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Departments of 1 Pediatrics, 2 Physiology and Neurosciences, and 3 Biochemistry, New York University Medical Center, New York, New York 10016
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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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
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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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.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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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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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 M
for the
current-passing electrode and 1-5 M 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(
-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 M. 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 M.
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.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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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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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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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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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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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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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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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 
-subunits (34) or
-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 -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-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 -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 -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 -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 -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 -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 -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
-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 |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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) or with a combination of
Kir2.1 cRNA and AS oligos (
; 40 fmol). Other oocytes (injected only
with Kir2.1 cRNA) were incubated in the presence of oligonucleotides in
the medium (3 µM each; 
). Experiments were performed with KD-96
bathing solution at room temperature. Clamping protocol consisted of
square step voltage-clamp pulses from 
, time-matched control experiments. D-600 (10 µM) and
4-aminopyridine (3 mM) were present in these experiments, to block
Ca2+ current and
Ito.
