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Am J Physiol Heart Circ Physiol 274: H892-H900, 1998;
0363-6135/98 $5.00
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Vol. 274, Issue 3, H892-H900, March 1998

Inhibition of rat ventricular IK1 with antisense oligonucleotides targeted to Kir2.1 mRNA

Tomoe Y. Nakamura1, Michael Artman1,2, Bernardo Rudy2,3, and William A. Coetzee1,2

Departments of 1 Pediatrics, 2 Physiology and Neurosciences, and 3 Biochemistry, New York University Medical Center, New York, New York 10016

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

The cardiac inward rectifying K+ current (IK1) is important in maintaining the maximum diastolic potential. We used antisense oligonucleotides to determine the role of Kir2.1 channel proteins in the genesis of native rat ventricular IK1. A combination of two antisense phosphorothioate oligonucleotides inhibited heterologously expressed Kir2.1 currents in Xenopus oocytes, either when coinjected with Kir2.1 cRNA or when applied in the incubation medium. Specificity was demonstrated by the lack of inhibition of Kir2.2 and Kir2.3 currents in oocytes. In rat ventricular myocytes (4-5 days culture), these oligonucleotides caused a significant reduction of whole cell IK1 (without reducing the transient outward K+ current or the L-type Ca2+ current). Cell-attached patches demonstrated the occurrence of multiple channel events in control myocytes (8, 14, 21, 35, 43, and 80 pS). The 21-pS channel was specifically knocked down in antisense-treated myocytes (fewer patches contained this channel, and its open frequency was reduced). These results demonstrate that the Kir2.1 gene encodes a specific native 21-pS K+-channel protein and that this channel has an essential role in the genesis of cardiac IK1.

potassium channels; inward rectifier current; inward rectifying potassium channels

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

INWARD RECTIFYING K+ channels are critically important in heart function because they set the maximum diastolic potential, which in turn helps to determine the upstroke velocity of the action potential and hence the conduction velocity. Furthermore, they carry a small but significant outward current during late stages of the action potential, thus contributing significantly to final repolarization (33). Comparatively less outward K+ current is passed than current in the inward direction; this property is termed "inward rectification."

Although the macroscopic phenotype of cardiac inward rectifying K+ current (IK1) is a strongly rectifying K+-selective current that is blocked by inorganic cations such as Ba2+ and Cs+ (12, 25), there is increasing evidence that IK1 actually consists of several different types of K+ channels. In isolated membrane patches under identical experimental conditions (symmetrical 140-150 mM K+ and at room temperature), IK1 channels have unitary conductances ranging between 8 and 35 pS (15, 17, 41). Furthermore, during development, there is an onset of different IK1 channels with distinct unitary conductance levels (4, 16, 40). These observations suggest that there may be a family of channels comprising whole cell IK1. However, the molecular composition of channels responsible for IK1 is presently unknown.

A family of genes coding for inward rectifying K+ (Kir) channels has recently been identified (19). These proteins all share the same basic structure of two putative membrane-spanning regions [called M1 and M2; analogous with S5 and S6 of voltage-gated K+ (Kv) channels] flanking a well-conserved pore region (H5 or P segment). Within the Kir family, most sequence similarity occurs in the membrane-spanning domains and pore regions, whereas the carboxy- and amino-termini are less well conserved. There are currently six Kir subfamilies (Kir1 to Kir6) classified according to similarities in amino acid sequence (6). These include 1) weakly inward rectifiers with ATP-binding domains [e.g., Kir1.1/ROMK1 (11)], 2) "classical" strongly Kir [such as Kir2.1/IRK1 (19)], 3) G protein-activated K+ channels [e.g., Kir3.1/GIRK1 (20)], and 4) members of ATP-inhibited K+ channels [Kir6 subfamily members (13)]. Other subfamily members are represented by Kir4.1/BIR10 (3) and Kir5.1/BIR9 (3). Of these, genes of the Kir2 subfamily are the most likely candidates coding for the various native IK1 channel proteins; like native IK1, Kir2 currents exhibit strong inward rectification, they do not require specific modulation for channel opening [such as by G proteins or the absence of ATP in the case of acetylcholine-activated or ATP-sensitive K+ channels (KATP), respectively], their single channel conductances resemble those of natively occurring IK1 channels, and Kir2 transcripts are found in heart tissue (19, 28, 35). In particular, the unitary conductance of Kir2.1 channels, which is 21 pS in heterologous expression systems (19), closely resembles that of the most predominantly occurring native IK1 channel [20-30 pS in ventricle (15, 24)]. However, any resemblance in single channel conductance between cloned channels (especially when expressed in oocytes) and native IK1 channels should be interpreted with caution, partly due to the different ionic conditions that often exist and partly because any resemblance in conductance between cloned and native channels does not necessarily imply a causal relationship. Other experimental techniques are therefore required to determine the functional contribution of a particular channel protein to a native whole cell current.

The rapid expansion of the use of antisense oligodeoxynucleotides (AS oligos) as inhibitors of specific gene transcripts over the past few years has led us to utilize this technology as a means of identifying the molecular components of specific native channels. We assessed the role of Kir2.1 channel proteins in rat ventricular IK1 using AS oligos specifically targeted to Kir2.1 mRNA. We examined the effect of these oligonucleotides on both whole cell IK1 and cell-attached single K+ channels recorded from isolated rat ventricular myocytes. Our results demonstrate a reduction of whole cell IK1 and a specific reduction of an abundantly occurring 21-pS channel (but not other K+ channels with different conductances), suggesting that Kir2.1 channel proteins are essential in generating a substantial component of native rat ventricular IK1.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Design of AS oligos. We designed two separate phosphorothioate oligonucleotides (AS oligos; which provide a high resistance to nuclease degradation) against different regions of Kir2.1 (Fig. 1). The first oligonucleotide (1022S; 22 mer with a 55% GC content) spanned the initiation codon (ATG). This oligonucleotide has sequence identity with the primary sequence of rat and mouse Kir2.1/IRK1 (with a single base mismatch in the human sequence). The interspecies identity in the first three bases of the 5'-untranslated region (Fig. 1) also predicts similarity in this region for rat Kir2.1 (for which this sequence is unavailable). The primary sequences of rat Kir2.2 and Kir2.3 have a low sequence similarity with the 1022S AS oligo (36 and 45%, respectively). The second AS oligo (1023S; 65% GC content) was targeted against a region 11 base pairs upstream of the termination codon of Kir2.1. Again, there was a high interspecies similarity in this region (only a single base mismatch occurs in rat and human sequences), whereas the similarity to rat Kir2.2 and Kir2.3 primary sequences across this region was <50%. The sequence of these two AS oligos therefore predicts specificity for Kir2.1 (even across several species). After synthesis, these oligonucleotides were purified by high-pressure liquid chromatography (GeneLink).


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  		Fig. 1.   Alignment of nucleotide sequences of antisense oligonucleotides (AS oligos) and the primary sequences of Kir2.1, Kir2.2, and Kir2.3. Two oligonucleotides [1022S (A) and 1023S (B)] were used. The following primary sequences were obtained from Genbank: rat Kir2.1 (accession no. L48490), mouse Kir2.1 (X73052), human Kir2.1 (U16861), rat Kir2.2 (X78461), and rat Kir2.3 (X87635). Nos. along the sequences represent the numerical position of the published sequence. Dots represent nucleotide identity with the oligonucleotides, and dashes represent missing nucleotides. Capital letters above the sequences represent corresponding amino acids of Kir2.1, and asterisks represent a region of noncoding sequence. Initiation codon (ATG) is underlined.

In vitro transcription and oocyte injection. cDNA was linearized, and capped cRNA was synthesized in vitro using the enzymes in parenthesis: Kir2.1 (IRK1) cloned from mouse macrophage (a kind gift from Dr. L. Jan; Not I/T7), Kir2.2 (IRK2) cloned from mouse brain (a kind gift from Dr. Y. Kurachi; Xho I/T3), and Kir2.3 (HIR or IRK3) cloned from human hippocampus (a kind gift from Dr. C. Vanderberg; Hind III/T3). The concentration of full-length cRNA was determined by electrophoresis on a denaturing agarose gel and by comparing the band density with a known standard. Adult female Xenopus laevis frogs (Nasco, Fort Atkinson, WI) were anesthetized with 0.17% 3-aminobenzoic acid ethyl ester (Sigma), and unfertilized oocytes were harvested after abdominal incision. Stage V-VI oocytes were stripped under a dissecting microscope and defolliculated enzymatically with 1 mg/ml collagenase (Sigma type I) in Ca2+-free ND-96 solution [in mM: 96 NaCl, 2 KCl, 1 MgCl2, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5 adjusted with NaOH] for 30 min at room temperature. Defolliculated oocytes were injected with 50 nl of cRNA (~3 fmol) and AS oligos (40 fmol each) or with diethyl pyrocarbomate-treated H2O using a 10-µl micropipette (Drummond Scientific, Broomall, PA). Injected oocytes were kept in 0.5× L-15 solution [1:1 dilution of GIBCO's Leibovits L-15 medium in filter-sterilized 50 mM HEPES buffer (pH 7.5 adjusted with NaOH) containing 50 U/ml nystatin (GIBCO-BRL) and 0.1 mg/ml gentamicin (GIBCO-BRL)] in the presence or absence of AS oligos (3 µM each) at 18-20°C. They were used for recording 2-5 days after injection.

Electrophysiological measurements in oocytes. Oocytes were voltage clamped using the two-microelectrode voltage-clamp technique. Microelectrodes were pulled from thin-walled glass with tip resistance of 0.7-1.2 MOmega for the current-passing electrode and 1-5 MOmega for the voltage-measuring electrode when filled with 3 M KCl solution. Recordings were obtained using a Geneclamp 500 amplifier (Axon Instruments) with data sampled at 5 kHz and filtered at 1 kHz. No leak subtraction was performed during the experiments. The recording chamber was continually perfused (1 ml/min). To avoid contamination with Ca2+-activated Cl- currents, a low-Cl- recording solution (KD-96) was used (in mM: 96 potassium glutamate, 2 sodium glutamate, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.5 adjusted with NaOH). All experiments were performed at room temperature (20 ± 2°C).

Isolation and culturing of ventricular myocytes. Single ventricular myocytes were isolated using standard enzymatic techniques under sterilized conditions. Briefly, adult Wistar rats (200-250 g) were anesthetized with pentobarbital sodium (50 mg/kg), and their hearts were removed and rapidly cannulated while bathed in Tyrode solution (in mM: 137 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 10 HEPES, and 10 glucose, pH 7.4 adjusted with NaOH). Hearts were perfused using a constant-pressure (60 mmHg) Langendorff perfusion system at 34°C with 1) Tyrode solution for 5 min, 2) nominally Ca2+-free Tyrode solution (in mM: 135 NaCl, 5.4 KCl, 0.33 NaH2PO4, 1.0 MgCl2, 10 HEPES, and 10 glucose, pH 7.2 adjusted with NaOH) for 5 min, 3) Ca2+-free Tyrode solution containing 0.1% collagenase (Sigma type I) and 0.014% protease (Sigma type XIV) for 10 min, and 4) Ca2+-free Tyrode solution for 5 min. All solutions were bubbled with 100% O2 during the perfusion procedure. The ventricles were separated and cut into small pieces in Kraftbrüfe (KB) solution [in mM: 20 taurine, 50 glutamic acid, 10 HEPES, 0.5 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 3 MgSO4, 30 KH2PO4, and 30 KCl, pH 7.2 adjusted with KOH], and myocytes were obtained by mechanical agitation. The cell suspension was filtered (200-µm mesh), centrifuged (50 g for 30 s), and resuspended in KB solution. Cells were kept in KB solution for 2 h at room temperature and then resuspended in Eagle's minimal essential medium containing 10% fetal bovine serum. They were plated on laminin (100 µg/ml)-coated glass coverslips (104 rod-shaped cells/cm2) and allowed to attach by incubation for 4 h at 37°C under a water-saturated atmosphere with 5% CO2. Debris and unattached myocytes were washed away by changing the medium to serum-free Eagle's minimal essential medium with or without AS oligos (3 µM). The medium was changed daily in the presence or absence of AS oligos.

Whole cell recordings of IK1 in myocytes. Standard patch-clamp techniques were used to obtain whole cell recordings using an Axopatch 200A amplifier and pCLAMP software (Axon Instruments). Patch electrodes were made from thin-walled glass capillaries (1.5 mm OD) using a horizontal puller (Universal Puller; Zeitz Instrumente, Augsburg, Germany) and heat polished. When filled with a pipette solution (in mM: 115 potassium aspartate, 5 KCl, 4 Na2ATP, 7 MgCl2, 5 EGTA, and 10 HEPES, pH 7.2 adjusted with KOH), electrode resistance ranged between 2 and 4 MOmega . Cell capacitance and pipette series resistance were compensated (usually >80%). Whole cell current density was expressed as picoampere per picofarad. The liquid junction potential was calculated using Axoscope (Axon Instruments).

Single channel recordings of IK1 channels in myocytes. The single channel current was recorded in the cell-attached or inside-out patch configurations. Patch electrodes were made from thick-walled glass capillaries (1.5 mm OD, 1.12 mm ID), heat polished, and Sylgard coated. When filled with a pipette solution (in mM: 140 KCl, 10 HEPES, and 1 CaCl2, pH 7.2 adjusted with KOH), electrode resistance ranged between 4 and 8 MOmega . The bath solution contained (in mM) 65 KCl, 60 potassium aspartate, 1 EGTA, and 10 HEPES, pH 7.2 adjusted with KOH. Thus, in cell-attached patches, the equilibrium potential for K+ is expected to be close to 0 mV, whereas that for Cl- is expected to be around -30 to -50 mV (assuming an intracellular Cl- concentration of 20-40 mM). For some inside-out patches, MgATP (3 mM) was added to the bath solution to block KATP channels. Currents were low-pass filtered (2 kHz; 8-pole Bessel response), acquired at 5 kHz (pCLAMP), and stored on a computer hard drive.

Data analysis. Data were analyzed using the pCLAMP suite of software (Axon Instruments) and Origin for Windows (Microcal Software, Northamptom, MA) software. For single channel recordings, the unitary current amplitude was determined by creating an events list followed by constructing an amplitude histogram of 20 s of recorded data. Curve fitting (a sum of Gaussian distributions) was performed on the amplitude histograms using pCLAMP or Origin. Data are expressed as means ± SE. Comparisons between data groups were performed using a t-test or Fisher's exact test. Differences at P < 0.05 were considered significant.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Specificity of AS oligos. To test the efficacy of Kir2.1 AS oligos, oocytes were injected with Kir2.1, Kir2.2, or Kir2.3 cRNA (3 fmol each) or coinjected with cRNA and a combination of the two AS oligos (1022S and 1023S; 40 fmol each). Two days after injection, oocytes injected with only Kir2.1 cRNA expressed large strongly rectifying currents (Fig. 2A). In contrast, when oocytes were coinjected with a combination of Kir2.1 cRNA and the two AS oligos, the current amplitudes were <10% of those not injected with AS oligos (Fig. 2A). A separate batch of oocytes was injected with Kir2.1 cRNA and bathed in medium supplemented with 1022S and 1023S (3 µM each). At 2 days after injection, there was no significant difference between Kir2.1 currents of these oocytes (Fig. 2A) and those without AS oligos in the medium. From day 2 to day 4 after injection, Kir2.1 currents increased in amplitude (Fig. 2B), but a substantial reduction in Kir2.1 current was observed over this period when oocytes were bathed in a combination of the two AS oligos (Fig. 2). This is not caused by a nonspecific decrease resulting from AS oligos, since we previously observed that oligos with a similar GC content to those used in this study, but which had no sequence similarity with Kir2.1, did not affect expression of Kir2.1 currents in oocytes (27). Thus Kir2.1 AS oligos inhibited Kir2.1 currents when applied in the incubating medium, suggesting that the AS oligos entered the oocyte through the plasma membrane. The relatively slow onset of action of extracellularly administered AS oligos seen in this study may be caused by a combination of a relatively slow entry of oligonucleotides through the plasma membrane and by the large volume of the oocytes, which may allow expression of high levels of channel protein before AS oligos could act on the target RNA. Under these conditions, any reductions in Kir2.1 current amplitude are expected to reflect the rate of protein turnover. The decrease of Kir2.1 current in the presence of AS oligos between days 2 and 4 is therefore consistent with a relatively short half-life of Kir2.1 protein.


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  		Fig. 2.   Antisense oligonucleotides inhibit Kir2.1 currents expressed in Xenopus oocytes. Oocytes were injected with Kir2.1 cRNA (3 fmol) and allowed to express Kir2.1 currents for 2 (A) or 4 (B) days after injection. Oocytes were injected with only Kir2.1 cRNA (bullet ) or with a combination of Kir2.1 cRNA and AS oligos (black-triangle; 40 fmol). Other oocytes (injected only with Kir2.1 cRNA) were incubated in the presence of oligonucleotides in the medium (3 µM each; square ). Experiments were performed with KD-96 bathing solution at room temperature. Clamping protocol consisted of square step voltage-clamp pulses from -120 to +50 mV from a holding potential of 0 mV (100-ms duration, 0.2 Hz).

To test the specificity of the AS oligos to Kir2.1, oocytes were injected with cRNA from closely related genes, Kir2.2 or Kir2.3. Some of these oocytes were coinjected with a combination of 1022S and 1023S AS oligos, in a concentration identical to that used above. Although coinjection with AS oligos almost totally abolished the Kir2.1 currents (Fig. 2), there was no significant effect on expression of either Kir2.2 or Kir2.3 currents (Fig. 3). These results therefore demonstrate that the combination of these AS oligos directed at the Kir2.1 transcript potently and specifically inhibited Kir2.1 currents in oocytes without having an effect on the closely related Kir2.2 or Kir2.3 transcripts.


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  		Fig. 3.   No effect of antisense oligonucleotides on Kir2.2 or Kir2.3 currents expressed in Xenopus oocytes. Oocytes were injected with only Kir2.2 (A) or Kir2.3 (B) cRNA (bullet ) or coinjected with a combination of cRNA and AS oligos (black-triangle). Experimental solutions and protocol were the same as described in Fig. 2.

AS oligos inhibit native rat ventricular IK1. To assess the effect of Kir2.1 AS oligos on native rat ventricular IK1, we incubated myocytes in the presence of 1022S and 1023S (3 µM each) for a period of 4-5 days. To eliminate Na+ and Ca2+ currents, whole cell inward rectifier K+ currents were recorded from a holding potential of -55 mV in the presence of a Ca2+ channel blocker (D-600; 10 µM). IK1 currents were recorded at -120 mV, followed by a recording of the transient outward current at +60 mV (where little IK1 flows). Time-matched control experiments were performed on myocytes incubated in the absence of oligonucleotides or in the presence of AS oligos having a comparable GC content but no sequence similarity to the Kir2.1 transcript. Peak inward current (measured near the beginning of the hyperpolarizing clamp) was identical in the two groups after 4 days of incubation (Fig. 4B). This inward current was blocked by removal of extracellular K+ or application of Ba2+ (1 mM; data not shown), thus exhibiting characteristics typical of IK1. When myocytes were incubated in the presence of Kir2.1 AS oligos, peak IK1 was significantly reduced compared with control myocytes, but peak transient outward current was not inhibited (Fig. 4). These results indicate that Kir2.1 AS oligos inhibit specific K+ currents in rat ventricular myocytes and suggest that Kir2.1 is a major molecular component of the channels responsible for native rat ventricular IK1.


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  		Fig. 4.   Kir2.1 antisense oligonucleotides inhibit native rat ventricular inward rectifying K+ current (IK1). A: adult rat ventricular myocytes were incubated in the presence or absence of 1022S and 1023S (3 µM each) or with control oligonucleotides [as used previously (27)] for a period of 4 days. Whole cell IK1 or transient outward currents (Ito) were recorded at the beginning of a 160-ms test pulse to -120 or +60 mV, respectively, from a holding potential of -55 mV. After the clamp step, the membrane was repolarized to -80 mV. Voltage-clamp episodes were repeated every 5 s. B: average current densities of IK1 (at -120 mV) and Ito (at +60 mV) of control myocytes (no oligonucleotides added; n = 10), Kir2.1 antisense-treated myocytes (n = 8), and myocytes treated with control oligonucleotides with no sequence similarity with Kir2.1 (control oligos; n = 7). Filled bars, control; open bars, Kir2.1 AS oligos; shaded bars, control oligos. * P < 0.05 vs. control.

We also examined the effects of Kir2.1 AS oligos on whole cell K+ and Ca2+ currents. In the presence of 4-aminopyridine (3 mM) to block transient outward current, the membrane potential was stepped to 0 mV (400-ms duration) from a holding potential of -45 mV, first in the absence and then in the presence of D-600 (10 µM), to obtain a measure of the L-type Ca2+ current density. Using this experimental protocol, we found no difference between the density of L-type Ca2+ current [-6.3 ± 1.24 (n = 7) vs. -7.8 ± 1.94 (n = 6) pA/pF in control and Kir 2.1 AS oligo groups, respectively]. While 4-aminopyridine and D-600 were maintained in the bath solution, a current-voltage relationship was constructed by clamp steps (+10-mV increments from -120 mV; 400-ms duration at a rate of 0.5 Hz) in the absence and presence of Ba2+ (1 mM). The current blocked by Ba2+ (1 mM) was obtained by subtraction and is plotted as a function of the clamp potential (Fig. 5). At voltages more negative than the zero current potential, inward current density was significantly smaller in the AS oligo-treated group compared with the control group [e.g., at -120 mV: -4.4 ± 0.61 (n = 5) vs. -11.8 ± 1.63 (n = 4) pA/pF in Kir2.1 AS oligo and control groups, respectively; P < 0.05]. The reversal potential of the Ba2+-senstive current was around -64 to -66 mV (for both experimental groups). Given a calculated liquid junction potential of -16.4 mV (see METHODS), the observed reversal potential of around -80 to -82 mV is close to the expected equilibrium potential for K+ under these experimental conditions (around -83 mV). We also performed similar experiments using a bath solution containing 140 mM K+ (NaCl was replaced by KCl) in which the Ba2+-senstive current density at -50 mV was -61.9 ± 16.8 (n = 3) in control vs. -16.7 ± 6.33 (n = 7) pA/pF in the Kir2.1 AS oligo group (P < 0.05). Under these conditions, the reversal potential was close to 0 mV (not shown), as expected for a K+-selective current.


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  		Fig. 5.   Effect of Kir2.1 antisense oligonucleotides on whole cell (Ba2+-sensitive) IK1 current-voltage relationship. From a holding potential of -45 mV, the membrane was stepped to -120 mV for 400 ms, followed by +10-mV increments repeated at 0.5 Hz. Current was recorded in the absence and presence of 1 mM Ba2+. The Ba2+-senstive current was obtained by subtraction and is plotted as a function of voltage. bullet , Results from the group of myocytes that were incubated for 5 days in the presence of antisense oligonucleotides (3 µM each of 1022S and 1023S); open circle , time-matched control experiments. D-600 (10 µM) and 4-aminopyridine (3 mM) were present in these experiments, to block Ca2+ current and Ito.

Effect of AS oligos on single channel events. We measured the single channel activity of inwardly rectifying K+ channels that open under steady-state conditions at negative membrane potentials and therefore potentially can contribute to native whole cell IK1. To examine the effects of Kir2.1 AS oligos on these channels, we performed cell-attached patch clamping.

Rat ventricular myocytes were cultured for 4-5 days in the presence or absence of Kir2.1 AS oligos (S1022S and 1023S; 3 µM each). Recordings were performed at pipette potentials of +100, +50, 0, and -50 mV. Representative recordings made at a pipette potential of +100 mV illustrate that, under control conditions, several channel events could be observed (Fig. 6A). The channel amplitudes were a direct function of the pipette potential. At 0 mV, no channel activity was observed, suggesting that, under our experimental conditions of symmetrical K+ but asymmetrical Cl- concentrations, K+ was the major charge carrier. Channel amplitudes were disproportionally smaller at a pipette potential of -50 mV, consistent with the property of inward rectification (data not shown). An amplitude histogram, constructed from an events list of a recording with a duration of 20 s, shows that three channels predominated in this representative patch (7.5, 12.0, and 21.4 pS; Fig. 6A, bottom). In a patch from a myocyte treated with AS oligos, the ~20-pS channel was rarely observed, although several other channels with varying single channel amplitudes could readily be observed (Fig. 6B). In this patch, an events list-constructed amplitude histogram shows the predominant occurrence of 6- and 11.8-pS channels, whereas a channel with a 20.9-pS conductance occurred relatively infrequently (Fig. 6B, bottom).


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  		Fig. 6.   Effects of Kir2.1 antisense oligonucleotides on IK1 single channel activity. Rat ventricular myocytes were incubated in the absence (A) or presence (B) of Kir2.1 antisense oligonucleotides (3 µM each of 1022S and 1023S). Examples of single channel activity are shown at top. Pipette potential was 100 mV. Patch current was filtered at 1 kHz and sampled at 5 kHz. Broken line indicates a single channel current of 2 pA (corresponding to a unitary conductance of ~20 pS under these experimental conditions). Scaling bars are 4 pA and 100 ms, respectively. An events list was constructed from recordings with a duration of 20 s, and the resulting amplitude histograms are plotted at bottom (for clarity, the closed state is not shown). Nos. above the peaks denote the single channel conductances for the various events.

The knockdown of the 21-pS channel by Kir2.1 AS oligos is confirmed when all data are combined (Table 1). In the control group, the 21-pS channel occurred in all of the patches and in ~36.2% of all sweeps. In contrast, this channel was only observed in 5 out of 14 patches in the AS oligos-treated group (P < 0.001 vs. control). More significantly, when patches contained the 21-pS channel, openings occurred only sporadically (<10% of the sweeps, P < 0.05). The occurrence or opening frequency of a 13.5-pS channel was not statistically different between the two groups (it occurred in ~25% of all sweeps). An 8-pS channel was also observed with equal abundance and frequency in patches from the control and antisense-treated groups (Table 1).

                              
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  		Table 1.   Single channel characteristics of channels recorded from myocytes cultured for 4 days in the presence or absence of Kir2.1 AS oligos

In addition to these more abundantly occurring channels, several larger conductance channels were also observed (34, 43, and 52 pS). These channels all occurred relatively infrequently (<10% of all sweeps; e.g., Fig. 6, top). Kir2.1 AS oligos appeared not to have an effect on the occurrence of any of these channels or on the frequency of opening of these channels (apart from the 43-pS channel, which occurred in slightly more sweeps than control patches; Table 1).

A larger conductance channel (77-80 pS) was inhibited when excising patches in ATP-containing solution, suggesting that they were caused by opening of KATP channels (data not shown). These channels occurred with surprising abundance; their appearance was most likely related to the culturing conditions. In cell-attached patches from control myocytes, 5 of 16 patches contained this channel (average: 80.8 ± 0.84 pS). Although KATP channels appeared to occur more frequently in the Kir2.1 antisense-treated group (11 of 19 patches; average conductance: 77.7 ± 1.27), this did not represent a statistically significant difference.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Antisense approaches in channel physiology. Most of the features of native cardiac IK1 channels can be found in many of the cloned Kir channels (K+ selectivity, rectification, block by cations, etc.). Because of the electrophysiological and pharmacological similarities between native channels and many of those expressed in heterologous expression systems, there are insufficient criteria to assign functional roles to cloned channels. This is especially difficult in the absence of specific modulators or blockers of IK1 channels. A much more convincing technique to confirm a causal relationship between the gene product and the native channel is by selective deletion of the cloned channel.

There have been several reports of specific inhibition of membrane channels or transporters by the use of AS oligos. For example, AS oligos targeted against mRNA of specific Ca2+ channel alpha -subunits (34) or beta -subunits (2) were able to confirm the function of these subunits in neural tissue. AS oligos also helped to establish the molecular identity of various other channels, including the slowly and rapidly inactivating K+ channels of a pituitary cell line (Kv1.5 and Kv1.4, respectively; see Ref. 5) and the A-type current of rat brain (Kv4 family; see Ref. 32). In cultured heart myocytes, AS oligos have also been used with success. As examples, Kv1.5 AS oligos inhibited the ultrarapid delayed rectifier K+ current in cultured adult human atrial cells (7), inhibition of beta -subunit expression by AS oligos prevented the formation of a mature Na+ current (21), and Kv4 AS oligos helped to confirm the molecular identity of rat ventricular transient outward current (9, 27). These reports demonstrate the usefulness of AS oligos in correlating specific channel proteins with native channels and currents. Using a similar approach, we have now identified a specific molecular constituent of the most predominant inward rectifier K+ channel in rat ventricle.

Genes encoding native IK1 channel proteins. The Kir family contains several subfamilies (Kir1-Kir6); each subfamily in turn contains several different members (6). On the basis of their unique modulation and/or their expression patterns, some Kir subfamilies can be disregarded as potential candidates coding for native IK1 channel proteins. The evidence implicating the involvement of Kir3 and Kir6 subfamily members in the genesis of G protein-activated K+ channels and KATP channels, respectively, is overwhelming (13, 18). Because members of the Kir1/ROMK and Kir4.1/KAB-2 subfamilies are poorly expressed in heart tissue (11, 37), they are also unlikely candidates for native IK1 channels.

The most obvious candidate genes coding for native IK1 channel proteins belong to the strongly rectifying Kir2/IRK subfamily. The first member of the Kir2 subfamily (Kir2.1/mmIRK1), which has been cloned from mouse macrophages by expression cloning (11), has a high degree of sequence similarity to heart Kir2.1 clones (1, 38, 42). The description of the Kir2.1 primary sequence was soon followed by homology cloning of two additional subfamily members [Kir2.2/IRK2 and Kir2.3/IRK3 (13, 28, 41)]. When expressed in oocytes, all three subfamily members express strongly rectifying K+ currents with a similar pharmacological phenotype (19, 28, 35). Because all three members of the Kir2 subfamily are expressed in heart (19, 28, 41), they can all be considered as possible candidate genes responsible for expressing native cardiac IK1 channel proteins. Most striking are the differences in the single channel conductances of the Kir2 subfamily members (21-25, 34-41, and 10-16 pS, respectively, for Kir2.1, Kir2.2, and Kir2.3; see Refs. 19, 28, 35). In the present study, we cultured rat ventricular myocytes in the presence of AS oligos that have been demonstrated to be specific against Kir2.1 mRNA. Using single channel recording techniques to analyze steady-state K+-channel activity at negative membrane potentials, we examined channel activity in terms of the number of patches containing the channel and frequency of opening. Using these criteria, we found that a native 21-pS channel was effectively and specifically reduced by AS oligos targeted against Kir2.1 transcripts. This observation, along with the resemblance in the unitary conductance of Kir2.1 channels, supports the concept that Kir2.1 codes for a protein that expresses a cardiac channel with a unitary conductance of 21 pS.

In addition to the 21-pS channel, we also observed K+ channels with conductances of ~8, 13.5, 35, 43, 53, and 80 pS. Based only on the single channel conductances of cloned channels, it is possible that the 13-pS channel is a product of the Kir2.3/IRK3 gene, which has been reported to have a unitary conductance of 10-16 pS under conditions of lower ionic strength (expression in oocytes; see Refs. 22 and 28).

The 43-pS channel may be a result of expression of Kir2.2 channels, which exhibit a unitary conductance 41 pS under recording conditions similar to that used in our study (140 mM K+; see Ref. 41). The 80-pS channel most likely represents openings of KATP channels; their openings showed a characteristic bursting behavior, and their activity was blocked by application of cytosolic ATP. Therefore, they probably are the products of coexpression of Kir6 subfamily members with members of the sulfonylurea receptors (13).

The molecular nature of the 53-, 35-, and 8-pS channels is presently unclear. It is possible that sporadic openings of G protein-activated K+ channels, which in rat ventricle have single channel conductances in the 53-pS range (36), may have occurred. If so, these channels are most likely heteromultimers consisting of channel alpha -subunits coded by the Kir3.1 and Kir3.4 genes (18). An intriguing possibility is that the 35-pS channel may be coded by TWIK-1, the first member of a novel structural group (4 transmembrane-spanning domains) of K+ channels that has recently been described in mammals (23). The nature of the 8-pS channel is totally obscure at present. The elucidation of the molecular nature of these channels awaits further experimentation using approaches such as those used in the present study.

Factors that may influence single channel conductance. Single channel conductance of the channels formed by Kir alpha -subunits may conceivably be altered by heteromultimeric assembly between Kir subfamily members or by accessory subunits. If so, this may have an impact on the unitary conductance of native IK1 channels and even lead to the existence of multiple conductance levels arising from a single Kir alpha -subunit.

It appears that a homomultimeric Kir channel is formed by the assembly of four subunits from the same subfamily (10, 44), whereas TWIK-1 may be formed by a dimeric subunit assembly (23). If heteromultimeric assembly of Kir alpha -subunits from different subfamilies occurs, then the resulting channels may have unique single channel conductances. However, little is known about heteromultimeric assembly within Kir subfamilies. In particular, it is not entirely clear whether members of the Kir2 subfamily coassemble. The colocalization of Kir2.1 and Kir2.3 in neural tissue (8) led to the suggestion that these two species may coassemble to form functional channels. However, purification of histidine-tagged Kir2.1 protein showed a virtual absence of association with FLAG-tagged Kir2.2 protein (39). Furthermore, in the same study, it has been shown that a dominant negative Kir2.1 construct (GYG in the pore replaced by AAA) prevents expression of Kir2.1 but not of Kir2.2 or Kir2.3 currents in oocytes. Taken together, these results strongly suggest that Kir2.1 does not normally coassemble with either Kir2.2 or Kir2.3 to form heteromultimeric K+ channels. However, these findings contrast with the observation that a different type of dominant negative construct (Kir2.3 NH2-terminus plus the core region and COOH-terminus of Kir3.2, K3G2G2) was able to abolish expression of both Kir2.1 and Kir2.3 currents, which has been interpreted as evidence that coassembly of members of the Kir2 subfamily can occur (8). It is not clear what the reasons are for these important differences. Further experiments are clearly warranted to determine possible coassembly between members of the Kir2 subfamily.

It is possible that heteromultimerization may occur between members of different Kir subfamilies. For example, Kir5.1 [which does not express current in oocytes (3)] is able to significantly alter expression levels and single channel conductance of an unrelated channel, Kir4.1 (29 and see above), but is not able to interact with Kir1.1, Kir2.1, Kir2.3, Kir3.1, Kir3.2, or Kir3.4, suggesting specific interaction between Kir4.1 and Kir5.1 [although interaction with Kir1.1 has previously been suggested (10)]. However, it is not clear whether Kir5.1 is expressed in heart or whether it can form heteromultimers with cardiac K+-channel alpha -subunits. More data are needed to conclude whether any heteromultimeric assemblies can give rise to native cardiac IK1 channels.

There are also many examples of regulatory proteins that alter the expression and behavior of a variety of Kv channels (14, 30, 31). If Kir channel alpha -subunits formed complexes with (as yet unknown) regulatory subunits, it might also conceivably alter the electrophysiological behavior (single channel conductance and pharmacological profile) and possibly even lead to altered expression of channels. Although there has been no description of, or the need to postulate the existence of, accessory subunits that modify Kir channels in a manner that would account for the features of native IK1 channels, this possibility remains to be explored.

Our experiments were not designed to test for the existence of heteromultimeric assemblies between Kir alpha -subunits or their modulation by accessory subunits. Nevertheless, our observation, which demonstrates that a 21-pS channel is specifically knocked down by Kir2.1 AS oligos, suggests that no channels with unique single channel conductances are formed even if coassembly occurs. Further experiments are clearly warranted to determine the effect that coassembly between alpha -subunit Kir subfamily members may have on unitary channel properties and the effect that such assemblies may have on native IK1 channels.

Subconductance states. Native IK1 channels may have three or four equal amplitude subconductance states that can be readily detected under certain experimental conditions, such as in the presence of the blocking cations Cs+ and Ba2+ (10-100 µM; see Refs. 25, 26, 43). Our data and the data of others (4, 16, 24) suggest that IK1 can be caused by opening of different K+ channels (with various conductances) that can exist independently from each other (not all patches contain the same channels). This suggests that these channel openings arise from distinct channels, rather than resulting from subconductance states of the same channel. Furthermore, the ability of Kir2.1 AS oligos specifically to knock down a certain channel suggests that at least some of the different channel conductances observed under our experimental conditions may result from the activity of different Kir channel proteins.

Possible compensatory responses. Although not statistically significant, the amplitude of the transient outward current appeared to be consistently larger in the Kir2.1 AS oligo-treated group. This observation, along with the observation of an increased opening frequency of a 41- to 44-pS channel (Table 1), could possibly be explained by a compensatory response of the myocyte to express K+ channels other than the targeted channels. However, the possibility of channel upregulation remains to be explored more rigorously.

Summary. Our data show that a native IK1 channel with a unitary conductance of 21 pS is specifically knocked down by AS oligos directed against Kir2.1 transcripts. This is consistent with the expectation that Kir2.1 channel proteins, which in heterologous expression systems have unitary channel conductances in the same order, are important molecular components of rat ventricular IK1 channels. Our results suggest that native IK1 may consist of several types of membrane K+ channels, which may be encoded by different genes. The exact contributions of the different channels to IK1 current, however, remain to be elucidated.

    ACKNOWLEDGEMENTS

This work was supported by the Seventh Masonic District Association, a Grant-in-Aid from the American Heart Association (New York City Affiliate, 97-GIA-053), Uehara Memorial Foundation, National Institute of Neurological and Communicative Disorders and Stroke Grant NS-30989, and National Science Foundation Grant IBN9630832.

    FOOTNOTES

Address for reprint requests: T. Y. Nakamura, Pediatric Cardiology, TH501, New York Univ. Medical Center, New York, NY 10016.

Received 18 August 1997; accepted in final form 21 November 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Ashen, M. D., B. O'Rourke, K. A. Kluge, D. C. Johns, and G. F. Tomaselli. Inward rectifier K+ channel from human heart and brain: cloning and stable expression in a human cell line. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H506-H511, 1995[Abstract/Free Full Text].

2.   Berrow, N. S., V. Campbell, E. M. Fitzgerald, K. Brickley, and A. C. Dolphin. Antisense depletion of beta-subunits modulates the biophysical and pharmacological properties of neuronal calcium channels. J. Physiol. Paris 482: 481-491, 1995.

3.   Bond, C. T., M. Pessia, X. M. Xia, A. Lagrutta, M. P. Kavanaugh, and J. P. Adelman. Cloning and expression of a family of inward rectifier potassium channels. Receptors Channels 2: 183-191, 1994[Medline].

4.   Chen, F., G. T. Wetzel, W. F. Friedman, and T. S. Klitzner. Single-channel recording of inwardly rectifying potassium currents in developing myocardium. J. Mol. Cell. Cardiol. 23: 259-267, 1991[Medline].

5.   Chung, S., D. B. Saal, and L. K. Kaczmarek. Elimination of potassium channel expression by antisense oligonucleotides in a pituitary cell line. Proc. Natl. Acad. Sci. USA 92: 5955-5959, 1995[Abstract/Free Full Text].

6.   Doupnik, C. A., N. Davidson, and H. A. Lester. The inward rectifier potassium channel family. Curr. Opin. Neurobiol. 5: 268-277, 1995[Medline].

7.   Feng, J. L., B. Wible, G. R. Li, Z. Wang, and S. Nattel. Antisense oligonucleotides directed against Kv1.5 mRNA specifically inhibit the ultrarapid delayed rectifier in cultured adult human atrial myocytes (Abstract). Circulation 94: I-528, 1996.

8.   Fink, M., F. Duprat, C. Heurteaux, F. Lesage, G. Romey, J. Barhanin, and M. Lazdunski. Dominant negative chimeras provide evidence for homo and heteromultimeric assembly of inward rectifier K+ channel proteins via their N-terminal end. FEBS Lett. 378: 64-68, 1996[Medline].

9.   Fiset, C., R. B. Clark, Y. Shimoni, and W. Giles. Shal-type channels contribute to the Ca2+-independent transient outward K+ current in rat ventricle. J. Physiol. (Lond.) 500: 51-63, 1997[Abstract/Free Full Text].

10.   Glowatzki, E., G. Fakler, U. Brändle, U. Rexhausen, H. P. Zenner, J. P. Ruppersberg, and B. Fakler. Subunit-dependent assembly of inward-rectifier K+ channels. Proc. R. Soc. Lond. B Biol. Sci. 261: 251-261, 1995[Medline].

11.   Ho, K., C. G. Nichols, W. J. Lederer, J. Lytton, P. M. Vassilev, M. V. Kanazirska, and S. C. Hebert. Cloning and expression of an inwardly rectifying ATP-regulated potassium channel. Nature 362: 31-38, 1993[Medline].

12.   Imoto, Y., T. Ehara, and H. Matsuura. Voltage- and time-dependent block of IK1 underlying Ba2+-induced ventricular automaticity. Am. J. Physiol. 252 (Heart Circ. Physiol. 21): H325-H333, 1987[Abstract/Free Full Text].

13.   Inagaki, N., T. Gonoi, J. P. Clement, N. Namba, J. Inazawa, G. Gonzalez, L. Aguilar-Bryan, S. Seino, and J. Bryan. Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270: 1166-1170, 1995[Abstract/Free Full Text].

14.   Jegla, T., and L. Salkoff. A novel subunit for shal K+ channels radically alters activation and inactivation. J. Neurosci. 17: 32-44, 1997[Abstract/Free Full Text].

15.   Josephson, I. R., and A. M. Brown. Inwardly rectifying single-channel and whole-cell K+ currents in rat ventricular myocytes. J. Membr. Biol. 94: 19-35, 1986[Medline].

16.   Josephson, I. R., and N. Sperelakis. Developmental increases in the inwardly-rectifying K+ current of embryonic chick ventricular myocytes. Biochim. Biophys. Acta 1052: 123-127, 1990[Medline].

17.   Kameyama, M., T. Kiyosue, and M. Soejima. Single channel analysis of the inward rectifier K current in the rabbit ventricular cells. Jpn. J. Physiol. 33: 1039-1056, 1983[Medline].

18.   Krapivinsky, G., E. A. Gordon, K. Wickman, B. Velimirovic, L. Krapivinsky, and D. E. Clapham. The G-protein-gated atrial K+ channel IKACh is a heteromultimer of two inwardly rectifying K+-channel proteins. Nature 374: 135-141, 1995[Medline].

19.   Kubo, Y., T. J. Baldwin, Y. N. Jan, and L. Y. Jan. Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature 362: 127-133, 1993[Medline].

20.   Kubo, Y., E. Reuveny, P. A. Slesinger, Y. N. Jan, and L. Y. Jan. Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel. Nature 364: 802-806, 1993[Medline].

21.   Kupershmidt, S., T. Yang, and D. M. Roden. Inhibition of beta-subunit expression prevents development of a mature sodium current phenotype in cultured heart cells (Abstract). Circulation 94: I-287, 1996.

22.   Lesage, F., F. Duprat, M. Ffink, E. Guillemare, T. Coppola, M. Lazdunski, and J. Hugnot. Cloning provides evidence for a family of inward rectifier and G-protein coupled K+ channels in the brain. FEBS Lett. 353: 37-42, 1994[Medline].

23.   Lesage, F., E. Guillemare, M. Fink, F. Duprat, M. Lazdunski, G. Romey, and J. Barhanin. TWIK-1, a ubiquitous human weakly inward rectifying K+ channel with a novel structure. EMBO J. 15: 1004-1011, 1996[Medline].

24.   Masuda, H., and N. Sperelakis. Inwardly rectifying potassium current in rat fetal and neonatal ventricular cardiomyocytes. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H1107-H1111, 1993[Abstract/Free Full Text].

25.   Matsuda, H., H. Matsuura, and A. Noma. Triple-barrel structure of inwardly rectifying K+ channels revealed by Cs+ and Rb+ block in guinea-pig heart cells. J. Physiol. (Lond.) 413: 139-157, 1989[Abstract/Free Full Text].

26.   Mazzanti, M., R. Assandri, A. Ferroni, and D. DiFrancesco. Cytoskeletal control of rectification and expression of four substates in cardiac inward rectifier K+ channels. FASEB J. 10: 357-361, 1996[Abstract].

27.   Nakamura, T. Y., W. A. Coetzee, E. Vega-Saenz de Miera, M. Artman, and B. Rudy. Modulation of Kv4 channels, key components of rat ventricular transient outward K+ current by PKC. Am. J. Physiol. 273 (Heart Circ. Physiol. 42): H1775-H1786, 1997[Abstract/Free Full Text].

28.   Perrier, F., C. M. Radeke, and C. A. Vandenberg. Primary structure and characterization of a small-conductance inwardly rectifying potassium channel from human hippocampus. Proc. Natl. Acad. Sci. USA 91: 6240-6244, 1994[Abstract/Free Full Text].

29.   Pessia, M., S. J. Tucker, K. Lee, C. T. Bond, and J. P. Adelman. Subunit positional effects revealed by novel heteromeric inwardly rectifying K+ channels. EMBO J. 15: 2980-2987, 1996[Medline].

30.   Sanguinetti, M. C., M. E. Curran, A. Zou, J. Shen, P. S. Spector, D. L. Atkinson, and M. T. Keating. Coassembly of KvLQT1 and minK (IsK) proteins to form cardiac IKs potassium channel. Nature 384: 80-83, 1996[Medline].

31.   Scott, V. E. S., J. Rettig, D. N. Parcej, J. N. Keen, J. B. C. Findlay, O. Pongs, and J. O. Dolly. Primary structure of a beta subunit of alpha-dendrotoxin-sensitive K+ channels from bovine brain. Proc. Natl. Acad. Sci. USA 91: 1637-1641, 1994[Abstract/Free Full Text].

32.   Serodio, P., C. Kentros, and B. Rudy. Identification of molecular components of A-type channels activating at subthreshold potentials. J. Neurophysiol. 72: 1516-1529, 1994[Abstract/Free Full Text].

33.   Shimoni, Y., R. B. Clark, and W. R. Giles. Role of an inwardly rectifying potassium current in rabbit ventricular action potential. J. Physiol. (Lond.) 448: 709-727, 1992[Abstract/Free Full Text].

34.   Snutch, T. P., J. P. Leonard, M. M. Gilbert, H. A. Lester, and N. Davidson. Rat brain expresses a heterogeneous family of calcium channels. Proc. Natl. Acad. Sci. USA 87: 3391-3395, 1990[Abstract/Free Full Text].

35.   Takahashi, N., K. Morishige, A. Jahangir, M. Yamada, I. Findlay, H. Koyama, and Y. Kurachi. Molecular cloning and functional expression of cDNA encoding a second class of inward rectifier potassium channels in the mouse brain. J. Biol. Chem. 269: 23274-23279, 1994[Abstract/Free Full Text].

36.   Takano, M., and A. Noma. Development of muscarinic potassium current in fetal and neonatal rat heart. Am. J. Physiol. 272 (Heart Circ. Physiol. 31): H1188-H1195, 1997[Abstract/Free Full Text].

37.   Takumi, T., T. Ishii, Y. Horio, K. Morishige, N. Takahashi, M. Yamada, T. Yamashita, H. Kiyama, K. Sohmiya, S. Nakanishi, and Y. Kurachi. A novel ATP-dependent inward rectifier potassium channel expressed predominantly in glial cells. J. Biol. Chem. 270: 16339-16346, 1995[Abstract/Free Full Text].

38.   Tang, W. M., A. Ruknudin, W. P. Yang, S. Y. Shaw, A. Knickerbocker, and S. Kurtz. Functional expression of a vertebrate inwardly rectifying K+ channel in yeast. Mol. Biol. Cell 6: 1231-1240, 1995[Abstract].

39.   Tinker, A., Y. N. Jan, and L. Y. Jan. Regions responsible for the assembly of inwardly retifying potassium channels. Cell 87: 857-868, 1996[Medline].

40.   Wahler, G. M. Developmental increases in the inwardly rectifying potassium current of rat ventricular myocytes. Am. J. Physiol. 262 (Cell Physiol. 31): C1266-C1272, 1992[Abstract/Free Full Text].

41.   Wible, B., M. De Biasi, K. Majumder, M. Taglialatela, and A. M. Brown. Cloning and functional expression of an inwardly rectifying K+ channel from human atrium. Circ. Res. 76: 343-350, 1995[Abstract/Free Full Text].

42.   Wood, L. S., T. D. Tsai, K. S. Lee, and G. Vogeli. Cloning and functional expression of a human gene, hIRK1, encoding the heart inward rectifier K+-channel. Gene 163: 313-317, 1995[Medline].

43.   Xie, L. H., M. Takano, and A. Noma. Development of inwardly rectifying K+ channel family in rat ventricular myocytes. Am. J. Physiol. 272 (Heart Circ. Physiol. 41): H1741-H1750, 1997[Abstract/Free Full Text].

44.   Yang, J., Y. N. Jan, and L. Y. Jan. Determination of the subunit stoichiometry of an inwardly rectifying potassium channel. Neuron 15: 1441-1447, 1995[Medline].

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