(Received for publication, August 28, 1995; and in revised form, September 26, 1995)
From the
Cardiac I is comprised of two inwardly rectifying
K
channel subunits, CIR and GIRK1 (Krapivinsky, G.,
Gordon, E. G., Wickman, K., Velimirovic, B., Krapivinsky, L., and
Clapham, D. E.(1995) Nature 374, 135-141). A cardiac
[Medline]
protein virtually identical to CIR, termed rcKATP-1 (Ashford, M. L. J.,
Bond, C. T., Blair, T. A., and Adelman, J. P.(1994) Nature 370, 456-459), was reported to form an ATP-sensitive
[Medline]
inwardly rectifying K
channel, I
. We
attempted to determine whether CIR alone or together with an unknown
protein(s) participated in the formation of cardiac I
.
Expression of CIR in insect, oocyte, and mammalian cell systems did not
increase the appearance of ATP-sensitive currents, but rather gave rise
to unique strongly inwardly rectifying, G protein-regulated
K
currents. CIR protein is found exclusively in atria,
in contrast to the predominance of I
functional activity
in ventricle. Also, CIR was completely depleted from heart membrane
after immunodepletion of GIRK1. We conclude that CIR/rcKATP-1 is not a
subunit of cardiac I
and that GIRK1 is the only channel
protein coassociating with CIR in heart.
Inwardly rectifying K channels (IRKs) (
)maintain resting membrane potential and control cell
excitability. Two of the most highly regulated IRK channels are the
cardiac atrial muscarinic-gated channel, I
, and the
ATP-sensitive channel,
I
(3, 4, 5, 6) .
I
is in part responsible for the vagally mediated
slowing of heart rate. Characteristic features of I
are
its gating by the G protein
dimer (G
),
single channel conductance of 35-40 picosiemens in symmetrical
140 mM K
, sharp inward rectification in the
presence of internal Mg
, and a mean single channel
open time of
1
ms(3, 7, 8, 9, 10, 11) .
Recently, it was shown that cardiac I
was comprised of
two homologous subunits, termed GIRK1 and CIR(1) . GIRK1 and
CIR are both members of a subfamily of IRKs regulated by G proteins
(GIRKs or Kir 3.0(12) ). In brain, GIRK1 may combine with a CIR
homolog, GIRK2, to form a channel with properties similar to
I
(13) . (
)
I is a
K
-selective, inward rectifier found in pancreatic
cells, heart, smooth muscle, skeletal muscle, brain, and kidney.
Evidence suggests that I
channels play roles in the
early repolarization of ischemic cardiac cells and insulin secretion.
Cardiac I
is blocked by intracellular ATP and
sulfonylureas, is modulated by intracellular nucleotide diphosphates
and activated
subunits of G proteins (G
),
rectifies weakly, and has a characteristic single channel bursting
profile(4, 5, 6) . A recently cloned cDNA,
termed rcKATP-1, was reported to form I
when expressed
in the human embryonic kidney cell line HEK293(2) . rcKATP is
virtually identical to CIR. The sequence for rcKATP-1 reported in
GenBank(TM) differs by only two amino acids from CIR (I188V, a
conserved substitution, and Q375E). Each difference can be explained by
a single base polymorphism. To reconcile the demonstration that CIR is
part of cardiac I
(1) and the demonstration that
CIR expression generates I
(2) , it was suggested
that CIR does not form I
alone, but may coassemble with
an unknown subunit(s) expressed in cardiac and HEK293 cells to form
I
. In this report we demonstrate that CIR is not part of
cardiac I
and appears to be wholly associated with GIRK1
in heart.
Plasma membranes from bovine atria and ventricle were isolated as described(15) . Two to 10 µg of membrane protein was separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (Millipore) film for Western blots. aCIRN2 antibodies raised against an N-terminal peptide (amino acids 19-32) were affinity-purified as described(1) . aCIRN2 specifically recognized CIR expressed in Sf9 cells and immunoprecipitated in vitro translated CIR (data not shown). Western blots were developed with anti-rabbit peroxidase (Pierce) and enhanced chemiluminescence (ECL; Amersham Corp.).
For
coimmunoprecipitation experiments, plasma membrane proteins were
biotinylated (16) and solubilized in a buffer containing 10
mM HEPES, pH 7.5, 300 mM NaCl, 1 mM EDTA, 1%
Triton X-100, 1 mM dithiothreitol, protease inhibitors (2
µg/ml aprotinin, leupeptin, and pepstatin, 0.2 mM
phenylmethylsulfonyl fluoride) and immunoprecipitated with aCIRN2 (7
µg/mg of solubilized protein) using 10 µl of protein
A-Sepharose FF (Pharmacia Biotech Inc.) for 2 h at 4 °C. After
washing of the immunoprecipitate with the same buffer, specifically
bound proteins were eluted with 2 25 µl of 100 µM CIRN2 peptide. Proteins were visualized by Western blotting with
streptavidin-peroxidase (Pierce) and ECL and probed with an anti-GIRK1
antibody, aCsh(1) .
The CIR coding region was subcloned into pCDNA1/Amp (Invitrogen) downstream from the cytomegalovirus promoter for use in mammalian transfection experiments. To maximize the chance of assaying HEK293 and CHO cells transiently expressing CIR, we included a construct driving expression of murine CD4, a T cell-specific cell-surface antigen, in all transfections(1) . CHO and HEK293 cells plated on 10-cm Petri dishes at 50% confluence were transfected via calcium phosphate precipitation with CIR or rcKATP-1 (10 µg) and 5 µg of the CD4 construct. 24 h post-transfection, CHO cells were detached, concentrated, and incubated at 4 °C for 10-15 min with 5 µl of (R)-phycoerythrin-conjugated anti-mouse CD4 monoclonal antibodies (Pharmingen, 0.4 mg/ml). Unbound antibody was removed by a 5-ml wash with phosphate-buffered saline. Cells were replated on coverslips in their respective media and allowed to reattach for 1-2 h prior to electrophysiological experiments. CD4-expressing CHO and HEK293 cells (observed by fluorescence) were analyzed in cell-attached and inside-out patch recording modes. CHO cells were cultured in Ham's F-12/Dulbecco's modified Eagle's medium (Life Technologies, Inc., 1:1), 10% dialyzed fetal bovine serum (Life Technologies, Inc.), 2 mM glutamine, penicillin (100 units/ml), and streptomycin (100 mg/ml). HEK293 cells (ATCC number CRL-1573) were cultured in an identical media supplemented with glycine and HT (Life Technologies, Inc.).
A CIR-containing baculovirus
construct was produced in the nonfusion baculovirus expression vector,
pBlueBac III (Invitrogen). The recombinant virus was generated,
isolated, and amplified as described (MaxBac, Invitrogen). For
infections, Sf9 cells grown in suspension were plated at 50%
confluence in 35-mm Petri dishes containing coverslips. Cells were
infected at an multiplicity of infection of
100 plaque-forming
units/cell and assayed for channel currents 24-40-h
post-infection.
The pipette and bath solutions for most Sf9, CHO,
and HEK293 patch clamp experiments were identical: 140 mM
K, 140 mM Cl
, 10 mM HEPES, 5 mM EGTA, and 2 mM Mg
,
pH 7.2. In several Sf9 experiments, bath Cl
was
reduced to 2 mM by replacement with methanesulfonate. Pipettes
were pulled from KG-12 glass, coated with Sylgard (Dow Corning), and
had resistances of 2-4 m
in the above solution. Currents
were recorded using an Axopatch 200A patch clamp amplifier, filtered at
5 kHz using an 8-pole Bessel filter, and digitized at 25 kHz using
PCLAMP6 software for analysis of amplitude histograms.
When CIR was infected or transfected into several host cells,
increased I channel activity was never observed. Sf9
cells infected with a CIR-containing baculovirus or mammalian cells
transfected with CIR, however, display novel currents unlike those of
I
. CIR in all expression systems (and rcKATP-1 in HEK293
cells) generates flickery, short-lived channels with variable
conductance (Fig. 1A; see also (1) ). Amplitude
histograms taken from data filtered at 5 kHz show a distribution of
amplitudes consistent either with many openings that are too brief to
be resolved or by a rapid block mechanism. The signal-to-noise ratio
did not allow us to determine the single channel conductance at larger
bandwidth where these brief openings might be resolved. However, within
many recordings are occasional longer openings of 32-36
picosiemens. Despite the fact that the range of conductances and brief
openings resulting from CIR expression are atypical of currents
expected in native tissues, the current fluctuations have properties
consistent with ion channels. The channels are
K
-selective, rectify sharply in the presence of
Mg
(Fig. 1), are blocked by 1 mM external Cs
and Ba
, and are
stimulated by GTP
S, G
, and ATP(1) .
Similar results are observed in Xenopus oocytes injected with
CIR mRNA. (
)
Figure 1:
Heterologous
expression of CIR or rcKATP-1 in Sf9, CHO, and HEK293 expression
systems gives similar channel currents. Recordings are from excised
patches at the voltages shown. An ``all-point'' amplitude
histogram was constructed for channels appearing upon infection of Sf9
cells with a recombinant CIR baculovirus (a), following
transfection of CHO cells with CIR (b), or rcKATP-1 in HEK293
cells (c). Holding membrane potential at positive voltages
reveals the strong inward rectification of these channels. With the
addition of 5 mM EDTA, which chelates the 2 mM bath
Mg, inward rectification was abolished. In all three
systems, amplitude histograms were generated from inside-out patches
held at -80 mV, stimulated by GTP
S (Sf9 cells) or ATP and
GTP
S (HEK293 and CHO cells).
In control cells a channel resembling that
described as I by Ashford et al.(2) was recorded. The channel was present in 1-3%
(CHO; n > 100) to 18% (HEK293; n = 78) of
control or mock-transfected cells. These endogenous channels were not
noted by Ashford et al.(2) .
I activity is higher in ventricular than atrial
cells(4, 11, 17, 18) , while
I
is found exclusively in atria. We used antibodies
specific for CIR to examine the distribution of this inward rectifier
subunit in bovine ventricle and atrial tissues. A unique peptide
corresponding to amino acids S19-Y32 of CIR (identical in rcKATP-1) was
synthesized to raise a CIR/rcKATP-1-specific polyclonal antibody. In
contrast to the functional distribution of I
, CIR
(rcKATP-1) was not detected in ventricular plasma membranes. CIR,
however, was readily apparent in atria, consistent with
I
expression (Fig. 2).
Figure 2: CIR (rcKATP-1) protein is expressed in atrial but not ventricular tissues. Plasma membranes were isolated from bovine heart atrial (A) and ventricular (V) tissues and Western blotted with 1 µg/ml anti-CIR antibody (aCIRN2, left panel). The right panel shows a Western blot with the same antibody in the presence of 20 µM antigenic peptide.
CIR was shown to coimmunoprecipitate with GIRK1 in atria(1) . To determine whether CIR associates with another protein(s) in heart tissue, we immunoprecipitated CIR directly. aCIRN2 immunoprecipitated the 45-kDa CIR as well as 58- and 72-kDa proteins from atrial membranes, but none were immunoprecipitated from ventricular plasma membranes (Fig. 3). In control experiments, aCIRN2 did not immunoprecipitate in vitro translated GIRK1 (data not shown). The proteins migrating at 72 and 58 kDa were identified as glycosylated and unglycosylated forms, respectively, of GIRK1 by the specific anti-GIRK1 antibody, aCsh (Fig. 3, see also (1) ). Thus, we did not find novel proteins interacting with CIR in atria. However, we could not rule out the possibility that other proteins comigrated with, and therefore could not be discriminated from, CIR and GIRK1 polypeptides.
Figure 3: aCIR antibody coimmunoprecipitates GIRK1 from bovine atrial plasma membranes. aCIRN2 immunoprecipitated three polypeptides from atrial plasma membranes and none from ventricular membranes. The 45-kDa polypeptide has an electrophoretic mobility identical to atrial CIR on Western blots. The other polypeptides (58 and 72 kDa) were recognized with anti-GIRK1 antibody (aCsh). In control experiments, aCIRN2 did not immunoprecipitate in vitro translated GIRK1.
To address the possibility that other proteins comigrated with CIR and GIRK1 polypeptides, we immunoprecipitated GIRK1 and measured depletion of CIR. If other proteins interacted with CIR, full depletion of GIRK1 would not eliminate all detectable CIR. GIRK1 was immunoprecipitated with an increasing amount of aCsh. The relative amounts of GIRK1 and CIR remaining in the supernatant and after immunoprecipitation were determined by Western blot with aCsh and aCIRN2 antibodies. As GIRK1 was depleted, CIR was exhausted; no CIR remained in the supernatant after removal of GIRK1/CIR complexes (Fig. 4). Therefore, we conclude that GIRK1 is the only channel protein associated with CIR in heart.
Figure 4: Atrial CIR interacts only with GIRK1. Atrial GIRK1 was immunoprecipitated with increasing amount of aCsh for 3 h at 4 °C. Numbers below the gel show the amount of antibody/mg of solubilized atrial protein. Relative amounts of GIRK1 and CIR remaining in the supernatant after immunoprecipitation were determined by Western blotting with aCsh and aCIRN2 antibodies. In control experiments, aCsh did not immunoprecipitate in vitro translated CIR. Depletion of GIRK1 from atrial proteins resulted in the complete and parallel depletion of CIR, demonstrating that GIRK1 is the only molecule interacting with CIR in atria.
We have shown that the inward rectifier subunit CIR
(rcKATP-1) initially proposed by Ashford et al.(2) to
comprise I does not participate in formation of cardiac
I
. Our evidence is summarized as follows. 1) Expression
of CIR alone in Sf9, CHO, and HEK293 expression systems did not yield
channels with properties similar to I
. Interestingly,
CIR channels rectify strongly, but have an asparagine, not an aspartic
acid residue, in their putative second transmembrane segment. This
contradicts the hypothesis that asparagine is a common feature of weak
inward rectifiers(19). 2) CIR was present only in cardiac atria, not
ventricle, whereas I
is abundant in both tissues. 3) CIR
protein does not associate with inward rectifier subunits other than
GIRK1 in heart. We speculate that the channels reported by Ashford et al.(2) in HEK293 cell were actually endogenous
channels, not novel currents appearing as a result of CIR or rcKATP-1
expression. It seems likely that an as yet undefined but homologous
inward rectifier subunits may form cardiac I
, perhaps in
conjunction with the sulfanylurea receptor(20) . In fact, an
IRK (uKATP-1) from a rat pancreatic cDNA library with only 43-46%
identity to any of ROMK1, GIRK1, CIR/rcKATP-1, and IRK1 has been
reported to constitute I
in HEK293 cells(14) .