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J Biol Chem, Vol. 273, Issue 34, 21542-21553, August 21, 1998
cDNA Cloning and Functional Characterization of the Mouse
Ca2+-gated K+ Channel, mIK1
ROLES IN REGULATORY VOLUME DECREASE AND ERYTHROID
DIFFERENTIATION*
David H.
Vandorpeabc,
Boris E.
Shmuklerabcd,
Lianwei
Jiangab,
Bing
Limbef,
James
Maylieg,
John P.
Adelmanh,
Lucia
de
Franceschii,
M. Domenica
Cappellinij,
Carlo
Brugnarakl, and
Seth L.
Alperabmn
From the a Molecular Medicine and Renal Units, Beth Israel
Deaconess Medical Center Boston, Massachusetts 02215, the
e Division of Hematology-Oncology, Beth Israel Deaconess Medical
Center and Harvard Institutes of Medicine, Boston, Massachusetts 02215, the g Department of Obstetrics and Gynecology and
h Vollum Institute, Oregon Health Sciences University, Portland,
Oregon 97201, the i Departments of Internal Medicine and General
Pathology, University of Verona, 37100 Verona, Italy, the
j Department of Internal Medicine, Maggiore Hospital IRCCS,
University of Milan, 20122 Milan, Italy, the k Department of
Laboratory Medicine, Children's Hospital, Boston, Massachusetts 02115, and the Departments of b Medicine, l Pathology, and
m Cell Biology, Harvard Medical School,
Boston, Massachusetts 02115
 |
ABSTRACT |
We have cloned from murine
erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding
the Ca2+-gated K+ (KCa)
channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia,
C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many
tissue types. mIK1 KCa channel activity expressed in
Xenopus oocytes closely resembled the Kca of
red cells (Gardos channel) and MEL cells in its single channel
conductance, lack of voltage-sensitivity of activation, inward
rectification, and Ca2+ concentration dependence. mIK1 also
resembled the erythroid channel in its pharmacological properties,
mediating whole cell and unitary currents sensitive to low
nM concentrations of both clotrimazole (CLT) and its
des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to
low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel,
Ca2+-dependent, CLT-sensitive regulatory volume
decrease response. Hypotonic swelling of voltage-clamped
mIK1-expressing oocytes increased outward currents that were
Ca2+-dependent, CLT-sensitive, and reversed
near the K+ equilibrium potential. mIK1 mRNA levels in
ES cells increased steadily during erythroid differentiation in
culture, in contrast to other KCa mRNAs examined. Low
nanomolar concentrations of CLT inhibited proliferation and erythroid
differentiation of peripheral blood stem cells in liquid culture.
 |
INTRODUCTION |
Terminal differentiation of erythroid precursor cells is marked by
enucleation and reduction in cell volume. A major component of cell
volume reduction is achieved by reduction of cell K+
content. Mature, circulating erythrocytes retain two major ion transport pathways mediating K+ efflux (1). These are: 1)
electroneutral K-Cl cotransport and 2) a voltage-insensitive,
Ca2+-activated potassium (K+) channel of
intermediate conductance (2-4), also known as the Gardos channel (5).
The Gardos channel is thought to play a major role in volume regulation
in normal (6) and sickle
(SS)1 human erythrocytes (7,
8).
Especially in the chronically hypoxic environment of adherent or
trapped sickle cells, the Gardos channel appears to mediate the major
component of K+ loss from the erythrocyte (9), leading to
an increased concentration of intracellular hemoglobin S, and
exponentially decreasing the lag time for accelerated hemoglobin S
polymerization (10). The Gardos channel's biophysical and
pharmacological properties have been characterized in excised
inside-out human red cell membrane patches, in which
Ca2+-activated K (KCa) currents show inwardly
rectifying properties with a unitary slope conductance ranging from 15 to 40 picosiemens, depending on the ionic conditions used (11-13). The
channel is sensitive to block by charybdotoxin (14-16), but
insensitive to the SK channel blocker, apamin, and to the
KATP channel blockers, the antihypoglycemic drugs
(17).2
Sickle cell disease is a lifelong illness in which severity varies for
poorly understood reasons not only among but within families, and even
during an individual patient's clinical course. It is very likely that
the pathological consequences of the autosomal recessive hemoglobin S
mutation underlying sickle cell disease (18) are influenced by the
polypeptide products of many yet undefined modifier genes. The central
role of the erythroid KCa channel in SS red cell
dehydration has suggested it as a strong candidate modifier gene in
sickle cell disease. We have hypothesized that KCa channel
blockade could serve as a useful adjunct therapy of sickle cell disease
(7, 8, 16, 19).
A subset of antifungal imidazole drugs was found potently to inhibit
K+ and Rb+ flux in normal and SS human red
blood cells. Clotrimazole was the most potent of those tested (7, 20),
blocking calcium ionophore A23187-induced 86Rb+
influx and displacing 125I-ChTX binding to red cells with
equivalent ID50 values of ~30 nM. The
combined results of 86Rb flux and 125I-ChTX
binding studies led to the proposal that CLT inhibited Ca2+-activated K transport by direct binding to the
external surface of the Gardos channel (7), in contrast to earlier (20)
and continuing suggestions (21) that CLT blocks K+
conductance via its inhibitory effects on cytochrome P450 enzymes. We
subsequently showed that an inwardly rectifying KCa channel from murine erythroleukemia (MEL) cells (22) was inhibited directly not
only by charybdotoxin and CLT, but also by the major des-imidazolyl metabolite of CLT, 2-chlorophenyl-bisphenyl-methanol, incapable of
inhibiting cytochromes P-450 (16).
The potency of CLT blockade of the erythroid KCa channel
and the status of CLT as a drug already long in clinical use for other
indications recommended consideration of CLT as an erythroid KCa channel blocker for adjunct therapy of sickle cell
disease (19). Indeed, oral administration of CLT was shown to inhibit the erythroid KCa channel in vivo, and to
diminish formation of dehydrated dense cells in a mouse model of sickle
cell disease (23) and in a phase I clinical trial with sickle cell
disease patients (8). More recently, a complementary approach to
prevention of red cell dehydration in patients with sickle cell disease
via inhibition of K-Cl cotransport (24) provided the first preliminary evidence for long term clinical benefit in a group of less stable patients (25), suggesting that therapeutic inhibition of the erythroid
KCa channel might yield similar (and possibly additive) clinical benefit.
Early attempts to clone the cDNA encoding the erythroid
KCa channel based on then-hypothesized homology to the
maxi-K (slo) family of KCa channels (26) were unsuccessful.
The subsequently cloned SK family of apamin-sensitive KCa
channels (27) also differed biophysically and pharmacologically from
the erythroid KCa channel. However, the SK-related human
IK1 channel more recently cloned by Ishii et al. (28) and
others (29, 30) has displayed many characteristics expected of the
erythroid KCa channel (2-4,7,11-17, 22).
In the present study, we have cloned the cDNA encoding mIK1, the
mouse homolog of hIK1 (28-30), and report the tissue distribution of
mIK1 mRNA, and the lack of N-glycosylation of in
vitro translated mIK1 polypeptide. We have functionally expressed
mIK1 in Xenopus oocytes, and studied its biophysical and
pharmacological properties at the levels of whole cell and unitary
currents. We have shown that hypotonic swelling of oocytes leads to
activation of mIK1 currents and to the novel volume regulatory response
of regulatory volume decrease (RVD). Both the currents and the volume
regulation are inhibited by CLT and require Ca2+. We have
also investigated the developmental profiles of mIK1 and other
K+ channel mRNAs in ES cells undergoing hematopoietic
differentiation along the erythroid lineage. Finally, we have
demonstrated that 10 nM CLT retards erythroid
differentiation of human peripheral blood stem cells, consistent with a
role of IK1 in this process.
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MATERIALS AND METHODS |
Inhibitors and Chemicals--
CLT was purchased from Sigma. The
CLT metabolite, 2-chlorophenyl-bisphenyl-methanol, was the kind gift of
R. Lombardy (Pharm-Eco Laboratories, Lexington, MA). Z1-Iodo-ChTX was
synthesized by the MIT Biopolymers Facility, using
N-Butoxycarbonyl-O-bromobenzyl-3-mono-iodo-L-tyrosine (Peninsula Laboratories, Belmont, CA) in place of tyrosine in position
36. The final product used was purified by high performance liquid
chromatography, confirmed by amino acid analysis, and by inhibition of
A23187-induced 86Rb influx into normal human red cells (15,
17). All salts were of analytical grade.
Reverse Transcriptase-Polymerase Chain Reaction
(RT-PCR)--
Total RNA from MEL cells and mouse tissues (freshly
resected kidney, stomach, spleen, distal colon, proximal colon, and
epididymis) was prepared using the RNeasy kit (Qiagen, Chatsworth, CA).
Total RNA from ES cells and ES-derived colonies was prepared using the RNAsol reagent (Biotecx, Houston, TX). A mouse tissue total RNA panel
(liver, brain, thymus, heart, testis, ovary, and embryo) was purchased
from Ambion (Austin, TX).
Reverse transcription was performed with the First Strand DNA Synthesis
Kit (Ambion) using 1 µg of total RNA. 5% of the reaction volume was
used for hot start PCR in a total reaction volume of 50 µl, using
either Taq DNA polymerase (Qiagen), Expand High Fidelity PCR
system (Boehringer Mannheim), or Taq DNA polymerase
(Promega, Madison, WI) in the suppliers' recommended buffers.
PCR mixes lacking only primers were preheated at 82 °C for 1 min,
after which appropriate primers (Table I) were injected into the mix
through mineral oil. The complete reaction mixes were denatured for 3 min at 95 °C, then cycled through these conditions: denaturation for
45 s at 94 °C, annealing for 2 min at 60 °C (or, as
indicated, in the presence of Q-solution (Qiagen), 52 °C), and
elongation for 2-3 min at 72 °C. Final extension of 10 min at
72 °C was terminated by rapid cooling to 4 °C after the indicated number of cycles. PCR products were separated in 1% agarose gels for
analysis and purified as necessary with the QIAquick Gel Extraction Kit
(Qiagen). Control amplification experiments were performed on RNA
samples in which reverse transcriptase was omitted.
DNA sequence analysis and data base searches were carried out with the
GCG suite of programs (University of Wisconsin Genetics Computing
Group, Madison, WI).
cDNA Cloning, Reconstruction, Transcription, and Translation
of mIK1--
With hIK1 sequence information (GenBank AF022150), four
degenerate oligonucleotides were designed (Table I), two corresponding to N- and C-terminal peptides (IK1.F, IK1.R) and two to internal peptide sequences of low degeneracy (IK1.IF, IK1.IR). With the combined
use of the Expand High Fidelity PCR system and of Q solution (Qiagen),
32 amplification cycles produced sufficient mIK1 cDNA for
sequencing and cloning.3
Overlapping PCR products for mIK1 were sequenced on both strands using
an ABI 373 DNA Sequencer, then cloned into the "T-vector" plasmid,
pCR 2.1 (Invitrogen, Carlsbad, CA).
Search of the EST data base identified 12 anonymous clones from mouse
embryo, lymph node, melanoma, and cultured myotubes that encoded parts
of mIK1: W30402, W45910, W79984, W82293, W99968, AA033013, AA042002,
AA140298, AA185916, AA185547, AA265296, and AA592555. These clones
helped verify nucleotide sequences encoding the N and C termini of mIK1 polypeptide. They also assisted in design of the primers IK1.5UTRF and
IK1.3UTRR (Table I), used for PCR
amplification of cDNAs from MEL cells and mouse stomach that
encompassed mIK1 initiator and terminator codon sequences. All PCR
products and their subclones were sequenced on both strands.
Subclones without mutations were selected to reconstruct the
full-length mIK1 polypeptide coding sequence between the
Xenopus -globin 5'- and 3'-untranslated regions in the
Xenopus oocyte expression vector, pXT7 (31).
EcoRI/BglII-cut pXT7 underwent a four-way
ligation with the following mIK1 cDNA fragments: the N-terminal
fragment ((vector-derived) EcoRI/MluI), the
central fragment (MluI/NcoI), and the C-terminal
fragment (NcoI/BamHI). The resulting recombinant
plasmid, pXmIK1, was linearized with XbaI to generate
transcription template for synthesis of capped cRNA from the T7
promoter (MEGAscript, Ambion).
In vitro translation of mIK1 from capped cRNA in the
presence of Tran35S-Label (ICN, Costa Mesa, CA) was
performed with the rabbit reticulocyte lysate (nuclease-treated) system
(Promega) in the presence or absence of canine pancreatic microsomal
membranes (Promega), according to the manufacturer's protocol.
N-Deglycosylation of in vitro-translated polypeptide with peptidyl-N-glycosidase F (PNGase F,
Promega), SDS-polyacrylamide gel electrophoresis, and autoradiography
were as described previously (32, 33).
Expression of mIK1 in Xenopus Oocytes--
Female
Xenopus laevis were purchased from NASCO (Madison, WI),
maintained at room temperature in running distilled water, and fed with
frog brittle (NASCO). Oocytes were manually defolliculated after
collagenase digestion of ovarian segments (34), then were microinjected
with water or with 10 ng mIK1 cRNA and incubated at 19 °C for 2-7
days in ND-96 with 2.5 mM sodium pyruvate and 500 µg/ml
gentamicin. ND-96 contained (in mM): 96 NaCl, 2 KCl, 1.8 MgCl2, 1 CaCl2, and 5 HEPES hemisodium, pH
7.40. All experiments with oocytes were conducted at 21 °C.
Two-microelectrode Studies--
Oocytes injected 2-7 days
previously with cRNA or with water were placed in a 1-ml chamber (Model
RC-11, Warner Instrument Corp, Hamden, CT) on the stage of a dissecting
microscope and impaled with microelectrodes under direct view.
Current-injecting and potential-sensing microelectrodes were pulled
from borosilicate glass with a Narishige puller, filled with 3 M KCl, and had resistances of 2-3 megohms. They were used
with a Geneclamp 500 amplifier (Axon Instruments, Foster City, CA)
interfaced to a Phoenix 386 computer with a TL-1 AD/DA board (Axon
Instruments). PCLAMP 6.0.3 software was used to control data
acquisition and to analyze data (Axon Instruments, Burlingame, CA). The
isotonic ND-96 solution was 212 mosM; hypotonic bath
solution (ND-70) was made by reducing [NaCl] from 96 to 70 mM, yielding an osmolarity of 160 mosM.
Membrane potentials and currents were expressed as means ± S.E.
Single-channel currents were recorded using standard techniques (35).
All voltages refer to the cell interior referenced to the patch
pipette. Currents were measured with a 10-gigohm headstage, low
pass-filtered at 1 kHz (Axopatch 1-D, Axon Instruments, Burlingame,
CA), digitized at 5 kHz, and stored on the hard drive of an Hewlett
Packard 486-DX computer. PCLAMP 6.0.3 software was used to control data
acquisition via a Digidata 1200 interface and to analyze data (Axon
Instruments, Burlingame, CA). Data were acquired continuously using
FETCHEX subroutines.
The pipette and bath solution compositions used in studies of
outside-out patches were as described (27). Inside-out or outside-out
recordings were made with symmetrical solutions containing 116 mM potassium gluconate, 4 mM KCl, 10 mM HEPES, adjusted to pH 7.2 with KOH. The intracellular
solution was supplemented with CaCl2 to give a free calcium
concentration of 5 µM (assuming a stability constant of
15.9 M 1 for calcium gluconate). To obtain
intracellular calcium concentrations below 1 µM, 1 mM EGTA was added to the bath solution and
CaCl2 was added as calculated using published stability
constants. After a cell-attached gigaseal patch was established, whole
cell configuration was attained by gentle suction. The outside-out
patch configuration was attained subsequently by slow withdrawal of the
micropipette from the cell body. Cell-attached patches were excised to
attain the inside-out configuration. A voltage ramp protocol (+100 mV to 100 mV, duration 2600 ms) was used to test sensitivity to activation by calcium ([Ca2+]i) in inside-out
patch configuration and sensitivity to inhibition by extracellular
antagonists in the outside-out patch configuration. Relative effects of
these agents (I/Io) were quantitated at a membrane potential
(Vm) of 100 mV. Absolute and relative currents
were expressed as means ± S.E. Values for inhibitor
ID50 were determined from best fit of the Langmuir isotherm
or Hill equation; ED50 and Hill coefficient for
Ca2+ were determined from best fit of the Hill equation
(28).
Control recordings were made for the generation of I-V plots and the
determination of NPo. NPo, the
product of N, the number of observed open channels, and
Po, the open probability, was calculated as follows.
|
(Eq. 1)
|
T was the record time, n was the number of
channels open, and tn was the time during which
n channels are open. Therefore, NPo can
be calculated without any assumptions about the total number of
channels in a patch or the open probability of a single channel.
NPo was calculated from 30-s records during control
and experimental periods, and expressed as means ± S.E.
Statistical analysis was by t test or ANOVA corrected by Bonferroni post-test.
Fluorescence Measurements of Oocyte Volume and Intracellular
Ions--
Oocytes were loaded in ND-96 medium containing 2-5
µM BCECF-AM (Molecular Probes, Eugene, OR) for 45 min,
then mounted in a superfusion chamber (36). 530 nm fluorescence
emission images resulting from timed excitations at 495 nm were
recorded at the equatorial plane of the oocyte. Acquired images were
loaded into Image-1 software and replayed with enhanced intensity and
contrast in contour display mode for measurement of oocyte diameter.
Acquired images were calibrated to the cross hatch lines of a standard hemocytometer as described previously (36). Oocyte volume was calculated from measured diameter, assuming spherical geometry, and was
expressed as mean % ± S.D. of original volume in isotonic medium.
Oocyte volume so calculated differed by <0.5% from that computed by
the Image-1 software from the equatorial spheroid area.
Global estimates of oocyte intracellular calcium concentration
([Ca2+]i) were obtained with two ion-sensitive
dyes. Oocytes loaded for 45 min with 5-10 µM Fura-2-AM
(Molecular Probes) were excited alternately at 340 and 380 nm (37, 38).
Excitation ratio images were collected at 15-90 s intervals at an
emission wavelength of 510 nm. In vitro calibration of the
Fura-2 free acid fluorescence ratio was performed (38) using a value
for the Kd of Ca2+ binding to Fura-2 of
224 nM, and with Rmin and
Rmax determined at 10 nM and 40 µM free Ca2+, respectively.
Alternatively, oocytes injected with Calcium Green-Dextran 70 kDa
(Molecular Probes) to a final intracellular concentration of 3.5-7
µM were excited at 490 nm and imaged at 530 nm. Oocyte [Ca2+]i was determined from relative
fluorescence intensity, and calculated from the equation below.
|
(Eq. 2)
|
Kapp is the apparent Ca2+
dissociation constant for the the dye, and values for
Fmax and Fmin were
determined in vitro. For Calcium Green, the resting oocyte
[Ca2+]i was assumed to be 80 nM, and
the limiting value for the ratio
Fmax/Fmin was assumed to
be <14 (39-41). Bleaching averaged <0.5% during 30 min of
intermittent 490 nm irradiation. Oocyte [Ca2+]i
was measured as relative changes from the fluorescence intensity in
isotonic medium.
With both methods, the transition from isotonic to hypotonic medium was
marked by small fluctuations in fluorescence ratio (Fura-2) or
intensity (Calcium Green), consistent with previously reported spatial
and temporal oscillations of [Ca2+]i (39, 40).
However, these oscillations could not be clearly resolved over the
large sampled areas within the nonconfocal equatorial plane. The
reported values of [Ca2+]i therefore represent
averages of global (putatively cytosolic) free Ca2+
concentrations, and were expressed as means ± S.D.
Culture and Erythroid Differentiation of ES Cells--
CCE
murine embryonal stem cells maintained on primary mouse embryo
fibroblasts were transferred onto gelatin-coated dishes in the presence
of 100 U/ml leukemia inhibitory factor (Genetics Institute, Cambridge,
MA) one day before a differentiation experiment. Two ES subclones, B2
and A20, were used (42).4
In vitro differentiation of ES cells at a density of 3000 cells/ml was performed as described previously (42-45) in 0.9% methyl
cellulose, 400 units/ml interleukin-1, 100 units/ml interleukin-3, 2 units/ml erythropoietin, 50 ng/ml Kit ligand, 5 × 10 4 M monothioglycerol, and 20% fetal calf
serum. The cell suspension was cultured in bacterial plates and
incubated at 37 °C in a humidified chamber with 5% CO2.
After the indicated times of differentiation in culture, embryoid
bodies were pooled from several dishes, rinsed several times in 150 mM NaCl, 20 mM sodium phosphate, pH 7.4, and
harvested for RNA extraction.
Culture and Differentiation of Human Peripheral Blood Stem
Cells--
Two-stage erythroid cultures derived from normal human
peripheral blood were set up as described by Fibach et
al. (46, 47). CLT was added at the beginning of the second phase
of culture (upon addition of erythropoietin), on culture day 5. Cells
were harvested and counted at the indicated times between culture days 9 and 19. Cell viability was determined by Trypan Blue exclusion. Cytocentrifuge preparations from the experiment of Fig. 10 were stained
with May Grunwald's Giemsa and benzidine-HCl for erythroid developmental staging (46, 47). In three additional experiments, staging assessed by morphological criteria in hematoxylin-eosin-stained cytocentrifuge preparations gave similar results.
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RESULTS |
Molecular Cloning of mIK1 cDNA--
The mIK1 cDNA was
cloned by homology-based RT-PCR supported by data base searches, as
described under "Materials and Methods." The compiled mIK1 sequence
has been assigned GenBank accession number AF042487. mIK1 nucleotide
sequences from MEL cells, mouse thymus, and mouse stomach were
identical.
The mIK1 amino acid sequence shares 88% identity with that of hIK1
(Fig. 1). The 425 amino acids of mIK1
conform to the topographical model of 6 transmembrane domains
plus pore region proposed by Kohler et al. for the SK family
of calcium-activated K channels (27), to which hIK1 (28-30) and mIK1
are most closely related. The putative pore region of mIK1 is identical
to that of hIK1, including the canonical signature sequence for
K+-selectivity, GYG (48). Indeed, only 1 amino acid
distinguishes mIK1 and hIK1 between the putative exofacial ends of
their S4 transmembrane helices until well beyond their endofacial ends of S6 (Fig. 1). The underlined N-glycosylation sequon
between S5 and the P-region is also conserved.

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Fig. 1.
Alignment of deduced amino acid sequences of
mIK1 and hIK1. Transmembrane spans (S1 S6) and the
pore (P) region are indicated above the sequences.
Underlined residues are those assigned to transmembrane
regions by all reports (28-30); boldface residues not
underlined are those so assigned by at least one report.
Boldface italics show putative leucine zipper
sequences.
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Major regions of sequence divergence between mIK1 and hIK1 include S2,
S3 and its adjacent second exofacial loop, and the putatively
cytoplasmic C-terminal domain. Though the potential 5-leucine zipper is
conserved in the divergent mIK1 C-terminal domain, the less extensive
amino acid changes in the short, putatively cytoplasmic mIK1 N-terminal
region do disrupt another potential leucine zipper (Fig. 1).
mIK1 mRNA and Polypeptide--
As has been previously noted
for hIK1 mRNA (28), mIK1 mRNA was detected in thymus, colon,
and stomach (Fig. 2A). With
the enhanced sensitivity of RT-PCR, mIK1 mRNA was also detected in kidney, liver, testis, ovary, heart, and brain. In addition, among tissues not tested in the human, mIK1 mRNA was present in whole embryo, epididymis, as well as in spleen, murine MEL cells (Fig. 2A), and murine ES cells (Fig. 9A).

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Fig. 2.
A, mIK1 mRNA expression in
mouse tissues as detected by RT-PCR (38 cycles) using primers IK1.IF
and IK1.R (Table I). Equal RNA loading was confirmed by RT-PCR of
glyceraldehyde-3-phosphate dehydrogenase cDNA (25 cycles, not
shown). B, 35S-labeled mIK1 polypeptide
translated from cRNA by rabbit reticulocyte lysate in the absence and
presence of dog pancreatic microsomes.
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mIK1 cRNA in vitro translated by reticulocyte lysate yielded
a homogeneous polypeptide of ~40 kDa (Fig. 2B), somewhat
lower than predicted from the calculated apoprotein molecular mass of 47,783 Da. The presence of dog pancreatic microsomes led to no increase
in mass (Fig. 2B). Incubation of the membrane-associated mIK1 polypeptide with PNGase-F led to no reduction in
Mr for mIK1 polypeptide, under conditions in
which the glycoprotein anion exchanger AE1 (33) was
N-deglycosylated (data not shown). Thus, in vitro
translation in the presence of microsomes provided no evidence for
utilization of the N-glycosylation sequon in mIK1. This
apparent absence of glycosylation may result from the sequon's distal
proximity to the P-region, at most 6 amino acids in contrast to the 12 amino acids on either side required for optimal
N-glycosylation (49).
Pharmacological Characterization of Recombinant mIK1 Expressed in
Xenopus Oocytes--
Xenopus oocytes were injected with water or with
10 ng of capped cRNA encoding mIK1, and examined 2-5 days later. mIK1
expression hyperpolarized resting membrane potential
(Vm) measured ~3-5 min after impalement
( 74 ± 2 mV; n = 49) compared with that of
water-injected oocytes ( 37 ± 1 mV; n = 13, p < 0.0001, unpaired t test). This change
was consistent with a shift toward EK of the
oocyte Vm normally dominated by Cl
conductance, and as predicted for increased expression of a
K+ channel with some basal activity.
hIK1 (28-30) and the murine Gardos (KCa) channel (16) were
activated by Ca2+ and inhibited by CLT. Therefore, we
assessed the response to CaCl2 injection into oocytes at a
holding potential of 50 mV chosen to discriminate K+ from
Cl currents in the ND-96 bath (27, 50). The peak currents
elicited by CaCl2 injection (arrows b) were
outward in mIK1-expressing oocytes (+600 ± 139 nA,
n = 6; Fig.
3A) and significantly differed from the inward currents ( 216 ± 63 nA, n = 4;
Fig. 3B) recorded in control oocytes (p = 0.002, unpaired t test). Similar results were observed in
oocytes expressing hIK1 (data not shown). These results suggest that an
outward K+ current developed in response to
CaCl2 injection in mIK1-expressing oocytes, likely greater
in magnitude than the inward Cl current stimulated in
control oocytes.5

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Fig. 3.
Intracellular Ca2+ activated
outward currents in a voltage-clamped oocyte previously injected with
mIK1 cRNA (A), and inward currents in an oocyte previously
injected with water (B). After introduction of
injection pipette (arrows a), injection of 50 nl
CaCl2 (20 mM, arrows b) was followed
by addition to bath of the CLT metabolite,
2-chlorophenyl-bisphenyl-methanol (10 µM, arrows
c). Vm = 50 mV.
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The identity of the mIK1-associated K+ current elicited by
Ca2+ injection was further tested pharmacologically. The
Gardos channel is potently inhibited by CLT (7, 8, 16). This property was subsequently demonstrated in outside-out patch for recombinant hIK1
(28) and confirmed in two-electrode voltage clamp recordings (data not
shown). We have shown that inhibition of the Gardos channel by
CLT-related drugs does not require inhibition of cytochrome P450 lipid
oxidases (16, 51). Therefore, 2-chlorophenyl-bisphenyl-methanol, the
major in vivo des-imidazolyl metabolite of CLT (51), was tested for its ability to inhibit mIK1. As shown in Fig. 3
(arrows c), the mIK1 current was significantly inhibited
( 781 ± 216 nA, n = 6; p = 0.015, paired t test) whereas control currents were unaffected (+66 ± 50 nA, n = 4; p = 0.63).
Inhibition of mIK1 by 2-chlorophenyl-bis-phenylmethanol was
investigated further in outside-out patch recordings. As shown in Fig.
4A, inwardly rectifying
currents were elicited by ramped voltages between +100 and 100 mV.
These currents were progressively inhibited by increasing
concentrations of 2-chlorophenyl-bis-phenylmethanol to the bath, with
an ID50 value of 14 ± 7 nM (Fig.
4B).

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Fig. 4.
A, currents elicited by 2.6-s voltage
ramps in outside-out patches pulled from an mIK1-expressing oocyte
exposed to the inhibitor, 2-chlorophenyl-bisphenyl-methanol at
concentrations (in nM) of 0, 10, 100, 1000, and 10,000. B, 2-chlorophenyl-bisphenyl-methanol half-maximally
inhibited mIK1 currents in Xenopus oocytes at a
concentration of 14 ± 7 nM (n = 4, Langmuir fit). C, synthetic nonradioactive iodocharybdotoxin
half-maximally inhibited mIK1 currents in Xenopus oocytes at
a concentration estimated at 4 ± 3 nM (n = 4, Hill fit).
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The Gardos channel is also inhibited by the scorpion venom component,
charybdotoxin (ChTX, 16, 28). However, whereas the maxi-K channel of
skeletal muscle (52) and whole cell K+ currents of
lymphocytes (53) were much less potently inhibited by
125I-ChTX than by ChTX, mIK1 was inhibited by synthetic
iodo-ChTX with an ID50 value of 4 ± 3 nM
(Fig. 4C; ID50 was 9 ± 5 nM
from a Langmuir fit). This value was very similar to those with which both cold iodo-ChTX (data not shown) and 125I-ChTX
inhibited A23187-activated 86Rb+ influx into
red cells (15) and with which bound 125I-ChTX was displaced
from intact red cells by ChTX (15). Thus, mIK1-mediated channel
activity in Xenopus oocytes was sensitive to inhibition not
only by CLT, but also by its major des-imidazolyl metabolite, and not
only to ChTX (data not shown) but also by iodo-ChTX. These results are
fully consistent with the pharmacological properties of the erythroid
Gardos channel.
Detection and Regulation by Ca2+ of mIK1 at the
Single-channel Level--
Single-channel events mediated by mIK1 in
inside-out patches were detected in symmetrical potassium gluconate
solutions (Fig. 5A).
NPo was 0.51 ± 0.04 at +50 mV, 0.44 ± 0.15 at +25 mV, 0.43 ± 0.08 at 25 mV, and 0.59 ± 0.18 at
50 mV. Thus, voltage dependence of NPo was not
present over the range tested (p = 0.66 by ANOVA). The
slope conductance (n = 3) measured between 50 to
100 mV was 35 picosiemens, and 9 picosiemens between 0 and +100 mV
(Fig. 5B). This degree of inward rectification was slightly
greater than that reported for the native Gardos channel (4), but was
almost exactly as reported for hIK1 expressed in oocytes (28).

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Fig. 5.
mIK1 currents measured in the inside-out
patch configuration. A, Sweeps displaying unitary
currents of mIK1 at the indicated membrane potentials. c,
closed; o, open conductance level. Result is representative
of three experiments. B, representative current-voltage
relationship for an inside-out patch containing mIK1. Error
bars are contained within the symbols. C, cytoplasmic
free Ca2+ dependence of mIK1 (n = 3).
K0.5 = 158 ± 8 nM. Hill
coefficient for Ca2+ = 0.9 ± 0.05.
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Inside-out patches containing mIK1 were voltage-ramped from +100 to
100 mV in the presence of 0.1, 0.2, 0.5, 1.0, and 10 µM
Ca2+. As shown in Fig. 5C, mIK1 was
half-maximally activated by 158 ± 8 nM free
Ca2+, and the Hill coefficient for activation was 0.9 ± 0.05. Thus, in voltage independence (see above) and in Ca2+
sensitivity, mIK1 resembled the erythroid Gardos channel (3). These
properties also resembled those of hIK1 (28) expressed in
Xenopus oocytes (K0.5 300 nM, Hill coefficient for activation by Ca2+ of
1.7), but differed more substantially from the Hill coefficients of 2.7 and 3.2 determined for hIK1 in HEK293 cells (29, 30).
mIK1 Expression Confers on Oocytes CLT-sensitive Regulatory Volume
Decrease--
Native oocytes lack the ability to respond to mild (36)
or extreme hypotonic swelling (54) with RVD. As shown in Fig. 6A, three oocytes previously
injected with water swelled upon exposure to mildly hypotonic medium,
and failed to return to their isotonic volumes. Whereas the resting
membrane potential of native oocytes is dominated by Cl
conductances, mIK1-expressing oocytes show a large shift in membrane potential toward EK. Therefore, we hypothesized that mIK1
expression might confer on oocytes a novel RVD response. Fig.
6B shows that mIK1-injected oocytes subjected to mild
hypotonic swelling indeed recovered their original isotonic volume in
the continued presence of hypotonic medium, but this RVD response was
prevented when exposure to hypotonicity occurred in the presence of 10 µM CLT (Fig. 6C). Moreover, mIK1-associated
RVD was abrogated in BAPTA-AM-loaded oocytes exposed to hypotonic
medium containing EGTA (Fig. 6D). These results and similar
results for oocytes expressing hIK1 are summarized in Table
II, and demonstrate that IK1 expression conferred on oocytes a novel, Ca2+-dependent,
CLT-sensitive RVD response.

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Fig. 6.
A, volume of water-injected oocytes
during transition from isotonic to hypotonic medium, with no evident
volume regulation. B, volume of five mIK1-expressing oocytes
during transition from isotonic to hypotonic medium, exhibiting RVD.
C, volume of five mIK1-expressing oocytes during transition
from isotonic to hypotonic medium in the presence of clotrimazole (10 µM). RVD has been inhibited. D, volume of five
mIK1-expressing oocytes loaded with BAPTA-AM and incubated in
EGTA-containing medium (<10 nM
[Ca2+]o) prior to and during the transition from
isotonic to hypotonic medium. Hypotonic swelling is enhanced, and RVD
is absent.
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mIK1-mediated, CLT-inhibited,
Ca2+-dependent K+ Conductance Is
Activated by Hypotonic Swelling of Xenopus Oocytes--
Hypotonic
swelling activates an endogenous Cl current in
Xenopus oocytes (50). However, this activation (Fig.
7A) is not accompanied by RVD (Fig.
6A). We reasoned that RVD in
IK1-expressing oocytes (Fig. 6B, Table II) likely was
mediated by concomitant activation of heterologous IK1 in coordination
with endogenous Cl channels. The left panels of Fig.
7A show that hypotonic swelling of control oocytes
previously injected with water led to enhanced current that displayed
no inhibition by subsequently added CLT. As summarized in Fig.
7B, currents in control oocytes (n = 9) measured at +20 mV were +88 ± 14 nA in isotonic medium, increased to +276 ± 50 nA in hypotonic medium (p < 0.01),
but were not reduced by addition of 10 µM CLT (317 ± 43 nA, p > 0.05, ANOVA).

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Fig. 7.
Hypotonic bath solutions activate
CLT-sensitive mIK1 currents. A, two-electrode voltage
clamp traces of hypotonicity-activated currents in control
(left) and mIK1-expressing ocytes (right). Each
oocyte was subjected to sequential I-V protocols in ND-96 (isotonic),
hypotonic medium, and in hypotonic medium containing 10 µM CLT for each oocyte. Hypotonic currents were sampled
after 8-min exposure to hypotonic medium. CLT currents were sampled 2 min after exposure to CLT. Whereas control oocyte currents were mildly
outwardly rectified and insensitive to CLT, mIK1-associated currents
were strongly outwardly rectified and CLT-sensitive. B,
current-voltage relationships for control (left,
n = 9) and mIK1-expressing oocytes (right,
n = 21) in isotonic ND-96 (open circles),
hypotonic medium (closed circles), and hypotonic medium plus
10 µM CLT (open triangles). Expression of mIK1
is associated with a negative shift of Erev of
~50 mV that is partially reversed by CLT. C,
current-voltage relationships of difference currents in control
(left) and mIK1-expressing oocytes (right).
Expression of mIK1 is associated with a negative shift of
Erev for hypotonic minus isotonic difference
currents (open diamonds) of ~60 mV. Hypotonic minus CLT
difference currents (closed diamonds) in mIK1-expressing
oocytes are significant at resting potential, but negligible in control
oocytes.
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In contrast, the right panels of Fig. 7A show
that outward currents measured in isotonic medium in oocytes previously
injected with mIK1 cRNA (n = 30) were larger than in
control oocytes (p < 0.03), were also activated by
hypotonicity, and displayed sensitivity to inhibition by clotrimazole.
As summarized in the right panel of Fig. 7B,
currents in mIK1 measured at +20 mV increased from +496 ± 100 nA
in isotonic medium to +1157 ± 269 nA in hypotonic medium
(p < 0.001), then fell to +657 ± 144 nA upon
exposure to CLT in hypotonic medium (p < 0.01, ANOVA).
Thus, CLT inhibited (at +20 mV) 75% of the mIK1-associated oocyte
current elicited by hypotonic swelling. It is also evident in the
right panels of Fig. 7 (A and B) that
whereas hypotonicity reduced total inward current in mIK1-expressing
oocyte, inward current was restored upon addition of CLT, likely
representing unmasking of the chloride current activated by hypotonic
swelling.
Fig. 7C plots the potential dependence of isotonic minus
hypotonic difference currents and of hypotonic minus clotrimazole difference currents. Note that, whereas Erev for
the hypotonicity-induced difference current was ~ 26 mV in control
oocytes, Erev was ~ 80 mV in mIK1-expressing
oocytes. Similarly, whereas the CLT-induced difference current was
negligible in control oocytes, the substantial CLT-induced difference
current in mIK1-expressing oocytes (Fig. 7C) displayed an
Erev of 103 mV (n = 30, r = 0.99) and in hIK1-expressing oocytes (data not
shown) an Erev of 99 mV (n = 6, r = 0.87). Both approximate the predicted
EK of 96 mV, supporting the hypothesis of
K+ selectivity for mIK1-mediated currents elicited by
hypotonic swelling of oocytes.
Fig. 8 demonstrates that the outward
current activated in mIK1-expressing oocytes by hypotonicity requires
Ca2+. mIK1-expressing oocytes bathed in isotonic
zero-Ca2+ medium exhibited an Erev
of 29 mV (n = 5). This value changed minimally to
33 mV upon exposure to hypotonic in the continued absence of
extracellular Ca2+, consistent with the activation of
Iclswell (50) and/or other Cl
conductances. However, subsequent addition of 2 mM
Ca2+ to the hypotonic extracellular medium elicited a
substantial outward current characterized by an
Erev of 88 mV (close to the predicted
EK) and by sensitivity to inhibition by 10 µM CLT (data not shown). Thus, the CLT-sensitive outward
current activated by hypotonic swelling of mIK1-expressing oocytes was
characterized by a Ca2+ requirement.

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Fig. 8.
Ca2+-dependence of
hypotonicity-activated steady-state currents in mIK-expressing
oocytes. MIK-expressing oocytes were exposed first to isotonic
EGTA-containing media (<10 nM
[Ca2+]o, open circles), then again
11 ± 1 min after the transition to hypotonic EGTA-containing
media (closed circles), and again 5 ± 1 min after
the subsequent transition to hypotonic medium containing 2 mM extracellular Ca2+ (open
triangles). Values are means ± S.E. (n = 5).
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Hypotonic Swelling of Oocytes Increases Intracellular
[Ca2+]i--
The ability of hypotonic
swelling to activate the Ca2+-sensitive,
voltage-independent mIK1 channel suggested that hypotonicity might also
elevate [Ca2+]i in the oocyte. The resting global
[Ca2+]i in isotonic medium as estimated by Fura-2
fluorescence ratio did not differ significantly (p > 0.1) between oocytes previously injected with water (106 ± 12 nM) or with mIK1 cRNA (94 ± 11 nM, n = 5). Both control and mIK1-expressing oocytes
responded to 25 min hypotonic swelling with gradual increases in global
[Ca2+]i (7.4-9.0% as reported by Calcium Green
Dextran and 3.9-4.5% as reported by Fura-2; p < 0.01 compared with isotonic values) that were indistinguishable in rate and
magnitude.
Regulation of mRNAs Encoding mIK1 and Other K Channels during
Erythroid Differentiation--
mIK1 mRNA was almost as abundant in
MEL cells as in spleen (Fig. 2A), did not differ
consistently among uninduced and dimethyl sulfoxide-induced MEL cells
of different clonal origins (data not shown), and so agreed with our
earlier report that CLT-sensitive K+ currents of
intermediate conductance were present in both induced and uninduced MEL
cells (16). However, even uninduced MEL cells are developmentally
committed to the erythroid lineage (16, 22). Therefore, to phenotype
expression of mIK1 and other K+ channel mRNA expression
in erythroid development, we turned to ES cells (42-44), comparing
independent erythroid inductions in two clonal cell lines, B-2 and A-20
(Fig. 9).

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Fig. 9.
A, mIK1 mRNA expression
in two lines of ES cells induced along the erythroid differentiation
pathway for the indicated times, as detected by two primer pairs and
compared with erythroid-specific ( -globin, AE1) and nonspecific
mRNAs ( -actin). Shown are two independent inductions of the B2
line, and one induction of the A20 line (normally with lower intensity
of benzidine reactivity). The IK1 fragment of 1279 bp was amplified
with the primer pair IK1.F and IK1.R; that of 711 bp with the primer
pair IK1.IF and IK1.R. B, changes in levels of mRNA
encoding other KCa channels during two independent
inductions of erythroid differentiation in the ES cell line, B2. The
Slo fragment of 1054 bp was amplified with the primer pair Slo.F
and Slo.R1; that of 1314 bp with the primer pair SloF and
Slo.R2.
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The ES cells formed embryoid bodies of which over 80% matured into
colonies containing hematopoietic cells detectable by phase contrast
microscopy. We and others have previously shown that early markers of
erythroid differentiation, such as GATA-1, are detected in embryoid
bodies as early as day 3-4 after initiation of in vitro
differentiation (42-45). Expression patterns of the erythroid-specific
later markers -globin and AE1 (band 3 anion exchanger) were nearly
identical in three inductions, evident at day 6 (accompanied by the
onset of microscopically evident hemoglobinization) and increasing
thereafter. The K+ channel mRNAs displayed uninduced
levels and patterns of induction that varied among independent
experiments to a greater degree than did those of the erythroid
specific markers. Nonetheless, they displayed distinct patterns.
IK1 mRNA was present at low (B2-1) or very low level in uninduced
cells (B2-2 and A20). After induction, however, IK1 mRNA levels
consistently increased between days 6 and 9 after induction and
remained elevated, as confirmed with two sets of primer pairs. This
pattern contrasted with the relatively unchanging mRNA levels of
-actin (Fig. 9A) and glyceraldehyde-3-phosphate
dehydrogenase (Fig. 9B) in uninduced and induced cells.
The pattern of increase in mIK1 mRNA during erythroid
differentiation of ES cells was not paralleled by most other tested K+ channel mRNAs, many of which were detectable at low
or very low level in ES cells (Fig. 9B). Among
KCa mRNAs, Slo was present in uninduced cells,
decreased as erythroid-specific gene expression was activated on day 6, and increased again by day 12. This biphasic pattern was shown with two
sets of primer pairs. In contrast, SK1 and SK2 mRNAs were unchanged
during the differentiation process (Fig. 9B), whereas SK3
(data not shown) was undetectable after 45 cycles amplification. Each
Kca PCR amplimer presented in Fig. 9 encompassed
exon-intron boundaries. In addition, no PCR products were amplified
from reactions from which reverse transcriptase was omitted (data
not shown).6
Effect of the IK1 Inhibitor, CLT, on Stem Cell Proliferation and
Erythroid Differentiation--
Two-stage liquid cultures of human
CD34 /CD38+ stem cells from peripheral blood
were examined to test the effect of CLT on the second stage of culture
in the presence of erythropoietin. As shown in Fig.
10A, increasing
concentrations of CLT proportionately inhibited cell proliferation at
all times tested. Cellular progression along the erythroid
differentiation pathway was retarded in the presence of the lowest
tested concentration of CLT, 10 nM (Fig. 10B).
Cell proliferation was only slightly inhibited at this concentration of
CLT at the times when retardation of developmental progression was most
pronounced on culture days 13 (CLT day 8) and 16 (CLT day 11).

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Fig. 10.
A, inhibition by increasing
concentrations of CLT of cell proliferation during erythroid
differentiation of human peripheral blood stem cells in liquid culture.
CLT exposure began on culture day 5. Cell numbers in the absence of CLT
(represented as 100% in graph) were (×10 6) 1.86 ± 0.10 at culture day 10 (diamonds), 1.72 ± 0.06 at culture day 15 (squares), and 2.30 ± 0.09 at
culture day 19 (triangles). Results are means ± S.D.
from four identical experiments. B, inhibition by 10 nM CLT of progression through the morphological stages of
erythroid differentiation: open bars, proerythroblasts;
stippled bars, basophilic erythroblasts; dark gray
bars, polychromatophilic erythroblasts; light gray
bars, orthochromatophilic normoblasts. Numbers within
bars represent percentage of total cells.
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DISCUSSION |
We have cloned the mIK1 intermediate conductance KCa
channel from MEL cells and from murine thymus and stomach (Fig. 1).
mIK1 mRNA was widely but not ubiquitously expressed in mouse
tissues (Fig. 2). mIK1 expressed in Xenopus oocytes mediated
whole oocyte K+-selective currents (Fig. 3), as well as
inwardly rectifying, Ca2+-activated, voltage-insensitive,
unitary K+ currents in inside-out patches (Fig. 5) similar
to those previously reported for the erythroid KCa (Gardos)
channel (2-4, 16) and for hIK1 (28). Inhibition of mIK1-mediated
currents by CLT did not require the presence of the imidazole group
responsible for cytochrome P450 inhibition by CLT. Moreover, inhibition
of mIK1 was not attenuated by the substitution of 3-iodotyrosine for
tyrosine at residue 36 of ChTX (Fig. 4), a critical residue for
inhibition of skeletal muscle maxi-K channels as defined by systematic
ChTX mutagenesis (55). Hypotonic swelling of oocytes activated
mIK1-mediated Ca2+-dependent outward currents
sensitive to CLT (Figs. 7 and 8) in parallel with small but significant
increases in global [Ca2+]i, and conferred on
oocytes the novel property of Ca2+-dependent,
CLT-sensitive RVD (Fig. 6, Table
II). mIK1
was present at low levels in uninduced ES cells, and exhibited a
sustained increase in abundance during erythroid differentiation of ES
cells (Fig. 9). The ability of 10 nM CLT to retard
erythroid differentiation (Fig. 10) is consistent with a physiological
role for IK1 KCa channel activity in this process.
Molecular Cloning and Tissue Distribution--
Several features of
the mIK1 sequence merit discussion. Three mIK1 EST clones from two
cDNA libraries encode reliable 5'-untranslated sequence to
nucleotide 98. Although this region lacks upstream in-frame
terminator codons, the residues surrounding the initiator codon of our
fully functional mIK1 cDNA constitute a favorable Kozak consensus
initiation site. It is also pertinent that an in-frame terminator
resides at nucleotide 252 in the hIK1 cDNA (30).
By analogy to other K+ channels, it is likely that IK1
forms a functional tetramer, and in the current study in
Xenopus oocytes a homotetramer. The N-terminal cytoplasmic
tail (56), among other channel regions (57), has been proposed to
contribute to subunit oligomerization of the Kv channels. However, the
lack of conservation of the candidate leucine zipper sequence in this region of mIK1 (L25 in hIK1 versus V25 in mIK1) suggests
some other mechanism for oligomerization possibly mediated through this
portion of the polypeptide. The conserved leucine zipper near IK1 C
termini may suggest a possible role for this region in protomer
oligomerization or in other protein-protein interactions. The S6 helix
is remarkable for conservation of four Cys residues spaced so as to
allow speculation of possible disulfide bonding within or between
channel protomers. The extreme C termini of mIK1 and hIK1 are not
conserved, and neither displays a consensus PZD binding domain (58).
Although mIK1 displayed only 88% identity to hIK1, this lower than
usual degree of identity between orthologous polypeptides from
different mammalian species arises almost entirely from several
delimited regions.
The presence of IK1 mRNA in mouse colon (Fig. 2) and in T84 cells
(data not shown), suggests that it may mediate or contribute to the
ChTX- and CLT-sensitive KCa current present in T84 cells (59, 60) required for Ca2+-mediated secretagogue-stimulated
transepithelial chloride secretion (59-61). The wide distribution of
IK1 mRNA suggests other physiological functions for this channel,
such as that suggested in lymphocyte blast transformation (30).
Relation between IK1 and the Erythroid KCa (Gardos)
Channel--
The strong functional similarities between recombinant
IK1 and the Gardos channel suggest that IK1 likely is or contributes to
erythroid KCa channel activity. This is especially true
with respect to the pharmacological profile of the recombinant and native channels. However, the differences between recombinant and
native function, including the more extreme inward rectification of
mIK1, demand consideration of two possibilities. It is possible that
mIK1 function is influenced by native oocyte K+ channels
such as GIRK or ISK (minK), or that overexpression of mIK1
elicits atypical expression of endogenous oocyte channels (62). This
possibility, as well as differences in kinase or phosphatase
activities, may relate to the different apparent cooperativities of
activation by Ca2+ in 293 cells with Hill coefficients of
2.7-3.2 (29, 30), compared with erythrocytes (3) and oocytes (28 and
this work) with Hill coefficients of 0.9-1.7. It is also possible that
IK1 must interact with another erythroid channel subunit (of or type) to reconstitute with complete fidelity the native Gardos channel
activity. Whatever the reason, one consequence of the less steep
[Ca2+ ] activation profile in the oocyte membrane is to
enhance the ability of resting values of [Ca2+]i
partially to activate IK1 in basal conditions.
IK1 and RVD--
RVD in many cell types has been associated with
swelling-associated elevations of [Ca2+]i (63).
The swelling-induced elevation in global oocyte [Ca2+]i though small, may nonetheless contribute
to the ability of hypotonic swelling further to activate IK1. This
small increase in global [Ca2+]i may have
reflected larger oscillatory changes in local [Ca2+]i (39-41) in the vicinity of plasmalemmal
IK1 channels. The importance of juxtaplasmalemmal
[Ca2+]i in activation of IK1 is further suggested
by its activation upon restoration of extracellular Ca2+
(Fig. 9). In parallel with hypotonic activation of IK1, several endogenous oocyte Cl channels may also be activated,
including IClswell (50) and two types of IClCa
(64). How might the combined activities of these channels lead to
RVD?
Heterologous expression in the RVD-deficient T lymphoid cell line
CTLL-2 of voltage-gated K+ channel Kv1.3 conferred the
capacity for RVD (65), likely by complementing a limiting endogenous
K+ conductance. RVD was elicited in Xenopus
oocytes expressing mIK1 likely by similar complementation of the
limiting endogenous K+ conductance. The net K+
efflux (3.77 nmol) required to achieve the measured 74% recovery from
a 6% peak increase in oocyte volume (Table
II) would require a time-averaged mean
outward K+ current of 284-331 nA, assuming
[K+]i = 91-104 mM in the maximally
swollen oocyte and in the effluent, an operational K+
space = 900 nl, and 20 min recovery time (Fig. 6). As shown in Fig. 7, mean mIK1-associated outward CLT-sensitive current at 60 mV
was 243 nA. Fig. 8 shows mean mIK1-associated outward
Ca2+-dependent currents at 60 mV of 309 nA.
Taken together with the concurrent ~100 nA of inward current
attributable to IClswell-mediated Cl efflux
(50), and the increased electrical driving force for Cl
efflux in hyperpolarized mIK1-expressing oocytes, we conclude that net
conductive flux of K+ plus Cl in
mIK1-expressing oocytes could reasonably account for most or all of the
observed RVD.
Red cell volume decrease following Gardos channel activation by
prostaglandin E2 also required parallel endogenous
K+ and Cl conductances, as suggested by
inhibition not only by ChTX and CLT, but also by DIDS (66). However,
though activation of the Gardos channel by A23187 or by PGE2 leads to
volume decrease, erythrocytes effect RVD in response to hypotonic
swelling via electroneutral K-Cl cotransport (67) rather than via the
Gardos channel. Thus, the cellular machinery required to transduce to the IK1 channel the major hypotonic swelling signal (whether elevation in [Ca2+]i or another signal) may be lacking in
the human erythrocyte.
A recent report has suggested that a large increase in oocyte
Cl conductance secondary to expression and
hyperpolarization-induced activation of the Cl channel
ClC-2 can also contribute to volume regulation in the voltage-clamped
Xenopus oocyte, attenuating or abolishing oocyte swelling
induced by extreme hypotonicity (68). However, the ability of ClC-2 to
confer RVD was not tested in this study.
Possible Role of IK1 in Erythroid Differentiation--
mIK1
mRNA increased in abundance during erythroid differentiation of ES
cells (Fig. 9A). Other KCa channel mRNAs
tested either decreased or remained unchanged during ES cell erythroid
differentiation (Fig. 9B). A possible requirement for or
influence of IK1 in erythroid differentiation was tested by assessing
the effect of increasing concentrations of CLT on the proliferation and
erythroid differentiation of human peripheral blood
CD34 /CD38+ stem cells. CLT inhibited stem
cell proliferation with an ID50 ~30 nM (Fig.
10A). The observed ID50 of ~30 nM
was >10-fold more potent than previously observed for inhibition by
CLT of mitogen-stimulated [3H]thymidine incorportation
and intracellular Ca2+ signaling in fibroblasts and tumor
cells (69), but close to that observed for inhibition of IK1 (16, 28, and this work). Stem cell differentiation along the erythroid pathway
was retarded by 10 nM CLT (Fig. 10B), a
concentration at which inhibition of proliferation was modest. In
addition, preliminary experiments indicate that 1 µM CLT
partially inhibits erythroid colony formation by murine ES
cells.7
IRK-like currents and unspecified IRK family mRNAs have also been
implicated in the in vitro cytokine-driven expansion of human CD33 /CD34+ hemopoietic progenitor cells
along both erythroid and myeloid pathways (70). IRK1 mRNA, was
indeed present at very low levels in resting ES cells, and showed a
modest, steady increase during erythroid differentiation (data not
shown).
Conclusion--
We have cloned the mIK1 KCa channel,
examined the tissue distribution of mIK1 mRNA, defined biophysical
and pharmacological characteristics of mIK1 expressed in
Xenopus oocytes, determined profiles of mIK1 and other
K+ channel transcripts in ES cells during erythroid
differentiation, and shown that pharmacological inhibition of IK1
inhibits both proliferation and erythroid differentiation of stem
cells. In its erythroid expression, Ca2+ dependence,
voltage independence, unitary conductance and rectification, pharmacological profile, and ability to mediate net volume decrease, mIK1 resembles the Gardos channel more closely than any other cloned
K+ channel. Future experiments will test whether mIK1 is
both necessary and sufficient to reconstitute Gardos channel function,
and test more extensively the role of IK1 in erythroid differentiation. We have also shown that mIK1 can confer upon oocytes the novel response
of RVD. Thus, in addition to its previously established utility in
studying RVI (36), the oocyte will be a useful system for the more
general study of cell volume regulation.
 |
ACKNOWLEDGEMENTS |
We thank Satoshi Takamatsu for ES cell RNA,
Paul Savin for technical assistance, and Leah Staffier for graphics
assistance.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants HL15157 (to S. L. A. and C. B.), DK51059
(to S. L. A.), DK47636 (to B. L.), and DK34854 (to the
Harvard Digestive Diseases Center), and American Cancer Society Grant
DHP-4B (to B. L.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF042487.
c
These authors contributed equally to this work.
d
Supported by National Institutes of Health Grant
R29-HL09853.
f
Scholar of the Leukemia Society of America.
n
Established Investigator of the American Heart Association.
To whom correspondence should be addressed: Molecular Medicine and
Renal Units, RW763, Beth Israel Deaconess Medical Center East Campus,
330 Brookline Ave., Boston, MA 02215. Tel: 617-667-2930; Fax:
617-667-2913; E-mail: salper{at}bih.harvard.edu.
The abbreviations used are:
SS, pertaining to or
describing homozygosity for the hemoglobin S mutation causing sickle
cell disease ChTX, charybdotoxinCLT, clotrimazoleMEL cell, murine
erythroleukemia cellES cell, murine embryonic stem cellI-ChTX, monoiodotyrosyl-36-ChTXPCR, polymerase chain reactionPNGase F, peptidyl-N-glycosidase FBCECF-AM, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylesterFura2-AM, Fura2-acetoxymethylester[Ca2+]i, intracellular calcium concentrationVm, transmembrane potential of the oocyte plasma membraneEN, the equilibrium transmembrane potential for
the ion "N"RT, reverse transcriptaseANOVA, analysis of
varianceRVD, regulatory volume decreasebp, base pair(s).
2
C. Brugnara and S. L. Alper, unpublished
results.
3
Not only these and other degenerate primers, but
also perfectly matched hIK1 primers failed to amplify hIK1 cDNA
from a positive control tissue, human placenta (28), under a range of
standard conditions. The same was true for many mIK1 primer pairs used with mouse cDNA. However, the addition of Q solution (Qiagen) to
the PCR reaction mix allowed successful amplification of mIK1 fragments
with multiple sets of primer pairs from many mouse tissue sources. The
unusually high GC content of mIK1 (62% overall, with clusters up to
82% of 79 nucleotides) is likely related to these observations.
4
The B2 and A20 ES cell clonal lines are /
and /+, respectively, for the GDI-D4 gene. They are completely normal
in adherence, spreading, proliferation, and erythroid differentiation
in methylcellulose, as judged by colony-forming efficiency,
histochemical indices of erythroid phenotype and hemoglobinization,
embryonic and adult globin mRNA induction, and secondary erythroid
colony formation. These cell lines are also normal in differentiation
of megakaryocyte, mast cell, and myeloid lineages (42).
5
The whole cell currents shown in the protocol
shown in Fig. 4 were recorded 2-3 days after cRNA injection. The
larger currents shown in Fig. 8 were recorded from oocytes 2-7 days
after cRNA injection. mIK1 current amplitude in the oocytes increased
with time post-cRNA injection.
6
Among Kir channel mRNAs (data
not shown), ROMK was undetectable after 38 cycles amplification, and
IRK1 (36 cycles) steadily increased from very low levels at day 0 to
low levels at days 10-12. In contrast, mRNA levels of IRK2 (33 cycles) and IRK3 (36 cycles) steadily decreased during the observed
periods. Though reproduced in three ES lines through multiple
amplifications, and with appropriate control RT-minus amplifications,
these results are to be interpreted with caution. The amplified regions
of these Kir cDNAs are encoded by genomic segments
uninterrupted by introns.
7
B. Lim, B. E. Shmukler, and S.L. Alper,
unpublished results.
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V. L. Lew, N. Daw, D. Perdomo, Z. Etzion, R. M. Bookchin, and T. Tiffert
Distribution of plasma membrane Ca2+ pump activity in normal human red blood cells
Blood,
December 1, 2003;
102(12):
4206 - 4213.
[Abstract]
[Full Text]
[PDF]
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Y. A. Assef, A. E. Damiano, E. Zotta, C. Ibarra, and B. A. Kotsias
CFTR in K562 human leukemic cells
Am J Physiol Cell Physiol,
August 1, 2003;
285(2):
C480 - C488.
[Abstract]
[Full Text]
[PDF]
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N. A. Tamarina, Y. Wang, L. Mariotto, A. Kuznetsov, C. Bond, J. Adelman, and L. H. Philipson
Small-Conductance Calcium-Activated K+ Channels Are Expressed in Pancreatic Islets and Regulate Glucose Responses
Diabetes,
August 1, 2003;
52(8):
2000 - 2006.
[Abstract]
[Full Text]
[PDF]
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J. F. Hoffman, W. Joiner, K. Nehrke, O. Potapova, K. Foye, and A. Wickrema
The hSK4 (KCNN4) isoform is the Ca2+-activated K+ channel (Gardos channel) in human red blood cells
PNAS,
June 10, 2003;
100(12):
7366 - 7371.
[Abstract]
[Full Text]
[PDF]
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M. Grunnet, T. Jespersen, N. MacAulay, N. K Jorgensen, N. Schmitt, O. Pongs, S.-P. Olesen, and D. A Klaerke
KCNQ1 channels sense small changes in cell volume
J. Physiol.,
June 1, 2003;
549(2):
419 - 427.
[Abstract]
[Full Text]
[PDF]
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M. N. Chernova, A. K. Stewart, L. Jiang, D. J. Friedman, Y. Z. Kunes, and S. L. Alper
Structure-function relationships of AE2 regulation by Ca2+i-sensitive stimulators NH+4 and hypertonicity
Am J Physiol Cell Physiol,
May 1, 2003;
284(5):
C1235 - C1246.
[Abstract]
[Full Text]
[PDF]
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M. A.G. van der Heyden, M. J.A. van Kempen, Y. Tsuji, M. B. Rook, H. J. Jongsma, and T. Opthof
P19 embryonal carcinoma cells: a suitable model system for cardiac electrophysiological differentiation at the molecular and functional level
Cardiovasc Res,
May 1, 2003;
58(2):
410 - 422.
[Abstract]
[Full Text]
[PDF]
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J. W. Stocker, L. De Franceschi, G. A. McNaughton-Smith, R. Corrocher, Y. Beuzard, and C. Brugnara
ICA-17043, a novel Gardos channel blocker, prevents sickled red blood cell dehydration in vitro and in vivo in SAD mice
Blood,
March 15, 2003;
101(6):
2412 - 2418.
[Abstract]
[Full Text]
[PDF]
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C. A. Syme, K. L. Hamilton, H. M. Jones, A. C. Gerlach, L. Giltinan, G. D. Papworth, S. C. Watkins, N. A. Bradbury, and D. C. Devor
Trafficking of the Ca2+-activated K+ Channel, hIK1, Is Dependent upon a C-terminal Leucine Zipper
J. Biol. Chem.,
February 28, 2003;
278(10):
8476 - 8486.
[Abstract]
[Full Text]
[PDF]
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K. Nehrke, C. C. Quinn, and T. Begenisich
Molecular identification of Ca2+-activated K+ channels in parotid acinar cells
Am J Physiol Cell Physiol,
February 1, 2003;
284(2):
C535 - C546.
[Abstract]
[Full Text]
[PDF]
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J. Wang, S. Morishima, and Y. Okada
IK channels are involved in the regulatory volume decrease in human epithelial cells
Am J Physiol Cell Physiol,
January 1, 2003;
284(1):
C77 - C84.
[Abstract]
[Full Text]
[PDF]
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T. Takahata, M. Hayashi, and T. Ishikawa
SK4/IK1-like channels mediate TEA-insensitive, Ca2+-activated K+ currents in bovine parotid acinar cells
Am J Physiol Cell Physiol,
January 1, 2003;
284(1):
C127 - C144.
[Abstract]
[Full Text]
[PDF]
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M. Simoes, L. Garneau, H. Klein, U. Banderali, F. Hobeila, B. Roux, L. Parent, and R. Sauve
Cysteine Mutagenesis and Computer Modeling of the S6 Region of an Intermediate Conductance IKCa Channel
J. Gen. Physiol.,
June 24, 2002;
120(1):
99 - 116.
[Abstract]
[Full Text]
[PDF]
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T. Ayabe, H. Wulff, D. Darmoul, M. D. Cahalan, K. G. Chandy, and A. J. Ouellette
Modulation of Mouse Paneth Cell alpha -Defensin Secretion by mIKCa1, a Ca2+-activated, Intermediate Conductance Potassium Channel
J. Biol. Chem.,
January 25, 2002;
277(5):
3793 - 3800.
[Abstract]
[Full Text]
[PDF]
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A. Rivera, P. Jarolim, and C. Brugnara
Modulation of Gardos channel activity by cytokines in sickle erythrocytes
Blood,
January 1, 2002;
99(1):
357 - 363.
[Abstract]
[Full Text]
[PDF]
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G. G. Goss, L. Jiang, D. H. Vandorpe, D. Kieller, M. N. Chernova, M. Robertson, and S. L. Alper
Role of JNK in hypertonic activation of Cl--dependent Na+/H+ exchange in Xenopus oocytes
Am J Physiol Cell Physiol,
December 1, 2001;
281(6):
C1978 - C1990.
[Abstract]
[Full Text]
[PDF]
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M. I. Niemeyer, L. P. Cid, L. F. Barros, and F. V. Sepulveda
Modulation of the Two-pore Domain Acid-sensitive K+ Channel TASK-2 (KCNK5) by Changes in Cell Volume
J. Biol. Chem.,
November 9, 2001;
276(46):
43166 - 43174.
[Abstract]
[Full Text]
[PDF]
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B. Nilius and G. Droogmans
Ion Channels and Their Functional Role in Vascular Endothelium
Physiol Rev,
October 1, 2001;
81(4):
1415 - 1459.
[Abstract]
[Full Text]
[PDF]
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R. Hosseini, D. C H Benton, P. M Dunn, D. H Jenkinson, and G. W J Moss
SK3 is an important component of K+ channels mediating the afterhyperpolarization in cultured rat SCG neurones
J. Physiol.,
September 1, 2001;
535(2):
323 - 334.
[Abstract]
[Full Text]
[PDF]
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E. Vázquez, M. Nobles, and M. A. Valverde
Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca2+-dependent potassium channels
PNAS,
April 12, 2001;
(2001)
91096498.
[Abstract]
[Full Text]
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H. Wulff, M. J. Miller, W. Hansel, S. Grissmer, M. D. Cahalan, and K. G. Chandy
Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: A potential immunosuppressant
PNAS,
July 5, 2000;
97(14):
8151 - 8156.
[Abstract]
[Full Text]
[PDF]
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C. A. Syme, A. C. Gerlach, A. K. Singh, and D. C. Devor
Pharmacological activation of cloned intermediate- and small-conductance Ca2+-activated K+ channels
Am J Physiol Cell Physiol,
March 1, 2000;
278(3):
C570 - C581.
[Abstract]
[Full Text]
[PDF]
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H. Rauer, M. D. Lanigan, M. W. Pennington, J. Aiyar, S. Ghanshani, M. D. Cahalan, R. S. Norton, and K. G. Chandy
Structure-guided Transformation of Charybdotoxin Yields an Analog That Selectively Targets Ca2+-activated over Voltage-gated K+ Channels
J. Biol. Chem.,
January 14, 2000;
275(2):
1201 - 1208.
[Abstract]
[Full Text]
[PDF]
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W. Su, B. E. Shmukler, M. N. Chernova, A. K. Stuart-Tilley, L. de Franceschi, C. Brugnara, and S. L. Alper
Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation
Am J Physiol Cell Physiol,
November 1, 1999;
277(5):
C899 - C912.
[Abstract]
[Full Text]
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C. B. Neylon, R. J. Lang, Y. Fu, A. Bobik, and P. H. Reinhart
Molecular Cloning and Characterization of the Intermediate-Conductance Ca2+-Activated K+ Channel in Vascular Smooth Muscle : Relationship Between KCa Channel Diversity and Smooth Muscle Cell Function
Circ. Res.,
October 29, 1999;
85
(9):
e33 - e43.
[Abstract]
[Full Text]
[PDF]
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A. Rivera, M. A. Rotter, and C. Brugnara
Endothelins activate Ca2+-gated K+ channels via endothelin B receptors in CD-1 mouse erythrocytes
Am J Physiol Cell Physiol,
October 1, 1999;
277(4):
C746 - C754.
[Abstract]
[Full Text]
[PDF]
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H. Rauer, M. Pennington, M. Cahalan, and K. G. Chandy
Structural Conservation of the Pores of Calcium-activated and Voltage-gated Potassium Channels Determined by a Sea Anemone Toxin
J. Biol. Chem.,
July 30, 1999;
274(31):
21885 - 21892.
[Abstract]
[Full Text]
[PDF]
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P. G. Gallagher and B. D. Smith
Dehydrated Hereditary Stomatocytosis Is Not Linked to the hIK1 Locus, a Gardos Channel Candidate, on Chromosome 19q13.2
Blood,
March 15, 1999;
93(6):
2134 - 2135.
[Full Text]
[PDF]
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C. M. Fanger, S. Ghanshani, N. J. Logsdon, H. Rauer, K. Kalman, J. Zhou, K. Beckingham, K. G. Chandy, M. D. Cahalan, and J. Aiyar
Calmodulin Mediates Calcium-dependent Activation of the Intermediate Conductance Channel, IKCa1
J. Biol. Chem.,
February 26, 1999;
274(9):
5746 - 5754.
[Abstract]
[Full Text]
[PDF]
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A. Mercado, L. Song, N. Vazquez, D. B. Mount, and G. Gamba
Functional Comparison of the K+-Cl- Cotransporters KCC1 and KCC4
J. Biol. Chem.,
September 22, 2000;
275(39):
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[Abstract]
[Full Text]
[PDF]
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D. H. Vandorpe, M. N. Chernova, L. Jiang, L. K. Sellin, S. Wilhelm, A. K. Stuart-Tilley, G. Walz, and S. L. Alper
The Cytoplasmic C-terminal Fragment of Polycystin-1 Regulates a Ca2+-permeable Cation Channel
J. Biol. Chem.,
February 2, 2001;
276(6):
4093 - 4101.
[Abstract]
[Full Text]
[PDF]
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A. C. Gerlach, C. A. Syme, L. Giltinan, J. P. Adelman, and D. C. Devor
ATP-dependent Activation of the Intermediate Conductance, Ca2+-activated K+ Channel, hIK1, Is Conferred by a C-terminal Domain
J. Biol. Chem.,
March 30, 2001;
276(14):
10963 - 10970.
[Abstract]
[Full Text]
[PDF]
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W. J. Joiner, R. Khanna, L. C. Schlichter, and L. K. Kaczmarek
Calmodulin Regulates Assembly and Trafficking of SK4/IK1 Ca2+-activated K+ Channels
J. Biol. Chem.,
October 5, 2001;
276(41):
37980 - 37985.
[Abstract]
[Full Text]
[PDF]
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H. Wulff, G. A. Gutman, M. D. Cahalan, and K. G. Chandy
Delineation of the Clotrimazole/TRAM-34 Binding Site on the Intermediate Conductance Calcium-activated Potassium Channel, IKCa1
J. Biol. Chem.,
August 17, 2001;
276(34):
32040 - 32045.
[Abstract]
[Full Text]
[PDF]
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E. Vazquez, M. Nobles, and M. A. Valverde
Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca2+-dependent potassium channels
PNAS,
April 24, 2001;
98(9):
5329 - 5334.
[Abstract]
[Full Text]
[PDF]
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Copyright © 1998 by the American Society for Biochemistry and Molecular Biology.
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