure (BP) and aldosterone production in humans.

Subjects and Study Design: Two groups of healthy European Americans (EA) and African Amer-
icans (AA) were studied: 1) a longitudinal study group (age ⬃14 yr when enrolled, 444 EA and 351
AA) and 2) an inpatient cross-sectional study group (age ⬃23 yr, 85 EA and 109 AA). Plasma renin
activity, plasma aldosterone concentration, and level of serum K were measured cross-sectionally;
BP was measured semiannually in the longitudinal study. SNP were selected to provide coverage
of the genes for both EA and AA (15 in KCNK3 and 74 in KCNK9).

Results: No associations with KCNK3 were observed. In the longitudinal study, multiple SNP in
KCNK9 associated with systolic BP in AA, whereas associations were primarily with aldosterone
production in EA. The direction of the changes was the same for aldosterone production and BP,
whereas serum K changed in the opposite direction. In the cross-sectional study, associations were
observed only in AA. Combining the two studies, one SNP in particular, rs888345, was strongly
associated with BP in AA and with indices of aldosterone production in AA and EA.

Conclusion: Results of an exploratory study suggest that BP and aldosterone production may be
affected by variations in KCNK9. The findings could have relevance to risk for hypertension. (J Clin
Endocrinol Metab 97: E2160 –E2167, 2012)

ldosterone stimulates reabsorption of sodium (Na)
A and secretion of potassium (K) in the kidney’s distal
nephron (1). These separate although interrelated actions
of aldosterone serve to, respectively, defend against vol-
ume depletion due to Na loss and prevent hyperkalemia.
The regulation of aldosterone production likely evolved
during an early ancestral period when diets contained little
Na and a surfeit of K. Today, aldosterone production may
exceed what is required for achieving Na and K homeo-
stasis, becoming instead pathogenic by participating in the
development of hypertension. Indeed, approximately
10% of individuals with hypertension have primary aldo-
steronism, the consequence of either adrenal tumor devel-
opment or adrenal hyperplasia (2), and 20% or more of
those with resistant hypertension [high blood pressure
(BP) refractory to multiple medications] have either pri-
mary aldosteronism (3) or normal aldosterone levels but
respond to treatment with antagonists of aldosterone (4).

ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2012 by The Endocrine Society
doi: 10.1210/jc.2012-2196 Received May 10, 2012. Accepted July 24, 2012.
First Published Online August 14, 2012
Abbreviations: AA, African-American; ARR, aldosterone to renin ratio; BMI, body mass
index; BP, blood pressure; EA, European-American; GCRC, General Clinical Research Cen-
ter; LD, linkage disequilibrium; MAF, minor allele frequency; PAC, plasma aldosterone
concentration; PRA, plasma renin activity; SNP, single-nucleotide polymorphism; TASK1,
TWIK-related acid-sensitive K⫹ channel 1; ZG, zona glomerulosa.

E2160 jcem.endojournals.org J Clin Endocrinol Metab, November 2012, 97(11):E2160 –E2167

Downloaded from https://academic.oup.com/jcem/article-abstract/97/11/E2160/2836322

by Universidad Autonoma de Madrid user
on 24 July 2018
 J Clin Endocrinol Metab, November 2012, 97(11):E2160 –E2167
Moreover, among individuals without hypertension, a
higher but normal plasma aldosterone concentration
(PAC) was shown to be predictive of an elevation in BP
within 4 yr (5). If indeed, given today’s dietary practices,
aldosterone production becomes relatively dissociated
from a need to retain Na, then identification of other de-
terminants of aldosterone production would make an im-
portant contribution to an understanding of the causes of
hypertension. In an earlier study, we demonstrated a her-
itability of aldosterone excretion rates (6), suggesting that
genetic studies could reveal important additional deter-
minants of human hyperaldosteronism.
Two pore domain K channel subunits, TWIK-related
acid-sensitive K⫹ channel 1 (TASK1) and TASK3 (en-
coded by KCNK3 and KCNK9, respectively) are promi-
nently expressed on the plasma membrane of rodent cell
zona glomerulosa (ZG) (7, 8). The subunits form homo- or
heterodimeric leak K channels and are important ionic
conductances that restrain electrical excitability and limit
the production of aldosterone. Extracellular K within the
physiological range of 3–5 mM acts both independently (9)
and synergistically with angiotensin II (10) to stimulate
aldosterone production in ZG cells. Its principle mecha-
nism of action is to depolarize the membrane potential of
the ZG cell, which permits the opening of voltage-depen-
dent calcium channels and the consequent increase of ex-
tracellular calcium entry that is essential for sustaining the
production of aldosterone. Indeed, the exquisite sensitiv-
ity of aldosterone production in vivo to incremental
changes in serum K (0.5–1.0 mmol/liter) (9) is a direct
measure of the strong dependence of the membrane po-
tential of the ZG cell on K permeability pathways. Thus,
modulation of K channel activity is a powerful means for
controlling the production of aldosterone. Deletion of
Kcnk3 in the mouse results in disruption of adrenal zo-
nation with hyperaldosteronism and elevated BP (11). The
tandem inactivation of Kcnk3 and Kcnk9 genes from the
mouse results in a 20-mV depolarization of the ZG cell
plasma membrane and a phenotype that closely resembles
human idiopathic primary aldosteronism: elevated aldo-
sterone levels, low renin levels, an increased aldosterone to
renin ratio (ARR), failure to suppress aldosterone produc-
tion with high salt or normalize with candesartan, and
hypertension (12). Thus, in the present study, we explored
the genetic association of single-nucleotide polymor-
phisms (SNP) in KCNK3 and KCNK9 with BP and indices
of aldosterone production in young normotensive subjects
that had over a period of years repeated measurements of
BP and had cross-sectional assessments of aldosterone
production.
Downloaded from https://academic.oup.com/jcem/article-abstract/97/11/E2160/2836322
by Universidad Autonoma de Madrid user
on 24 July 2018
jcem.endojournals.org E2161
Subjects and Methods
Subjects and study design
All subjects were healthy (normotensive) and either European
American (EA) or African American (AA). They were partici-
pants in a longstanding cohort study of BP regulation (13). Race
was based on the subject’s self-report. There were two groups
that differed from each other by the study protocols employed.
The first group consisted of individuals who had measurements
made longitudinally in an outpatient setting (13). The second
group consisted of individuals who had measurements made in
an inpatient facility [General Clinical Research Center (GCRC)]
cross-sectionally. They are here referred to as the longitudinal
study and the cross-sectional study. In the longitudinal study,
444 EA and 351 AA had measurements made over the age range
of 5.6 –25 yr. In the cross-sectional study, there were 106 AA and
85 EA who ranged in age from 18 –36 yr. The latter group had
been participants in an inpatient study of racial differences in Na
reabsorption (14); only data collected at baseline were used in the
present study. Nineteen subjects participated in both the longi-
tudinal study and the cross-sectional study. The studies were
approved by the Indiana University-Purdue University of Indi-
anapolis Institutional Review Board. Each subject provided con-
sent or assent with a parent or guardian providing informed
consent.
Measurements
In the longitudinal study, weight, height, and BP were mea-
sured every 6 months, in the morning, either at the school they
attended or in the GCRC. Blood samples were collected after
subjects had been in the sitting position for 10 min. After an
additional 10 min of sitting, BP was measured in the right arm
three times (with intervals of 2 min between readings) using a
random zero sphygmomanometer (Hawksley and Sons, Lanc-
ing, West Sussex, UK); the average of the last two readings was
used in the analyses. The number of measurements ranged from
two to 27 per subject with a mean of 16. Plasma renin activity
(PRA), PAC, and serum K were measured once but in some cases
twice.
In the cross-sectional study, subjects were admitted to the
GCRC the afternoon of the day before when measurements were
made. They consumed a standardized diet for dinner and evening
snack and for breakfast the following morning. After breakfast,
subjects were supine for 1 h before measurements were made and
blood samples drawn. BP was measured three times (2 min be-
tween readings) in the right arm using a Dynamap Pro Series 300
machine.
Assay procedures
K was measured using a COBAS MIRA analyzer (Roche Di-
agnostics, Indianapolis, IN); PRA was measured using a RIA for
generated angiotensin I (Clinical Assays, GammaCoat RIA kit
from Diasorin, Stillwater, MN); and PAC was measured by RIA
(Coat-A-Count kit from Diagnostic Products Corp., Los Ange-
les, CA). The intraassay and interassay coefficients of variation
were, respectively, for PRA, 4.6 and 7.6%, and for PAC, 5.4 and
13.1%.
SNP selection and genotyping
KCNK3 spans 40 kb on chromosome 2p23, and KCNK9
spans 92 kb on chromosome 8q24.3. Tagging SNP of KCNK3
 E2162 Jung et al. Potassium Channel Genes, BP, and Aldosterone J Clin Endocrinol Metab, November 2012, 97(11):E2160 –E2167

TABLE 1. Characteristics of subjects at time of enrollment in the longitudinal study group (mean ⫾ SD)
Males Females
Characteristic EA (n ⴝ 229) AA (n ⴝ 150) P value EA (n ⴝ 215) AA (n ⴝ 201) P value
Age (yr) 14.0 ⫾ 4.1 14.7 ⫾ 4.2 0.71 14.5 ⫾ 4.4 15.0 ⫾ 4.7 0.52
BMI (kg/m2) 20.9 ⫾ 4.8 23.8 ⫾ 6.2 ⬍0.0001 21.2 ⫾ 4.6 24.5 ⫾ 6.9 ⬍0.0001
SBP (mm Hg) 107.1 ⫾ 12.7 109.4 ⫾ 12.5 ⬍0.0001 102.8 ⫾ 10.1 105.2 ⫾ 10.2 ⬍0.0001
DBP (mm Hg) 62.2 ⫾ 10.7 64.1 ⫾ 10.8 ⬍0.0001 61.7 ⫾ 9.7 62.9 ⫾ 9.7 0.0004
PAC (ng/dl) 14.0 ⫾ 9.0 8.8 ⫾ 6.5 0.0001 14.7 ⫾ 9.6 9.2 ⫾ 6.8 0.0001
PRA (ng/liter 䡠 sec) 3.3 ⫾ 2.3 2.6 ⫾ 2.0 0.002 3.3 ⫾ 2.5 2.8 ⫾ 2.1 0.0008
ARR 5.30 ⫾ 3.6 4.9 ⫾ 6.8 0.001 5.8 ⫾ 4.5 5.1 ⫾ 7.9 0.001
K (mmol/liter) 4.2 ⫾ 0.4 4.3 ⫾ 0.7 0.88 4.2 ⫾ 0.4 4.3 ⫾ 1.0 0.32
DBP, Diastolic BP; SBP, systolic BP.

and KCNK9 were selected from Single Nucleotide Polymor-
phism Database (http://www.ncbi.nlm.nih.gov/projects/SNP/)
using the criteria of R2 ⫽ 80% and minor allele frequency over
10% based on HapMap YRI (Yoruba from Nigeria) data. Sev-
enty-four tagging SNP of KCNK9 and 15 of KCNK3 were se-
lected. All SNP were in Hardy-Weinberg Equilibrium (P value
⬎0.05). SNP location was determined from the annotations in
the NCBI human genome assembly Build 36.2.
Genotyping was carried out using a modified single-nucle-
otide extension reaction with allele detection by mass spectrom-
etry (Sequenom MassArray system; Sequenom, San Diego, CA).
The assays were designed and run with either of two formats,
hME or iPLEX. The genotyping success rate for each genotype
was over 95%, and minor allele frequencies were over 10%. SNP
coverage across both genes was evaluated using the program
Haploview version 4.2, which examines the extent of linkage
disequilibrium (LD) between pairs of SNP to estimate how well
the selected SNP represented the genetic information contained
in nongenotyped SNP.
Quality control and population stratification
Due to potential differences in minor allele frequencies and
LD patterns, EA and AA were studied separately. We genotyped
57 ancestry-informative SNP markers (12, 13) 10 on the X-chro-
mosome to correct for population stratification and exclude in-
dividuals who were misclassified as to ancestry. In addition, we
performed principal component analysis on the ancestry-infor-
mative SNP markers using EIGENSTRAT and obtained the first
10 eigenvectors.
Statistical analysis
Longitudinal study
All BP data, which spanned the ages 5–25 yr, were used in the
analyses. A semiparametric mixed-effect model for repeated
measurement analysis of the longitudinal data were used to test
for associations of each SNP with systolic and diastolic BP. The
model incorporates genetic (SNP) effects on BP as a fixed effect
at the population level and subject-specific growth curves of BP
level as random effects. Nonparametric growth curve models
with random subject effects were fitted using penalized splines,
and additive genetic effects were tested after adjusting for sex,
age, body mass index (BMI), and four principal component
scores (15, 16) for accommodation of subpopulation differences.
The code for these analyses was written using R software (version
2.13.1). For biochemical measurements such as PRA, PAC,
ARR, and serum K, we used a parametric mixed-effect model
with a random effect for data analysis. Genetic variations were
included in the models of main factors of interest. We adjusted
PAC for PRA and serum K as well as for sex, age, BMI, and
principal component score (reflecting the magnitude of popula-
tion stratification for each subject). The mixed-effect model anal-
ysis was implemented using SAS PROC MIXED (version 9.2).
Many of the SNP were in LD, and thus, we estimated a gene-wide
significance threshold using Nyholt’s method (17) that takes into
account LD among SNP and calculates an effective number of
independent tests for a given gene. The estimated significance
thresholds for KCNK9 that took into account the multiplicity of
comparisons was P ⫽ 0.0011 for AA and 0.0014 for EA.

TABLE 2. Characteristics of subjects in cross-sectional study group (mean ⫾ SD)

Males Females
Characteristic EA (n ⴝ 38) AA (n ⴝ 49) P value EA (n ⴝ 47) AA (n ⴝ 60) P value
Age (yr) 22.3 ⫾ 3.8 22.8 ⫾ 4.0 0.6 23.5 ⫾ 4.6 24.0 ⫾ 4.9 0.6
BMI (kg/m2) 25.4 ⫾ 4.5 26.9 ⫾ 5.6 0.17 25.9 ⫾ 5.4 30.8 ⫾ 8.6 0.0004
SBP (mm Hg) 122.0 ⫾ 9.3 124.1 ⫾ 10.8 0.36 110.0 ⫾ 8.8 113.7 ⫾ 8.7 0.03
DBP (mm Hg) 68.1 ⫾ 6.6 67.9 ⫾ 6.7 0.88 65.0 ⫾ 7.1 64.2 ⫾ 5.4 0.54
PAC (ng/dl) 9.0 ⫾ 4.5 5.2 ⫾ 3.1 0.0002 8.3 ⫾ 5.3 5.9 ⫾ 3.4 0.0004
PRA (ng/liter 䡠 sec) 1.4 ⫾ 1.0 1.0 ⫾ 0.9 0.03 1.5 ⫾ 1.0 1.0 ⫾ 0.8 0.0002
ARR 9.4 ⫾ 6.8 12.6 ⫾ 21.9 0.67 7.0 ⫾ 6.0 12.0 ⫾ 13.4 0.03
K (mmol/liter) 4.0 ⫾ 0.2 4.2 ⫾ 0.4 0.01 4.0 ⫾ 0.3 4.1 ⫾ 0.3 0.47
DBP, Diastolic BP; SBP, systolic BP.

Downloaded from https://academic.oup.com/jcem/article-abstract/97/11/E2160/2836322

by Universidad Autonoma de Madrid user
on 24 July 2018
 J Clin Endocrinol Metab, November 2012, 97(11):E2160 –E2167
FIG. 1. Pairwise LD structure of KCNK9 (R2). Top panel, AA LD; bottom panel, EA LD. The
KCNK9 gene structure is illustrated in the middle in scale; the black box represents exons; the
arrow represents the direction of transcription. Red dots indicate the locations of nine SNP
significantly associated with systolic BP in AA in the longitudinal study.
Cross-sectional study
There were nine AA sib pairs and 15 EA sib pairs. No signif-
icant correlation of phenotypes between siblings was observed
based on the Spearman correlation test, and thus data collected
from siblings were used in the analyses. A mixed-effect model
was employed to test for additive genetic effects of each SNP with
PRA, PAC, ARR, and serum K after adjusting for sex, BMI, and
age including principle component scores as well as relevant phe-
notypes described above in Longitudinal study. Logarithms of
PRA, PAC, and ARR were used because normality assumptions
were not met.
Results
Characteristics of subjects
In the longitudinal study (Table 1), AA were heavier
(P ⬍ 0.0001) and had higher BP (P ⬍ 0.0001) than EA.
PRA, PAC, and ARR were lower in AA (P ⬍ 0.0001 to
0.002). For subjects in the cross-sectional study (Table 2),
female AA had higher BMI (P ⬍ 0.0001) and higher sys-
tolic BP (P ⫽ 0.03) than female EA, and again PRA and
PAC were lower in AA than in EA (P ⫽ 0.0002 to 0.03),
but ARR was higher in AA than in EA (P ⫽ 0.03). Male AA
had a higher serum potassium level than male EA (4.2 vs.
4.0; P ⬍ 0.01).
Association studies using individual SNP
The pattern of LD differed between AA and EA for both
KCNK3 and KCNK9 (Fig. 1). For KCNK9, EA have a
large LD block consisting of 18 SNP (from rs7833765 to
rs1619829, R2 ⬎ 70%, D⬘ ⬎80%, where D⬘ is the mag-
nitude of linkage disequilibrium between two SNPs) as
well as five pair-wise LD blocks. By contrast, AA had a
Downloaded from https://academic.oup.com/jcem/article-abstract/97/11/E2160/2836322
by Universidad Autonoma de Madrid user
on 24 July 2018
jcem.endojournals.org E2163
smaller LD block containing four pair-
wise LD blocks. Because of the LD, the
74 genotyped SNP provided information
across the entire gene region of KCNK9,
including 10 kb upstream and 10 kb
downstream of the coding region. The
average R2 for CEU (Utah residents with
Northern and Western European ances-
try) was 0.976, capturing 83% (60 of 72)
of known HapMap alleles with minor al-
lele frequency (MAF) higher than 0.1 in
the region at R2 higher than 0.7. The av-
erage R2 for YRI (Yoruba in Ibadan, Ni-
geria) was 0.938, capturing 67% (53 of
78) of known HapMap alleles with
MAF higher than 0.1 at R2 higher
than 0.5. The 15 SNP of KCNK3 cap-
tured a similar amount of the varia-
tion for CEU (nine SNP capturing
89%) and YRI (11 SNP capturing
65%). Because a number of SNP we
genotyped were not in the HapMap database (HapMap
Data Phase III), the actual coverage of the region with
SNP is underestimated.
We found no association for any SNP in KCNK3 with
BP or with any of the indices of aldosterone production in
either AA or EA, and thus, only results of association stud-
ies with KCNK9 are reported in detail. The P values of
individual SNP that showed associations with KCNK9 are
presented in Tables 3 and 4. The direction of the arrow
accompanying the superscript nucleotide depicts either an
increase or decrease in the measured parameter.
Longitudinal study
We found associations of several SNP with each of the
phenotypes measured, although often an association was
found only in EA or only in AA (Table 3). Nine SNP in
KCNK9 were associated with systolic BP in AA (in four
instances, the SNP associated with a lower BP); none of
them associated with BP in EA. An additional SNP was
marginally associated (rs13256087, P ⫽ 0.04). Two of
those nine SNP were also associated with diastolic BP in
AA, along with two other SNP; in EA, the single SNP
associated with systolic BP was also associated with dia-
stolic BP. In AA, rs888345 showed an association with all
five measures. In contrast to AA where associations were
primarily with BP, the associations in EA were related
primarily to parameters of aldosterone production, pri-
marily PAC, ARR, and serum K. Among the five SNP
associated with PAC in EA, four were also associated with
ARR.
 E2164 Jung et al. Potassium Channel Genes, BP, and Aldosterone J Clin Endocrinol Metab, November 2012, 97(11):E2160 –E2167

TABLE 3. Associations of selected SNPs in KCNK9 in subjects from the longitudinal study group (P values)
AA (n ⴝ 351) EA (n ⴝ 444)
SNP NT MAF SBP DBP PAC ARR Serum K MAF SBP DBP PAC ARR Serum K PRA
rs2542424 C/T 0.30 0.07 0.026C1 0.20 0.12 0.56 0.44 0.05 0.09 0.16 0.74 0.87 0.02C2
rs2545462 T/G 0.47 0.02T1 0.11 0.12 0.64 0.08 0.40 0.33 0.21 0.92 0.67 0.66 0.23
rs888345 G/A 0.15 0.009G2 0.02G2 0.01G2 0.003G2 0.02G1 0.20 0.29 0.15 0.005G2 0.009G2 0.56 0.19
rs13256087 A/G 0.27 0.90 0.91 0.70 0.40 0.56 0.37 0.04G1 0.01G1 0.37 0.46 0.58 0.50
rs1469039 A/G 0.19 0.03A2 0.26 0.99 0.87 0.22 0.17 0.78 0.87 0.0009A2 0.0005A2 0.53 0.15
rs2545460 A/G 0.39 0.24 0.026A2 0.87 0.89 0.15 0.47 0.73 0.52 0.10 0.03A2 0.76 0.04A1
rs12114521 A/G 0.49 0.02A2 0.11 0.89 0.46 0.62 0.04 0.43 0.98 0.01A2 0.08 0.31 0.82
rs3780045 T/C 0.13 0.01T1 0.05 0.50 0.05 0.38 0.03 0.07 0.58 0.93 0.71 0.22 0.31
rs3824281 A/G 0.33 0.004A1 0.10 0.51 0.23 0.05 0.06 0.45 0.87 0.01G1 0.010G1 0.54 0.73
rs7004779 T/C 0.15 0.31 0.63 0.16 0.11 0.01T2 0.03 0.92 0.29 0.006T1 0.004T1 0.27 0.15
rs3780037 T/G 0.30 0.01T1 0.02T1 0.86 0.91 0.02T1 0.23 0.11 0.17 0.73 0.29 0.04T2 0.46
rs10110946 C/T 0.23 0.01C1 0.11 0.73 0.91 0.11 0.24 0.10 0.14 0.77 0.36 0.03C2 0.50
rs7828107 A/C 0.21 0.004A2 0.83 0.49 0.09 0.29 0.49 0.11 0.37 0.50 0.38 0.01A1 0.30

Superscript letters depict nucleotides associated with risk alleles; direction of the arrow depicts whether level of phenotype increases or decreases.
DBP, Diastolic BP; NT, nucleotides with minor allele followed by major allele; SBP, systolic BP.

Cross-sectional study rs888345 was approximately 1.5 mm Hg with two copies

In this study group, significant associations were
observed only in AA (Table 4). Five of nine SNP that
associated with systolic BP in AA from the longitu-
dinal study also associated with systolic BP in subjects
in the cross-sectional study (rs888345, rs1469039,
rs3780037, rs10110946, and rs7828107). The direc-
tion of the change in BP for all five SNP was the same in
the two study groups. Five SNP (rs4736287, rs741463,
rs1469039, rs2542481, and rs888349) associated with
serum K, four of which also associated with BP. Five SNP
(rs741463, rs13256087, rs882555, rs3780037, and
rs10110946) showed associations with PAC and/or ARR.
Because the study protocol for the cross-sectional study
included collection of urine samples, urinary K was also
analyzed. Two SNP showed the direction of the associa-
tion with urinary K excretion to be the same as the change
in systolic BP (rs3780037 and rs10110946).
Effect size on BP
Effect size was estimated separately in EA and AA using
a semiparametric mixed-effect model unadjusted for pop-
ulation stratification. The effect size for systolic BP of
of the risk allele (homozygous for the risk allele, A), com-
pared with homozygous and heterozygous groups of the
nonrisk allele, G. The effect size of rs888345 on diastolic
BP was approximately 2–3 mm Hg with at least one copy
of the risk allele.
Gene interactions
Although we observed no associations with KCNK3 in
the current study, deletion of both Kcnk3 and Kcnk9 from
the mouse genome resulted in a greater state of hyperal-
dosteronism than removal of either gene alone. We there-
fore looked for evidence of a genetic interaction that might
enhance associations with BP, serum K, PAC, or ARR. In
an exploratory analysis, we tested whether certain poly-
morphisms in KCNK9 might permit and/or augment the
phenotypic expression of genetic variations in KCNK3.
The three SNP in KCNK9 that showed the most significant
associations (rs888345, rs3780037, and rs3824281) and
the SNP in KCNK3 were studied. The analysis was limited
to AA. Using an additive interaction model, we found that
rs34292597 in KCNK3 and rs888345 and rs3780037 in
KCNK9 were associated with serum K interactively (P ⬍

TABLE 4. Associations of selected SNP in KCNK9 in subjects from the cross-sectional study group (P values)
AA (n ⴝ 106) EA (n ⴝ 83)
SNP NT SBP DBP PAC ARR Serum K Urine K SBP DBP PAC ARR Serum K Urine K
rs4736287 G/C 0.01G1 0.06 0.40 0.53 0.04G2 0.83 0.94 0.95 0.22 0.96 0.28 0.38
rs2545432 A/G 0.01A1 0.01A1 0.37 0.89 0.13 0.90 0.70 0.62 0.46 0.34 0.03 0.12
rs741463 C/G 0.96 0.62 0.25 0.01C2 0.04C1 0.94 0.08 0.55 0.77 0.60 0.94 0.36
rs888345 G/A 0.03G2 0.10 0.67 0.57 0.82 0.32 0.85 0.84 0.92 0.05 0.26 0.44
rs13256087 A/G 0.46 0.95 0.04A1 0.006A1 0.08 0.72 0.13 0.67 0.85 0.46 0.90 0.16
rs1469039 A/G 0.04A2 0.53 0.97 0.56 0.03A2 0.35 0.76 0.69 0.98 0.09 0.20 0.34
rs882555 T/C 0.57 0.32 0.02T2 0.17 0.50 0.04T2 0.98 0.40 0.06 0.79 0.71 0.07
rs2542481 A/G 0.03A2 0.67 0.27 0.15 0.01A1 0.30 0.21 0.52 0.67 0.66 0.85 0.99
rs888349 G/T 0.04G2 0.39 0.50 0.38 0.02G2 0.39 0.59 0.27 0.22 0.67 0.33 0.05
rs3780037 T/G 0.02T1 0.29 0.45 0.23 0.89 0.005T1 0.69 0.68 0.19 0.22 0.37 0.42
rs10110946 C/T 0.02C1 0.09 0.20 0.10 0.13 0.01C1 0.53 0.61 0.53 0.19 0.95 0.35
rs7828107 A/C 0.02A2 0.27 0.22 0.35 0.36 0.73 0.83 0.29 0.95 0.56 0.53 0.69

Superscript letters depict nucleotides associated with risk alleles; direction of the arrow depicts whether level of phenotype increases or decreases.
DBP, Diastolic BP; NT, nucleotides with minor allele followed by major allele; SBP, systolic BP.

Downloaded from https://academic.oup.com/jcem/article-abstract/97/11/E2160/2836322

by Universidad Autonoma de Madrid user
on 24 July 2018
 J Clin Endocrinol Metab, November 2012, 97(11):E2160 –E2167
0.005), whereas rs34292597 alone was not. In addition,
there was evidence to suggest that any of eight SNP in
KCNK3 and rs3824281 in KCNK9 interacted to enhance
associations with BP, serum K, PAC, and ARR. Thus,
there was suggestive evidence for an interaction of the two
channel subunits in humans.
Additive effects
To look for additive effects, we incorporated the nine
SNP into a single model as additive terms. In so doing we
found that two of the nine SNP remained significant:
rs3780037 (P ⫽ 0.0023) and rs7828107 (P ⫽ 0.046). The
remaining SNP appeared to function nonadditively. The
individual effect sizes of rs3780037 and rs7828107 were
1.7 and 2.8 mm Hg, respectively. We estimated the addi-
tive effect size of the two SNP when homozygous for the
risk alleles (TT for rs3780037 and CC for rs7828107)
would be 3.8 mm Hg higher than those heterozygous (GT
for rs3780037 and AC for rs7828107). The heterozygous
group was estimated to be 3.5 mm Hg higher than the
group homozygous for the nonrisk alleles (GG for
rs3780037 and AA for rs7828107).
Discussion
Although it is widely accepted that BP variability, includ-
ing the development of hypertension, has a strong genetic
basis (6, 18), BP genes have been exceptionally elusive
(19). Most hypertension is accompanied by an exagger-
ated retention of Na (20). It therefore follows that genetic
contributions frequently encode for proteins involved
with the reuptake of Na in the kidney. What has become
increasingly apparent is that aldosterone with its Na-re-
taining properties participates in the pathogenesis of hy-
pertension including common forms (4, 5). Production of
aldosterone is highly sensitive to the transmembrane po-
tential of the ZG cells, a characteristic coupled to the ac-
tivity of K channels (21, 22). We report on translational
studies, taking findings in the mouse (deletion of Kcnk3
and Kcnk9 led to hyperaldosteronism and hypertension)
(12) and exploring those genes in human subjects. Our
sample sizes were not of optimal size, but this was miti-
gated in part by the refined phenotypic measurements and
the choice of young subjects. In the longitudinal study
group (n ⫽ 795), BP was measured biannually over a pe-
riod of years, and in the cross-sectional study group (n ⫽
189), assessments were carried out in the GCRC under
tightly controlled and standardized conditions.
We found common variations in KCNK9 that associ-
ated with BP and production of aldosterone. In mice, de-
letion of Kcnk9 alone also produces hyperaldosteronism,
Downloaded from https://academic.oup.com/jcem/article-abstract/97/11/E2160/2836322
by Universidad Autonoma de Madrid user
on 24 July 2018
jcem.endojournals.org E2165
albeit with a milder phenotype [aldosterone excretion
rates increased 37%, whereas they increased 400% in
Kcnk9/Kcnk3 double-knockout mice (23); BP also in-
creased more in the double knockout than in the Kcnk9].
In the present study, variations in KCNK3 showed no
associations; however, an interaction of the genes was sug-
gested in that selected SNP from each gene showed stron-
ger associations with BP, serum K, PAC, and ARR than did
individual SNP.
Associations were observed in both the longitudinal
and the cross-sectional study groups and in AA and EA
with P values ⬍0.05. We analyzed 74 SNP to capture most
of the variability in KCNK9, in keeping with the explor-
atory intent of the study. One SNP (rs1469039 in EA in the
longitudinal group) met the stringent threshold for signif-
icance given the multiple testing. Several others were nom-
inally significant and worth targeted exploration in other
samples.
The findings were also noteworthy in that individual
SNP associated with multiple phenotypes whose direc-
tional change was consistent with the known biology
(PAC and ARR increased or decreased as BP increased or
decreased with serum K going in the opposite direction; an
exception was rs3780037 in AA, Table 3). In the longi-
tudinal group, rs888345 showed associations with five
phenotypes in AA and two in EA. Additionally, six SNP
that showed an association in the longitudinal study group
showed an association in the cross-sectional study. There
were, therefore, elements of replication when one consid-
ers associations with the same SNP in two separate race
groups as well as in separate study groups. These inter-
nally and externally consistent findings suggest that the
associations go beyond simply mere coincidence, despite
their only nominal significance given the low sample size.
The observations should be tested in more directed future
studies.
In the longitudinal study group, where BP had been
measured repeatedly over time and thus from the sheer
numbers of observations could be more informative, mul-
tiple SNP in KCNK9 showed an association with BP in AA
(nine SNP associated with systolic BP) (Table 3). At the
same time, there was no similar degree of association with
aldosterone production. However, levels of aldosterone
(and PRA) were measured cross-sectionally with fewer
overall actual assessments, and thus as phenotypes, they
may have been less robust. In addition, because BP in AA
is characteristically salt sensitive (24), minor increases in
Na reabsorption from small differences in the level of al-
dosterone may have been enough to increase BP. Evidence
for a genetic influence on aldosterone production was, on
the other hand, apparent in EA but without a comparable
change in BP. In this case, BP may simply have been less
 E2166 Jung et al. Potassium Channel Genes, BP, and Aldosterone
responsive to the Na retention evoked by aldosterone. In
the cross-sectional study, no SNP in EA associated with
any of the phenotypes. The findings are in keeping with
AA residing closer to a threshold wherein only a small
increment in Na uptake may be sufficient to affect BP.
Although the genetic variations in KCNK9 reported
here in healthy individuals are not contained within the
coding sequences of the gene, it is possible that they are in
LD with coding variants or that intronic variants modify
the expression of TASK3 subunits to produce changes in
the K conductance of the ZG cell. If the reduction in K
conductance is small, the baseline membrane potential of
the ZG cell may not change significantly and the expressed
phenotype, aldosterone production, may be normal.
However, a reduced K conductance would render the ZG
cell more susceptible to depolarizing influences (such as
angiotensin II and ACTH) and, thus, we predict may mod-
estly amplify responses to physiological concentrations of
aldosterone secretagogues and thus increase the risk for
hypertension. By contrast, mutations in the coding region
of KCNJ5 channels found in a subset of patients with
tumorigenic primary aldosteronism that change the ion
selectivity of the channel pore itself are responsible for
large overproduction of aldosterone observed in some pa-
tients with severe primary aldosteronism (25). The same
group also found KCNK9 and KCNK3 expressed in al-
dosterone-producing adrenal adenomas (25).
Our subjects were healthy and for the most part too
young to have hypertension. Although this could be con-
sidered a limitation, at the same time, it provided the ad-
vantage of having eliminated the confounding influences
that accompany age and/or the hypertensive state, includ-
ing the attendant use of antihypertensive medications. Al-
though genetic associations were observed in AA and EA,
providing some measure of replication, confirmation of
our findings in other populations will be necessary to fully
establish a role for KCNK9 in causing BP variability.
In summary, genetically derived differences in the ac-
tions of a K channel that is expressed in ZG cells and that
normally functions to restrain aldosterone production
showed associations with BP and level of plasma aldoste-
rone in this exploratory study. The findings will hopefully
encourage additional testing of associations in other pop-
ulation groups.
Acknowledgments
We greatly appreciate the excellent technical support provided
by Mary Anne Wagner.
Downloaded from https://academic.oup.com/jcem/article-abstract/97/11/E2160/2836322
by Universidad Autonoma de Madrid user
on 24 July 2018
J Clin Endocrinol Metab, November 2012, 97(11):E2160 –E2167
Address all correspondence and requests for reprints to: J.
Howard Pratt, M.D., CL365, 541 Clinical Drive, Indianapolis,
Indiana 46202-5111. E-mail: johpratt@iupui.edu.
This work was supported by National Institutes of Health
Grants HL095086 (to W.T. and J.H.P.) and HL089717 (to
P.Q.B.) and the Indiana Clinical and Translational Science In-
stitute, by the Department of Veterans Affairs, by the Regenstrief
Institute, and by the Center for Medical Genomics at Indiana
University School of Medicine.
Disclosure Summary: The authors have nothing to disclose.
References
1. Meneton P, Loffing J, Warnock DG 2004 Sodium and potassium
handling by the aldosterone-sensitive distal nephron: the pivotal role
of the distal and connecting tubule. Am J Physiol Renal Physiol
287:F593–F601
2. Funder JW, Carey RM, Fardella C, Gomez-Sanchez CE, Mantero F,
Stowasser M, Young Jr WF, Montori VM 2008 Case detection,
diagnosis, and treatment of patients with primary aldosteronism: an
Endocrine Society clinical practice guideline. J Clin Endocrinol
Metab 93:3266 –3281
3. Calhoun DA, Jones D, Textor S, Goff DC, Murphy TP, Toto RD,
White A, Cushman WC, White W, Sica D, Ferdinand K, Giles TD,
Falkner B, Carey RM 2008 Resistant hypertension: diagnosis, eval-
uation, and treatment: a scientific statement from the American
Heart Association Professional Education Committee of the Council
for High Blood Pressure Research. Circulation 117:e510 – e526
4. Nishizaka MK, Zaman MA, Calhoun DA 2003 Efficacy of low-dose
spironolactone in subjects with resistant hypertension. Am J Hyper-
tens 16:925–930
5. Vasan RS, Evans JC, Larson MG, Wilson PW, Meigs JB, Rifai N,
Benjamin EJ, Levy D 2004 Serum aldosterone and the incidence of
hypertension in nonhypertensive persons. N Engl J Med 351:33– 41
6. Manatunga AK, Reister TK, Miller JZ, Pratt JH 1992 Genetic in-
fluences on the urinary excretion of aldosterone in children. Hyper-
tension 19:192–197
7. Czirják G, Enyedi P 2002 Task-3 dominates the background potas-
sium conductance in rat adrenal glomerulosa cells. Mol Endocrinol
16:621– 629
8. Czirják G, Fischer T, Spät A, Lesage F, Enyedi P 2000 Task (twik-
related acid-sensitive K⫹ channel) is expressed in glomerulosa cells
of rat adrenal cortex and inhibited by angiotensin II. Mol Endocrinol
14:863– 874
9. Dluhy RG, Axelrod L, Underwood RH, Williams GH 1972 Studies
of the control of plasma aldosterone concentration in normal man.
II. Effect of dietary potassium and acute potassium infusion. J Clin
Invest 51:1950 –1957
10. Pratt JH 1982 Role of angiotensin II in potassium-mediated stim-
ulation of aldosterone secretion in the dog. J Clin Invest 70:667– 672
11. Heitzmann D, Derand R, Jungbauer S, Bandulik S, Sterner C,
Schweda F, El Wakil A, Lalli E, Guy N, Mengual R, Reichold M,
Tegtmeier I, Bendahhou S, Gomez-Sanchez CE, Aller MI, Wisden
W, Weber A, Lesage F, Warth R, Barhanin J 2008 Invalidation of
Task1 potassium channels disrupts adrenal gland zonation and min-
eralocorticoid homeostasis. EMBO J 27:179 –187
12. Davies LA, Hu C, Guagliardo NA, Sen N, Chen X, Talley EM, Carey
RM, Bayliss DA, Barrett PQ 2008 Task channel deletion in mice
causes primary hyperaldosteronism. Proc Natl Acad Sci USA 105:
2203–2208
13. Manatunga AK, Jones JJ, Pratt JH 1993 Longitudinal assessment of
blood pressures in black and white children. Hypertension 22:
84 – 89
14. Chun TY, Chander PN, Kim JW, Pratt JH, Stier Jr CT 2008 Aldo-
sterone, but not angiotensin II, increases profibrotic factors in kid-
 J Clin Endocrinol Metab, November 2012, 97(11):E2160 –E2167
ney of adrenalectomized stroke-prone spontaneously hypertensive
rats. Am J Physiol Endocrinol Metab 295:E305–E312
15. Ruppert D, Wand MP, Carroll RJ 2003 Semiparametric regression.
Cambridge, UK: Cambridge University Press
16. Durbán M, Harezlak J, Wand MP, Carroll RJ 2005 Simple fitting of
subject-specific curves for longitudinal data. Stat Med 24:1153–
1167
17. Li J, Ji L 2005 Adjusting multiple testing in multilocus analyses using
the eigenvalues of a correlation matrix. Heredity (Edinb) 95:221–
227
18. Ward R 1990 Familial aggregation and genetic epidemiology of
blood pressure. In: Laragh JH, Brenner BM, eds. Hypertension:
Pathophysiology, diagnosis, and management. New York: Raven;
81–100
19. Harrap SB 2003 Where are all the blood-pressure genes? Lancet
361:2149 –2151
20. Hall JE, Guyton AC 1990 Control of sodium excretion and arterial
pressure by intrarenal mechanisms and the renin-angiotensin sys-
tem. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiol-
Downloaded from https://academic.oup.com/jcem/article-abstract/97/11/E2160/2836322
by Universidad Autonoma de Madrid user
on 24 July 2018
jcem.endojournals.org E2167
ogy, diagnosis, and management. New York: Raven Press; 1105–
1129
21. Lotshaw DP 2001 Role of membrane depolarization and T-type
Ca2⫹ channels in angiotensin II and K⫹ stimulated aldosterone se-
cretion. Mol Cell Endocrinol 175:157–171
22. Aguilera G, Catt KJ 1986 Participation of voltage-dependent cal-
cium channels in the regulation of adrenal glomerulosa function by
angiotensin II and potassium. Endocrinology 118:112–118
23. Guagliardo NA, Yao J, Hu C, Schertz EM, Tyson DA, Carey RM,
Bayliss DA, Barrett PQ 2012 Task-3 channel deletion in mice re-
capitulates low-renin essential hypertension. Hypertension 59:999 –
1005
24. Weinberger MH, Miller JZ, Luft FC, Grim CE, Fineberg NS 1986
Definitions and characteristics of sodium sensitivity and blood pres-
sure resistance. Hypertension 8(6 Pt 2):II127–II134
25. Choi M, Scholl UI, Yue P, Björklund P, Zhao B, Nelson-Williams C,
Ji W, Cho Y, Patel A, Men CJ, Lolis E, Wisgerhof MV, Geller DS,
Mane S, Hellman P, Westin G, Åkerström G, Wang W, Carling T,
Lifton RP 2011 K⫹ channel mutations in adrenal aldosterone-pro-
ducing adenomas and hereditary hypertension. Science 331:768 –
772