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J Biol Chem, Vol. 273, Issue 34, 21542-21553, August 21, 1998
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
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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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.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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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 |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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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.
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-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.
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(Eq. 1) |
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.
![[<UP>Ca<SUP>2+</SUP></UP>]<SUB>i</SUB>)=K<SUB><UP>app</UP></SUB>×<FENCE>F−F<SUB><UP>min</UP></SUB></FENCE>/<FENCE>F<SUB><UP>max</UP></SUB>−F</FENCE>](/content/vol273/issue34/fulltext/21542/img002.gif)
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(Eq. 2) |
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 × 104 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 |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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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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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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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.
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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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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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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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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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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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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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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-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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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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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).
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?
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
/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.
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ACKNOWLEDGEMENTS |
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We thank Satoshi Takamatsu for ES cell RNA, Paul Savin for technical assistance, and Leah Staffier for graphics assistance.
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FOOTNOTES |
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* 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
S6) and the
pore (P) region are indicated above the sequences.
Underlined residues are those assigned to transmembrane
regions by all reports (
