(Received for publication, December 5, 1994; and in revised form, January 9, 1995)
From the
ATP-sensitive K (K
)
channels play a crucial role in coupling metabolic energy to the
membrane potential of cells. We have isolated a cDNA encoding a novel
member (uK
-1) of the inward rectifier K
channel family from a rat pancreatic islet cDNA library. Rat
uK
-1 is a 424-amino acid residue protein (M
= 47,960). Electrophysiological studies
of uK
-1 expressed in Xenopus laevis oocytes show
that uK
-1 is a weak rectifier and is blocked with
Ba
ions. Single-channel patch clamp study of clonal
human kidney epithelial cells (HEK293) transfected with
uK
-1 cDNA reveals that uK
-1 closes in
response to 1 mM ATP and has a single channel conductance of
70 ± 2 picosiemens (n = 6), indicating that
uK
-1 is an ATP-sensitive inward rectifier
K
channel. In addition, uK
-1 is
activated by the K
channel opener, diazoxide. RNA blot
analysis shows that uK
-1 mRNA is expressed ubiquitously
in rat tissues, including pancreatic islets, pituitary, skeletal
muscle, and heart, suggesting that uK
-1 may play a
physiological role as a link between the metabolic state and membrane
K
permeability of cells in almost every normal tissue.
Since uK
-1 shares only 43-46% amino acid identity
with members of previously reported inward rectifier K
channel subfamilies, including ROMK1, IRK1, GIRK1, and
cK
-1, uK
-1 is not an isoform of these
subfamilies and, therefore, represents a new subfamily of the inward
rectifier K
channel family having two transmembrane
segments.
The ATP-sensitive potassium (K) (
)channel, discovered originally in cardiac
muscle(1) , has a feature of regulation of channel openings by
intracellular ATP or the ATP/ADP ratio(2) . Recent
electrophysiological and pharmacological studies have indicated that
K
channels are present in pancreatic
-cells(3, 4, 5, 6) ,
pituitary(7) , skeletal muscle(8) , brain(9) ,
and vascular smooth muscle (10) as well as in cardiac muscle.
They play a key role in cellular functions such as secretion and muscle
contraction by coupling metabolic status to the membrane potential of
cells(2) . It has been shown that the properties of K
channels vary among different tissues, suggesting molecular
heterogeneity of K
channels(2) .
Recently,
inward rectifier K channels of a new class of
K
channel have been
identified(11, 12) . These channels contain two
putative transmembrane segments and correspond to the inner core
structure of voltage-gated K
channels(11, 12, 13, 14) . A
cardiac K
channel (cK
-1) belonging to this
class of K
channel also has been
identified(15) . Inward rectifier K
channels
have thus far been divided into four subfamilies, ROMK1(11) ,
IRK1(12) , GIRK1(16, 17) , and
cK
-1(15) , based on the amino acid sequence
identity between subfamilies. In addition, several isoforms of IRK1 (18, 19, 20, 21, 22) and
GIRK1 (23) subfamilies have also been described.
In the
present study, we have identified a novel K channel
(uK
-1), which represents a new subfamily of the inward
rectifier K
channel family. Interestingly,
uK
-1 mRNA is expressed in all rat tissues examined,
suggesting that uK
-1 may play an important role in the
regulation of membrane potential by metabolic energy in cells of most
tissues.
Figure 2:
Whole-cell currents recorded from Xenopus oocytes expressing uK-1. A,
representative traces of currents elicited by voltage steps from
-150 to +60 mV in 15-mV increments (holding potential is
-17 mV) in a uK
-1 cRNA-injected (left) or
H
O-injected (right) oocyte. Ba
(300 µM) inhibition of the expressed currents in a
cRNA-injected oocyte is shown (middle). B,
current-voltage relationships in bath solutions containing 90 mM (
), 45 mM (
), 20 mM (
), and 4
mM (
) K
ions. The holding potential
was set at the zero current level in these solutions, as described
under ``Materials and Methods.'' C, external
K
dependence of reversal potentials (E
) for uK
-1 currents in
cRNA-injected oocytes. Each point represents measured E
(mean ± S.E.) of three determinants.
Measured E
varies linearly with ln
[K
]
.
Figure 3:
Single channel currents of
uK-1 on HEK293 cell membranes. A, representative
current traces from inside-out patches at various pipette potentials in
the presence (2 mM)(1, 2, 3, 4, 5, 6, 7) or absence
of Mg
(8) are indicated. B, a
representative current-voltage relationship in the presence (2
mM) (
) or absence (
) Mg
. The
conductance was 74 picosiemens in inward current. C, the
effect of 1 mM ATP, 0.1 mM AMP-PNP, and 100
µM diazoxide on uK
-1 channel activity. The
channel recordings were done at -60 mV in inside-out patch.
AMP-PNP and diazoxide were added in open and closed states of channels,
respectively. Mean patch currents (pA) at the time indicated by arrows are: a, 0.20; b, 0; c, 1.62; d, 0.03; e, 0.20; f, 0; g, 0.24; h, 0; and i, 0.48. D, the effect of
glibenclamide on uK
-1 channel activity in inside-out
patches. The trace was recorded at -60 mV in the presence of 1
µM ATP.
Using a P-labeled GIRK cDNA fragment as a probe,
a rat pancreatic islet cDNA library was screened, and five positive
clones were obtained. DNA fragments isolated from a
clone
carrying the longest insert, designated
rIK-5, were subcloned and
sequenced. The sequence of 2389 base pairs contains a single open
reading frame beginning with the third ATG in the cDNA sequence (there
is an in-frame termination codon upstream of this ATG, and the first
and second ATG are followed by termination codons), which predicts the
amino acid sequence of a 424-amino acid residue protein (M
= 47,960) (Fig. 1). The predicted
amino acid sequence of rIK-5 shows 43, 43, 44, and 46% identity with
ROMK1, GIRK1, cK
-1, and IRK1, respectively, each of which
represents a different subfamily of the inward rectifier K
channel
family(11, 12, 13, 14, 15, 16, 17) .
These amino acid identities are much lower than those found among
various isoforms belonging to the same subfamily, where more than 60%
amino acid identity is
found(18, 19, 20, 21, 22, 23) .
This strongly suggests that rIK-5 is not an isoform of previously
described inward rectifier K
channels but represents a
new subfamily. Although the central region of rIK-5 protein shows a
high homology with other inward rectifier K
channels,
the N- and C-terminal regions do not. A hydropathy plot of rIK-5
reveals two hydrophobic regions, suggesting that it has two putative
transmembrane segments, a feature characteristic of inward rectifier
K
channels(13, 14) . There are two
potential cAMP-dependent protein kinase phosphorylation sites (Thr-234
and Ser-385) and seven protein kinase C-dependent phosphorylation sites
(Ser-224, Thr-345, Ser-354, Ser-379, Ser-385, Ser-391, and Ser-397) in
the second intracellular region. There are also one (Thr-63) and four
(Thr-234, Ser-281, Thr-329, and Ser-354) potential casein kinase
II-dependent phosphorylation sites in the first and second
intracellular regions, respectively. Unlike
cK
-1(15) , there is no potential N-linked glycosylation site in the extracellular region.
Figure 1:
Comparison of the amino acid sequences
of the five members of inward rectifier K channel
family. Amino acids are indicated in single-letter code. The
identical amino acid residues among these proteins are shown in boldface. Gaps introduced to generate this alignment are
represented by dots. Predicted transmembrane (M1 and M2) and
pore (H5) segments are indicated.
We
also examined the electrophysiological properties of rIK-5 expressed in X. laevis oocytes. Fig. 2shows the results of the two
microelectrode voltage-clamp experiments in Xenopus oocytes
injected with cRNA for rIK-5 or with water. rIK-5 cRNA-injected oocytes
showed inward currents at extracellular K ([K
]
) of 45 mM (Fig. 2A, left), which are blocked by
external Ba
(300 µM) (Fig. 2A, middle), while water-injected
oocytes had negligible inward currents under the same conditions (Fig. 2A, right). The effects of various
concentrations of [K
]
on rIK-5
currents are shown in Fig. 2B. As
[K
]
was lowered from 90 to 4
mM, the slope conductance was decreased. A weak inward
rectification was clearly observed. The reversal potential of rIK-5
currents was in good agreement with the equilibrium potential for
K
values predicted from the Nernst equation at various
[K
]
(Fig. 2C).
To further characterize the properties of rIK-5, we have performed
single-channel recordings of HEK293 cells transiently transfected with
the rIK-5 expression vector. Under symmetrical K
conditions of 140 mM, the current-voltage relationship
showed inward rectification in the presence of 2 mM Mg
in the intracellular solution and a reversal
potential of 0 mV (Fig. 3, A and B). The
inward current was ohmic, and its channel conductance was calculated to
be 70 ± 2 picosiemens (n = 6). In the case of 5
mM K
in the pipette solution, the reversal
potential was shifted to -79 ± 2 mV (n =
3), almost identical to that predicted by the Nernst equation
(-85.6 mV at 25 °C). The single channels observed on the
cell-attached and inside-out patch membranes had large fluctuations in
open frequency, as shown in Fig. 3, C and D,
and a flickering block was observed at -60 and -80 mV (Fig. 3A). Open time and closed time histograms of
rIK-5 channel activity at -60 mV within a burst were well fitted
with a single exponential, resulting in the time constants of open and
closed times of 3.31 ± 0.40 and 0.91 ± 0.13 ms (n = 4), respectively. The degree of rectification was
enhanced when intracellular Mg
concentration was
raised from 0 to 2 mM (Fig. 3, A and B). Under symmetrical K
conditions of 140
mM at +80 mV, unit amplitude of rIK-5 was 3.83 ±
0.08 (n = 4), 2.73 ± 0.07 (n =
5), and 1.59 ± 0.07 (n = 3) pA at 0, 2, and 5
mM intracellular Mg
concentrations,
respectively. Some rectification is still observed even upon removal of
intracellular Mg
. Recent mutagenesis studies have
suggested that the aspartate in the second transmembrane segment (amino
acid residue 172 of IRK1) is a crucial determinant of
rectification(28, 29) . IRK1 and GIRK1, both of which
are strong rectifiers(12, 16, 17) , have
aspartate at this position, while ROMK1 and cK
-1, both of
which are weak rectifiers(11, 15) , have asparagine.
Consistent with this, uK
-1, which shows weak
rectification, has an asparagine at the corresponding position.
ATP
sensitivity was examined using patches protected from channel run-down
by 1 µM ATP. As shown in Fig. 3C, rIK-5
channel activity was completely suppressed by application of 1 mM ATP and also inhibited by 100 µM nonhydrolyzable ATP
analog AMP-PNP; these effects were reversed on washout of the agents (n = 8). Diazoxide (0.1 mM), a potent opener
of K channels of pancreatic
-cells(30) ,
activated rIK-5 channels on inside-out and cell-attached patch
membranes (Fig. 3C) (n = 3), while 200
µM pinacidil, a cyanoguanidine that activates cK
channels in the presence of intracellular
ATP(30, 31) , failed to reopen the rIK-5 channels
inhibited by 1 mM ATP (n = 5) (data not
shown), indicating a property different from that of
cK
-1(15) . In addition, rIK-5 channel activity
was not inhibited (n = 6) by 1 µM glibenclamide, the sulfonylurea that blocks K
channels (30) (Fig. 3D). These
electrophysiological studies demonstrate that rIK-5 is an ATP-sensitive
potassium channel, and it was designated uK
-1
accordingly.
RNA blotting studies (Fig. 4) reveal that 2.7-
and 1.7-kilobase transcripts are expressed in all rat tissues examined.
uK-1 mRNAs are expressed at high levels in the heart,
ovary, and adrenal, at moderate levels in the skeletal muscle, lung,
brain, stomach, colon, testis, thyroid, and pancreatic islets, and at
low levels in the kidney, liver, small intestine, and pituitary. An
additional 2.9-kilobase transcript also is detected in adrenal.
However, uK
-1 is not expressed in any of the endocrine
tissue-derived clonal cells examined, including the insulin-secreting
cell lines RINm5F (rat), MIN6 (mouse), and HIT-T15 (hamster), the mouse
glucagon-secreting cell line
TC (data not shown), the rat
catecholamine-secreting cell line PC12, the rat growth
hormone-secreting cell line GH3, the mouse ACTH-secreting cell line
AtT-20, or in HEK293 cells. In addition, uK
-1 is not
expressed in vascular endothelium-derived cell lines, the calf
pulmonary artery endothelial cell line CPAE, or the bovine carotid
artery endothelial cell line HH. The absence of uK
-1 mRNA
in clonal insulin-secreting cells suggests that another K
channel is expressed in these cells.
Figure 4:
RNA blot analysis of uK-1
mRNA in various rat tissues, endocrine tissue- and vascular
endothelium-derived clonal cells, and HEK293 cells. For autoradiography
the nylon membrane was exposed to x-ray film with an intensifying
screen at -80 °C for 3 days. The sizes of the hybridizing
transcripts are indicated. Below the autoradiographs are the ethidium
bromide-stained gels before transfer. 28 and 18 S ribosomal RNAs are
shown.
Since intracellular ATP
is the essential carrier of metabolic energy for all mammalian cells,
it seems reasonable to assume that uK-1, expressed
ubiquitously in normal tissues, may play a fundamental role in the
regulation of K
permeability in almost every cell by
coupling metabolic energy to the membrane potential of the
cell(14) . Thus, it should be interesting to examine how the
activation and inactivation processes of uK
-1 are
regulated in altered metabolic states such as diabetes mellitus,
starvation, and ischemia. In addition, since the metabolic process
occurs in a glucose-dependent manner in certain cells such as
pancreatic
-cells, uK
-1 may be regulated by glucose
in these cells.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D42145[GenBank].