Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel

Bo Skaaning Jensen1, Dorte Strøbæk1, Palle Christophersen1, Tino Dyhring Jørgensen1, Claus Hansen2, Asli Silahtaroglu2,3, Søren-Peter Olesen1, and Philip Kiær Ahring1

1 NeuroSearch A/S, DK-2600 Glostrup; 2 Department of Medical Genetics, Institute of Medical Biochemistry and Genetics, University of Copenhagen, DK-2200 Copenhagen N, Denmark; and 3 Department of Genetics, Division of Biomedical Sciences, Cerrahpasa Medical Faculty, Istanbul University, TR-34303 Istanbul, Turkey

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The human intermediate-conductance, Ca2+-activated K+ channel (hIK) was identified by searching the expressed sequence tag database. hIK was found to be identical to two recently cloned K+ channels, hSK4 and hIK1. RNA dot blot analysis showed a widespread tissue expression, with the highest levels in salivary gland, placenta, trachea, and lung. With use of fluorescent in situ hybridization and radiation hybrid mapping, hIK mapped to chromosome 19q13.2 in the same region as the disease Diamond-Blackfan anemia. Stable expression of hIK in HEK-293 cells revealed single Ca2+-activated K+ channels exhibiting weak inward rectification (30 and 11 pS at -100 and +100 mV, respectively). Whole cell recordings showed a noninactivating, inwardly rectifying K+ conductance. Ionic selectivity estimated from bi-ionic reversal potentials gave the permeability (PK/PX) sequence K+ = Rb+ (1.0) > Cs+ (10.4) >>  Na+, Li+, N-methyl-D-glucamine (>51). NH+4 blocked the channel completely. hIK was blocked by the classical inhibitors of the Gardos channel charybdotoxin (IC50 28 nM) and clotrimazole (IC50 153 nM) as well as by nitrendipine (IC50 27 nM), Stichodactyla toxin (IC50 291 nM), margatoxin (IC50 459 nM), miconazole (IC50 785 nM), econazole (IC50 2.4 µM), and cetiedil (IC50 79 µM). Finally, 1-ethyl-2-benzimidazolinone, an opener of the T84 cell IK channel, activated hIK with an EC50 of 74 µM.

intermediate-conductance calcium-activated potassium channel; charybdotoxin; clotrimazole; fluorescent in situ hybridization; radiation hybrid mapping; patch clamp; Diamond-Blackfan anemia

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CALCIUM-ACTIVATED POTASSIUM channels are almost ubiquitously distributed in mammalian cells and constitute a major link between second messenger systems and the electrical activity of the cell. On the basis of their electrophysiological characteristics, three major classes of Ca2+-activated K+ channels have been described: voltage-dependent, large-conductance channels (BK); voltage-independent, small-conductance channels (SK); and inwardly rectifying, voltage-independent, intermediate-conductance channels (IK). BK (13) and SK (22) channels are widely distributed in excitable cells as well as in some nonexcitable cells (e.g., Ref. 13). In neurons, their activation underlies the generation of fast and slow afterhyperpolarizations, respectively, indicating a major role in the regulation of neuronal excitability. The human BK channel gene has been cloned from brain and functionally expressed in both Xenopus oocytes and mammalian cells (36). Three genes encoding the class of SK channels have been cloned from human brain (hSK1) and rat brain (rSK2 and rSK3) (22). Through the use of the sequence information from these genes, human IK (hIK1 or hSK4) has recently been cloned from placenta (21) and pancreas (20). The predicted amino acid sequence of hIK is related to but distinct from the neuronal SK channels.

IK channels are apparently absent in excitable tissues but are present in various blood cells (15), endothelial cells (31, 34), and cell lines of epithelial origin (7, 9). Physiologically, IK channels are strongly activated by release of intracellularly stored Ca2+, induced by agonists such as ATP, bradykinin, and histamine. The activation of IK is followed by long-lasting or oscillatory hyperpolarizations of the cell membrane, which closely reflect the intracellular Ca2+ activity. In the present study, we define the chromosomal localization and describe the expression level of hIK in 50 different human tissues. The pharmacology and ion selectivity of the hIK channel after stable transfection of the gene into mammalian HEK-293 cells is examined.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cloning and Sequencing of hIK

A TBLASTN search of the GenBank database at the National Center for Biotechnology Information (NCBI) returned 12 human expressed sequence tag (hEST) sequences with homology to hSK1. Alignment of the sequences with hSK1 suggested that an hEST from placenta (GenBank no. N56819) could encode a full-length K+ channel. This hEST was obtained from the American Tissue Culture Collection.

The cDNA of the hEST clone was sequenced bidirectionally by automated sequencing (ABI 377, Applied Biosystems) using a standard dideoxy chain-termination sequencing kit (Perkin-Elmer). Sequence assembly and analysis were carried out using Lasergene software (DNASTAR). The hEST clone contains a 1284-bp open reading frame, and when sequenced the hEST clone was found to correspond to hIK1/hSK4 (20, 21).

RNA Dot Blot Analysis

A 32P-labeled cDNA probe was generated using random priming (Amersham, Little Chalfont, UK) and used to probe a commercially obtained human RNA master blot (Clontech, lot 7061008). Prehybridizations were for 30 min at 38°C in ExpressHyb (Clontech). Hybridization of the dot blot was for 16 h at 68°C with 109 dpm/µg probe in ExpressHyb. The blot was washed for 45 min at 25°C in 2× saline sodium citrate (SSC)-0.05% SDS, for 40 min at 50°C in 0.1× SSC-0.1% SDS, and finally for 30 min at 65°C in 0.1× SSC-0.1% SDS and was analyzed by autoradiography.

Radiation Hybrid Mapping

DNA from the subset of 86 GeneBridge4 clones for radiation hybrid (RH) mapping (18) was obtained from the Human Genome Mapping Project Resource Center (United Kingdom Medical Research Council). Primers (T-A-G-Copenhagen) located at positions 1474-1690 in the 3' untranslated region of the hIK gene and amplifying a 238-bp fragment were used for RH mapping. PCR amplification was performed for 40 cycles in a PTC-225 DNA engine tetrad thermocycler (MJ Research) at 95, 59, and 72°C. PCR-generated fragments were separated on a 2% agarose gel, and amplification products of correct length were scored as positives, whereas absent products or PCR products of incorrect length were scored as ambiguities or negatives. Mapping was performed by direct submission to the RH mapping facility at The Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research.1 After mapping, neighboring markers of the RH data vector were identified using the ENTREZ genome database at NCBI, and the chromosomal location was documented.

Fluorescent In Situ Hybridization

Fluorescent in situ hybridization (FISH) with corresponding 4',6'-diamidine-2'-phenylindole dihydrochloride (DAPI)-banding and measurement of the relative distance from the long arm telomere to the signals [fractional length from the short arm telomere (FLpter value)] was performed essentially as described previously (25), using 100 ng biotin-labeled 1.9-kb hIK. The metaphases were visualized on a Leica DMRB epifluorescence microscope equipped with a Sensys 1400 charge-coupled device camera (Photometrics) and IPLab Spectrum imaging software (Vysis).

Stable Expression

hIK was excised from pT3T7 using EcoR I and Not I and subcloned into the mammalian expression vector pNS1Z (NeuroSearch), a custom-designed derivative of pcDNA3Zeo (InVitrogen). HEK-293 cells were grown in DMEM (Life Technologies) supplemented with 10% FCS (Life Technologies) at 37°C in 5% CO2. One day before transfection, 106 cells were plated in a cell culture T25 flask (Nunc). Cells were transfected with 2.5 µg of the plasmid pNS1Z_hIK using Lipofectamine (Life Technologies) according to the manufacturer's instructions. Cells transfected with pNS1Z_hIK were selected in media supplemented with 0.25 mg/ml Zeocin. Single clones were picked and propagated in selection media until sufficient cells for freezing were available. Thereafter the cells were cultured in regular medium without selection agent. Expression of functional hIK channels was verified by patch-clamp measurements.

Electrophysiology

Experiments were performed at room temperature with an EPC-9 amplifier (HEKA Electronics, Lambrecht, Germany). Pipettes (1.5-3.0 MOmega ) were pulled from borosilicate glass and, for single-channel experiments, coated with Sylgard and fire polished. A custom-made perfusion chamber (volume 15 µl) with a fixed AgCl-Ag pellet electrode was mounted on the stage of an inverted microscope equipped with Hoffman interference contrast.

A coverslip with transfected HEK-293 cells was transferred to the perfusion chamber and continuously superfused at a rate of 1 ml/min with an extracellular K+ solution with a composition (in mM) of 144 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES (pH 7.4). The pipettes were filled with intracellular solutions consisting of (in mM) 144 KCl, 10 HEPES, 1 or 10 EGTA, 9 or 0 nitrilotetraacetic acid, and MgCl2 and CaCl2 in the concentrations calculated (EqCal, Cambridge, UK) to give free Ca2+ concentrations of 0.1, 0.3, and 3 µM, respectively. Free Mg2+ concentration was always 1 mM, and pH was adjusted to 7.2 before experiments. These intracellular solutions were also used in the bath in inside-out experiments.

In the selectivity study, extracellular solutions consisting of 150 mM of the desired cation (Cl- salts) and 2 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES [pH adjusted to 7.4 with 10 mM N-methyl-D-glucamine (NMDG+)] were used.

The electrode potentials were always zeroed with the open pipette in a high-K+ solution. No zero current or leak current subtraction was performed during the experiments. In whole cell experiments, the cell capacitance and the series resistance (Rs) were updated before each pulse application. Rs values ranged from 3 to 6 MOmega and remained stable during all experiments (10-30 min). At least 80% compensation of Rs was obtained. The current signals were low-pass filtered at 3 kHz (whole cell recordings) or 0.5-1 kHz (single-channel recordings) and digitized at a sample rate of at least three times the filter cutoff frequency. All analyses and drawings were performed with IGOR software (WaveMetrics, Lake Oswego, OR).

The IC50 values reported for the tested compounds were calculated from the kinetics of the block. The time course of the decrease in current (I) was fitted to the equation
<IT>I</IT> = <IT>I</IT><SUB>0</SUB>{1 − C/[C + (<IT>K</IT><SUB>off</SUB>/<IT>K</IT><SUB>on</SUB>)]}{1 − exp[−(C<IT>K</IT><SUB>on</SUB> + <IT>K</IT><SUB>off</SUB>)<IT>t</IT>]}
where Koff is off rate (in s-1), Kon is on rate (in M-1 · s-1), I0 is basal current, C is drug concentration, and t is time. IC50 Koff/Kon, and this is the value that is reported.

Clotrimazole, charybdotoxin, iberiotoxin, apamin, econazole, and miconazole were from Sigma. Ketoconazole was from Research Biochemicals International. 1-Ethyl-2-benzimidazolinone (1-EBIO) was from Aldrich. Kaliotoxin, margatoxin, and Stichodactyla toxin were from Alomone Lab. Stock solutions were prepared in DMSO (clotrimazole, econazole, miconazole, ketoconazole, and 1-EBIO) or water (peptides) and diluted to final concentrations in the appropriate salt solutions. BSA (0.01% wt/vol) was present in all external solutions when peptides, clotrimazole, or clotrimazole analogs were used during the experiment. None of the vehicles had any effect on the recorded currents.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cloning and Sequencing of hIK

The cloning of hIK was based on the identification of clones in the GenBank EST database with low but significant homology to hSK1. Although most of the retrieved ESTs with homology to hSK1 were identical to or highly similar to the channels SK1, SK2, and SK3, the rest exhibited rather low homology (<50%) to known K+ channels and were potentially members of a new branch of the six-transmembrane K+ channel family. The majority of these ESTs are part of the IMAGE Consortium Washington University-Merck EST project (27). Three of the hESTs with low homology to hSK1 were obtained, and two pancreatic hESTs were found to be partial clones by sequencing. The last of these three hESTs was from placenta, and this cDNA clone includes a full-length open reading frame encoding a polypeptide of 427 amino acids. The initiating methionine was assigned to an in-frame ATG within a strong Kozak consensus site (GXXGCC<UNL>ATG</UNL>G) (24) in the most 5' end of the clone. The predicted molecular mass of hIK is 48 kDa.

Tissue Expression of hIK

Northern blot analysis of eight human tissues revealed a prominent 2.3-kb and a 2.9-kb transcript expressed in placenta (not shown). To estimate the tissue distribution of hIK, the expression of hIK in 50 different human tissues was examined by RNA dot blot analysis (Fig. 1). Significant amounts of transcript were evident in several nonexcitable tissues, including salivary gland, placenta, trachea, and lung, but not in any neuronal tissue (Fig. 1). The widespread distribution of hIK in tissues rich in epithelia suggests that hIK may represent an isoform primarily involved in secretion and absorption in salt-transporting epithelia.


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Fig. 1.   RNA dot blot analysis of expression of human intermediate-conductance, Ca2+-activated K+ channel (hIK) in human tissue. Left: human RNA master blot [2 µg of poly(A)+-selected RNA, Clontech] was probed with a 32P-labeled cDNA probe from hIK. Right: schematic representation of dot blot analysis of hIK expression in human tissues: +, detectable levels of transcript (relative expression indicated by no. of +); -, none detectable. Letters and numbers refer to columns and rows, respectively, of master blot at left.

Chromosomal Localization of hIK

Mapping by RH analysis. To define the chromosomal localization of the hIK gene, PCR was performed on DNA from an RH panel that included 86 cell lines (18). Using RH mapping, the hIK-encoding gene was mapped to within 2.74 centiRays (lod score >3.0) of marker WI-6526 and centromeric to D19S420 (17) and is thus contained within chromosomal region 19q13.2-19q13.3.

Mapping by FISH. Specific FISH signals were observed on the distal part of the long arm of chromosome 19, with 58 of 60 analyzed metaphases (97%) displaying at least one specific signal. In total, 139 of the 240 chromatids (58%) were labeled; there were 2 cells with 0, 5 cells with 1, 28 cells with 2, 19 cells with 3, and 5 cells with 4 labeled chromatids. The FLpter value derived from measurements of 37 signal-bearing chromosomes was 0.132 ± 0.021, corresponding to a localization at 19q13.13-19q13.31 (3). The localization of the FISH signals on elongated metaphase chromosomes suggested a finer localization to the border of bands 19q13.13-19q13.2 (Fig. 2).


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Fig. 2.   Fluorescent in situ hybridization (FISH) mapping of hIK. Left: partial metaphase with specific FISH signals on 19q (arrows). Right: ideogram (12) showing localization of hIK based on DAPI-band pattern and mean FLpter value (arrow). Horizontal lines above and below arrow indicate variation in FLpter values among individual signal-bearing chromosomes.

Stable Expression of hIK in HEK-293 Cells

Functional expression of hIK channels was revealed by single-channel as well as whole cell recordings. Figure 3A shows single-channel recordings from an inside-out patch exposed to symmetrical K+-solutions with 0.3 µM free Ca2+ at the intracellular side. Single-channel openings were observed at both positive and negative membrane potentials, and the gating showed no significant voltage dependency. However, the channel exhibited clear open-channel inward rectification. In the experiment depicted in Fig. 3, the unitary inward current fluctuations estimated at -100 mV were 2.9 pA, whereas the corresponding outward currents were only 1.1 pA. The mean values of five independent experiments were 3.0 ± 0.2 and 1.1 ± 0.1 pA at -100 and +100 mV, respectively, corresponding to chord conductances of 30 ± 2 and 11 ± 1 pS, respectively. The single-channel current-voltage (I-V) relation is plotted in Fig. 3B.


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Fig. 3.   A: hIK single-channel currents from an inside-out patch exposed to symmetrical (144 mM) K+ solutions. "Intracellular" free Ca2+ concentration = 0.3 µM. Membrane potential was stepped from -100 mV to 100 mV in increments of 10 mV (15 ms at start and end of current traces was blanked to remove residual capacitive transients). Low-pass filtering: 500 Hz. B: single-channel current-voltage (I-V) curve obtained in symmetrical K+-solutions.

Figure 4A shows a typical whole cell experiment with a slightly inwardly rectifying current developing during the first 30 s after break-in to the whole cell mode (Fig. 4A). Only a linear leak (trace 1) of a maximum of 100 pA was seen when a voltage ramp (-100 to +100 mV, 200 ms in duration) was applied at the very moment that the whole cell configuration was obtained. The hIK current (trace 2 and inset) was activated as the cytosol was exchanged by the pipette solution due to the increase in intracellular Ca2+ concentration (3 µM in pipette solution). The reversal potential (Vr) shifted from 0 to -91 ± 3 mV (mean ± SE, n = 12; trace 3) on reduction of the extracellular K+ concentration from 144 to 4 mM, indicating a high K+/Na+ selectivity (see also below). Furthermore, with the physiological ion gradient, the I-V curve became almost linear in the range from -100 to 50 mV, and a decrease in current was obtained at potentials >50 mV, as previously described for IK currents in human red blood cells (8).


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Fig. 4.   A: whole cell currents (I) recorded on application of voltage ramps from -100 mV to +100 mV (200 ms in duration). Traces were recorded just after break-in (trace 1), 15 s later (trace 2), and after exchange of extracellular solution from a solution with 144 mM K+ to a solution containing only 4 mM K+ (trace 3). Vm, membrane potential. Inset: development of current (Iramp) was measured at +80 mV from voltage ramps, and time course is shown, with 1 and 2 indicating times at which traces were obtained. B: currents recorded from another cell on application of voltage steps from -100 mV to +100 mV (20-mV increments, 100 ms in duration). Extracellular solution contained 144 mM KCl. Free Ca2+ concentration in pipette solutions was 3 µM in both A and B.

Figure 4B shows the immediate appearance of a noninactivating whole cell hIK current as voltage steps were applied. The observed whole cell inward rectification therefore represents an instantaneous property of the channels (open channel rectification or voltage-dependent block) and is not related to voltage-dependent gating, in accordance with the single-channel data.

Selectivity of hIK

The selectivity of the hIK channel was addressed in bi-ionic whole cell experiments. Voltage ramps (-130 to +70 mV, 200 ms in duration) were applied, and control traces were recorded with the same high-K+ solution in the pipette and in the bath. The bath solution was then changed to a solution containing 150 mM XCl, with X being Rb+, Cs+, Na+, Li+, NH+4, or NMDG+. At the end of the experiment, 200 nM charybdotoxin was added to confirm the identity of the current. Figure 5 shows representative traces obtained with extracellular K+ compared with those obtained in Rb+, Na+, Li+, NMDG+, Cs+, and NH+4. Exchange of the bath solution from K+ to Rb+ did not alter the Vr (0 ± 0.3 mV, n = 15 for K+; 1 ± 0.5 mV, n = 4 for Rb+; means ± SE), indicating an equal permeability of K+ and Rb+. With extracellular Cs+, the Vr shifted toward negative potentials, with a small but distinct (charybdotoxin-sensitive) inward current at potentials negative to -59 ± 1.4 mV (mean ± SE, n = 4), indicating a significant Cs+ permeability. However, with Na+, Li+, or NMDG+ in the bath, the Vr shifted from 0 mV to potentials more negative than -100 mV (often more than -130 mV, n = 3-7) within 30 s, indicating virtually no permeation of these ions. It was not possible to obtain an estimate of the permeability to NH+4, since this ion completely blocked the inward as well as the outward currents (n = 16). The permeability ratios (PK/PX) were calculated from the Vr values using the equation PK/PX = exp(-VrF/RT), where F is Faraday's constant, R is the gas constant, and T is absolute temperature. The following permeability sequence was obtained: K+ (1.0) = Rb+ (1.0) > Cs+ (10.4) >>  Na+, Li+, NMDG+ (>51). In the Eisenman theory for equilibrium selectivity, the observed sequence for the alkali metal ions corresponds to binding to a site of intermediate field strength (series III or IV).


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Fig. 5.   Currents recorded on application of voltage ramps from -130 mV to +70 mV to transfected cells in whole cell configuration. Pipettes contained an intracellular high-K+ solution with free Ca2+ concentration buffered to 300 nM. Each trace shows a new cell. Solid lines, traces obtained with K+ solution in bath; dashed lines, traces recorded with indicated cations (150 mM) in bath. Note that dashed trace for NH+4 is almost covered by x-axis. Currents recorded in K+ solution at -130 mV in individual cells were normalized for comparison.

Pharmacology of hIK

The sensitivity of the expressed hIK channel to three K+ channel blockers is shown in Fig. 6. Ramp currents were elicited every 5 s, and the time course of the current at +80 and -80 mV is shown in Fig. 6A. Addition of 1 µM clotrimazole, a blocker of the IK channel in human erythrocytes (1), blocked the current in a reversible way. The SK channel inhibitor apamin (100 nM) was added for 2 min but failed to influence the hIK current. Finally, a nearly total block of the current was obtained after a shift to an extracellular solution containing 100 nM charybdotoxin. Figure 6B shows the control traces compared with the traces obtained after application of each of the three compounds for 1-2 min. IC50 values of 153 ± 33 nM (n = 11) for clotrimazole and 28 ± 3 nM (n = 19) for charybdotoxin were obtained. It is apparent from the time courses at ±80 mV as well as from the current traces that neither the block by clotrimazole nor the block by charybdotoxin was voltage dependent. The high sensitivity to block by charybdotoxin and clotrimazole, as well as the insensitivity toward the selective SK2 and SK3 channel blocker apamin, is consistent with the current being conducted by voltage-independent, Ca2+-activated K+ channels closely related to the human erythrocyte IK channel.


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Fig. 6.   A: time course of a whole cell experiment after application of clotrimazole (CLT; 1 µM), apamin (100 nM), and charybdotoxin (ChTx; 100 nM). hIK currents were measured at +80 mV (open circle ) and at -80 mV (triangle ) from traces obtained after application of voltage ramps. B: control (solid lines) and wash (dotted lines) traces, as well as traces recorded in presence of 3 compounds (dashed lines) after application of voltage ramps.

To strengthen this view further, we extended the pharmacological study to include 1) other peptides with well-known K+ channel blocker profiles, 2) imidazole antimycotics considered to be analogs of clotrimazole, and 3) various other compounds previously reported to modulate native IK channels. Figure 7 illustrates the pharmacological experiments. Among the peptides, Stichodactyla toxin (100 nM; Fig. 7A) and margatoxin (100 nM; Fig. 7B) blocked the current, although less potently than charybdotoxin. The mean IC50 values obtained were 291 ± 50 nM (n = 3) for Stichodactyla toxin and 459 ± 34 nM (n = 3) for margatoxin. Also shown in Fig. 7B is the lack of effect of the SK channel blocker dequalinium chloride (1 µM) (6). Furthermore, kaliotoxin (50 nM, n = 3) and iberiotoxin (300 nM, n = 5) had no discernible effects (Fig. 7C). This potency order is consistent with that obtained by Brugnara et al. (4) on Ca2+-activated K+ transport in erythrocytes, although the IC50 values obtained with erythrocyte suspensions were somewhat lower than the values obtained in the present study.


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Fig. 7.   A-F: time course of whole cell currents at +80 mV measured from voltage ramps (±100 mV every 5 s, 200 ms in duration) under control conditions (extracellular K+ solution in bath) and during application of indicated compounds. Each trace represents a new cell. Concentrations used were 100 nM Stichodactyla toxin (StK), 50-100 nM charybdotoxin, 100 nM margatoxin (MgTx), 1 µM dequalinium chloride (Deq.Cl), 50 nM kaliotoxin (KTx), 300 nM iberiotoxin (IbTx), 5 µM econazole, 5 µM ketoconazole, 1 µM nitrendipine, 100 µM cetiedil, and 10 µM 1-ethyl-2-benzimidazolinone (1-EBIO). Free Ca2+ concentration in pipette solutions was 300 nM, and increase in currents apparent at beginning of each trace is activation of current by Ca2+ in pipette solution.

In addition to clotrimazole, the Ca2+-dependent K+ channels in human erythrocytes are blocked by several imidazole antimycotics (1). Figure 7D shows that hIK is blocked by econazole (IC50 = 2.4 ± 0.5 µM, n = 7), whereas ketoconazole is without effect (5 µM; n = 4). Miconazole blocked hIK with an IC50 = 785 ± 107 nM (n = 6).

Several classical Ca2+ channel antagonists were found to inhibit the Gardos channel (11), and the dihydropyridine nitrendipine is the most potent compound in this chemically diverse group of compounds. The effect of 300 nM nitrendipine is shown in Fig. 7E. The IC50 for the block by nitrendipine was 27 ± 6 nM (n = 6); nifedipine, a nitrendipine analog, was much less potent (1.5 ± 0.2 µM, n = 4), as were verapamil (72 ± 20 µM, n = 4) and diltiazem (154 ± 22 µM, n = 4).

Finally, cetiedil, a compound previously tested for its effect in sickle cell anemia, blocked the current with an IC50 value of 79 ± 44 µM (Fig. 7F; n = 4). Figure 7F also shows an activation of hIK by 10 µM 1-EBIO, which has been reported to activate Ca2+-activated K+ currents in colonic T84 epithelial cells and to stimulate short-circuit current and secretion in epithelia (10).

The activation of hIK by 1-EBIO was further elucidated by running cumulative dose-response experiments like the one shown in Fig. 8. In these experiments, the intracellular Ca2+ concentration was buffered at 0.1 µM, which is too low for significant activation of the channels. However, superfusion with 1-EBIO dose dependently activated the hIK current. The time course of a typical experiment is shown in Fig. 8A; the corresponding I-V curves are plotted in Fig. 8B. From the dose-response curve in Fig. 8C, an EC50 value of 74 ± 11 µM (n = 3) was estimated.


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Fig. 8.   A: time course of whole cell current as in Fig. 7. B: corresponding whole cell currents measured with 0 (control; con), 5, 15, 50, 150, and 500 µM 1-EBIO in bath. C: mean dose-response relationship from 3 cells.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In summary, we have cloned, expressed, and functionally characterized an hIK from human placenta. The gene was localized at chromosome 19 at region 19q13.2-19q13.3, and mRNA expression was detected at the highest density in salivary gland, placenta, lung, and trachea and in smaller quantities in liver, colon, thymus, kidney, and bone marrow. It was absent from excitable tissues, such as brain and skeletal and heart muscle, as well as from most embryonic tissues except liver. Recently, data on the cloning and expression of a pancreatic IK channel with similar characteristics were presented by Ishii et al. (20), whereas Kaczmarek and co-workers (21) designated the very same channel hSK4 due to the similarity of hIK to the SK channel family (~45% identity on the amino acid level). However, the functional and pharmacological characteristics, as well as the tissue distribution of hIK expression, strongly suggest that the cloned channel is identical to the well-defined type of IK channel present in a number of nonexcitable cells, such as T and B lymphocytes (14, 32), red blood cells (8, 15), and cells derived from epithelia (7, 35) and endothelia (34). Logsdon et al. (28) recently cloned an IK channel from T lymphocytes that is identical to hIK.

The unifying functional characteristics of this group of native channels are the single-channel conductance, which exhibits weak inward rectification in symmetrical K+ (30-40 pS at -100 mV; 10-15 pS at 100 mV), the weak or absent voltage dependency of gating, and the high and steep sensitivity to intracellular Ca2+ (8, 10, 15, 31, 35). In comparison, the estimated single-channel chord conductance for the cloned channel is 30 pS (present study) or ~33 pS (20).

The cloned channel was found to be highly selective for K+, with demonstrable permeability (PX/PK) to Rb+ (1.0) and Cs+ (10.4), whereas Li+, Na+, and NMDG+ were largely impermeable (>51). The permeability sequence obtained for hIK closely resembles that of the prototype channel within this group, the erythrocyte K+ channel (8). The notable differences are 1) that Cs+ in the present study was demonstrated to conduct inward current, 2) the powerful block of the cloned channel by NH+4, and 3) a tendency of hIK toward decreased selectivity after prolonged exposure to K+-free solutions. Regarding difference 1, inward single-channel currents conducted by Cs+ were impossible to demonstrate in the erythrocyte channel study, most likely due to very small single-channel currents. However, the extrapolated value of the Vr in that study corresponds to the value determined here. Regarding difference 2, the erythrocyte channel was not completely blocked by NH+4, and genuine permeability measurements were possible at the single-channel level (PK/PX = 8.5). However, the gating of the erythrocyte channel was pronouncedly changed in NH+4 solutions (short openings, low open state probability) (2). Qualitatively, the complete block seen in the present study may reflect the same phenomenon. Regarding difference 3, after a longer period in Na+, Li+, or NMDG+, the decrease of the outward current at positive potentials became more prominent, a small inward current developed at very negative potentials, and the Vr shifted toward less negative potentials. This could indicate that the channels become permeable to these ions in the total absence of external K+, as described by Korn and Ikeda (23) for Kv1.5. However, although mechanistically interesting, the phenomenon has not been further elucidated in this study.

In addition to the difference in open channel properties, the SK and IK channels can be distinguished pharmacologically. It is well known that charybdotoxin blocks IK channels with high affinity (low nanomolar range, e.g., Ref. 4), whereas SK channels are not affected. Conversely, apamin blocks certain SK channels (equivalent to the cloned SK2 and SK3), whereas there is no effect on IK channels. Because the cloned hIK is potently blocked by charybdotoxin and is insensitive to apamin [and to dequalinium, which binds to the apamin-site (6)], the basic pharmacology points toward a classical IK channel. The experiments with the K+ channel-blocking peptides clearly show that charybdotoxin is the most potent, with a potency order exactly as described for erythrocytes. Clotrimazole, nitrendipine, cetiedil, and 1-EBIO are classical modulators of IK channels, but their selectivity properties are largely unknown. However, the overall correlation between the potency of these compounds (and their analogs) on native IK channels and the cloned channel supports the conclusion of a closely related pharmacology.

The physiological significance of IK channels has been described in various tissues. The erythrocyte K+ channel was the first Ca2+-activated K+ channel ever described, but despite this the function of IK channels in normal erythrocytes is obscure, whereas pathological activation is known to be critical in the development of crises in sickle cell anemia (5). The hIK gene is mapped to chromosome 19q13.2 at the same localization as Diamond-Blackfan anemia (16), a disease with the clinical hallmarks of a selective decrease in erythroid precursors and anemia. In some salt (and water) secreting epithelia, activation of basolateral IK channels is essential for maintaining the driving force for luminal Cl- efflux (10). In the lung, where hIK is expressed relatively abundantly, a charybdotoxin-sensitive IK channel is thought to hyperpolarize the airway epithelia in response to agonists that elevate intracellular cAMP and Ca2+, thus facilitating apical Cl- secretion (29, 37). Possibly a pharmacological activator of hIK might therefore be of clinical interest in the treatment of mild forms of cystic fibrosis. Furthermore, hIK is expressed in colon (Fig. 1), and activation of Ca2+-activated K+ channels has been shown to be coupled to the apical Cl- secretion (9, 33). Blockers of hIK may thus prove efficient in the treatment of secretory diarrhea. The high level of mRNA expression detected in salt-transporting tissues (Fig. 1) may indicate a universal role of IK channels in epithelial function. In T and B lymphocytes, the number of IK channels is upregulated on activation with antibodies or the protein kinase C activator phorbol 12-myristate 13-acetate (14, 32), suggesting important functions of this class of channels in the activation of the immune system.

A number of native channels with characteristics different from those of the erythrocyte-type IK channel have traditionally been classified as IK channels. Thus the IK channel group is quite heterogeneous, comprising channels that differ widely in single-channel conductances (20-150 pS), degree of rectification, voltage dependency of gating, pharmacology, and Ca2+ sensitivity (19, 26, 30). Many of these channels deviate strongly from the channel described in the present paper, and some are even expressed in excitable cells. It is therefore possible that a whole family of IK channels exists and that more types of Ca2+-activated K+ channels with intermediate conductance will be cloned in the future.

    ACKNOWLEDGEMENTS

We gratefully acknowledge L. G. Larsen and H. Z. Andresen for excellent technical assistance.

    FOOTNOTES

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. §1734 solely to indicate this fact.

1 RH mapping can be performed via the World Wide Web (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl).

Address for reprint requests: B. S. Jensen, NeuroSearch A/S, 26B Smedeland, DK-2600 Glostrup, Denmark.

Received 2 March 1998; accepted in final form 8 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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