(Received for publication, May 22, 1995; and in revised form, December 20, 1995)
From the
Activity of the recently cloned ATP-sensitive epithelial
K channel, ROMK (Ho, K., Nichols, C. G., Lederer, W.
J., Lytton, J., Vassilev, P. M., Kanazirska, M. V., and Hebert, S.
C.(1993) Nature 362, 31-38), is regulated by
phosphorylation-dephosphorylation processes with cAMPdependent protein
kinase (PKA)-dependent phosphorylation events being required for
maintenance of channel activity in excised membrane patches
(McNicholas, C. M., Wang, W., Ho, K., Hebert, S. C., and Giebisch,
G.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8077-8081;
Kubokawa, M., McNicholas, C. M., Higgins, M. A., Wang, W., and
Giebisch, G.(1995) Am. J. Physiol. 269, F355-F362). To
determine whether this channel is a substrate for PKA, ROMK tagged with
the hemagglutinin epitope was transiently transfected into HEK293
cells. In vitro labeling of immunoprecipitated proteins from
transfected cells showed that ROMK could be phosphorylated by PKA.
Metabolic labeling of ROMK resulted in a significantly increased
phosphorylation upon pretreatment of the cells with forskolin,
consistent with an action of cAMP-dependent protein kinase.
Phosphoamino acid analyses of the ROMK phosphoproteins revealed that
phosphate was attached exclusively to serine residues. Three putative
PKA phosphorylation sites containing serine residues in the predicted
ROMK proteins are shown directly to be substrates for PKA.
Site-directed mutagenesis of each of these sites or double mutation of
any two sites showed that ROMK proteins retained the ability to be
phosphorylated by PKA both in vivo and in vitro to a
variable extent, while triple mutation of all three PKA sites abolished
the phosphorylation induced by cAMP agonists in transfected cells.
Two-electrode voltage clamp experiments showed that PKA-dependent
phosphorylation was required for ROMK channel activity and that at
least two of the three sites were required for channel function when
expressed in X. laevis oocytes. Taken together, these results
provide strong evidence that direct phosphorylation of the channel
polypeptide by PKA is involved in channel regulation and PKA-dependent
phosphorylation is essential for ROMK channel activity.
ATP-sensitive (K) potassium channels have been
identified in apical membranes of several renal epithelial cells, where
they are in a position to play critical roles in mediating and
regulating K
secretion (Misler and Giebisch, 1992).
These epithelial secretory K
channels are characterized
by a high open probability, inward rectification, exquisite pH
sensitivity, and inhibition by cytosolic ATP (Misler and Giebisch,
1992; Wang et al., 1992). The low conductance (25-35 pS) (
)K
channel in apical membranes of thick
ascending limbs of Henle is critical to NaCl absorption, as it ensures
that adequate luminal potassium is provided for efficient function of
the Na
:K
:Cl
cotransporter (Hebert and Andreoli, 1984; Wang, 1994b). A similar
apical K
channel has been identified in principal cells
in the cortical collecting duct, where it facilitates potassium
secretion (Frindt and Palmer, 1987; Wang et al., 1990; Misler
and Giebisch, 1992).
An inwardly rectifying, ATP-regulated
K channel, ROMK1 was recently cloned from the outer
medulla of rat kidney (Ho et al., 1993). ROMK, along with
other subsequently identified K
channel genes (Dascal et al., 1993; Kubo et al., 1993a, 1993b; Ashford et al., 1994; Suzuki et al., 1994; Zhou et
al., 1994. Takumi et al., 1995) define a new family of
inward rectifying K
channels. The inward rectifying
K
channel protein contains an H5-like
``pore-forming'' region related to the voltage-gated
K
channels and exhibits a characteristic topology
featuring only two potential membrane-spanning segments. ROMK1 channels
expressed in Xenopus laevis oocytes display properties similar
to those of the low conductance K
channels identified in
renal epithelia (Ho et al., 1993; Nichols et al.,
1994; McNicholas et al., 1994; Kubokawa et al.,
1995). Recently, splice variants of ROMK1 denoted ROMK1-3 have
been identified (Zhou et al., 1994; Boim et al.,
1995), which display alternative splicing at the 5` end and give rise
to channel proteins differing in their amino-terminal amino acid
sequences. These isoforms are differentially expressed along the loop
of Henle and distal nephron in the kidney; functional expression in X. laevis oocytes showed that they all form functional
Ba
-sensitive K
channel (Boim et
al., 1995).
Both the secretory K channel in renal
epithelia (Wang and Giebisch, 1991a, 1991b) and ROMK channels expressed
in X. laevis oocytes are regulated by phosphorylation and
dephosphorylation processes, with activation of channel activity by
cAMP-dependent protein kinase (PKA) (McNicholas et al., 1994).
The predicted ROMK channel protein contains three PKA consensus
phosphorylation sites, suggesting that ROMK may be a substrate for PKA
and that direct phosphorylation of the channel polypeptide may play a
role in channel regulation by this serine-threonine kinase.
Phosphorylation of specific amino acid residues on other ion channels
is one mechanism of regulating channel properties (for review, see
Levitan(1988, 1994). Thus, in the present report we investigated
whether the modulation of ROMK channel activity by PKA is associated
with direct phosphorylation of the ROMK channel polypeptide. A
functional HA-tagged ROMK1 channel cDNA construct was transiently
expressed in HEK293 cells and expression confirmed biochemically. We
observed phosphorylation of ROMK1 protein both in vivo and in vitro, as indicated by PKA-induced
[
P]phosphate (
P
)
incorporation. We also found that phosphate is attached exclusively to
serine residues and the extent of
P
incorporation is enhanced by preincubation of cells with
forskolin. Site-directed mutagenesis, coupled with phosphopeptide
mapping, identified three serine phosphorylation sites in ROMK2.
Expression of these serine mutants of ROMK2 channels in X. laevis oocytes showed that at least two sites were required for channel
function. Mutation of all three PKA phosphorylation sites rendered the
channel inactive and abolished the phosphorylation of channel protein
induced by cAMP agonists in transfected HEK293 cells.
Figure 1:
ROMK1 constructs tagged at the carboxyl
terminus with the HA epitope. A, schematic representation of
the HA epitope-tagged ROMK1 coding region (box) plus 3` and 5`
untranslated regions. The oligonucleotide and amino acid sequences of
the HA epitope are also shown with a stop codon, as indicated by the dot. B, in vitro translation of the
wild-type and HA-tagged ROMK1 cDNA. Both cDNA constructs were in
vitro translated using TNT(TM)-coupled reticulocyte lysate and
[S]methionine. Produced proteins were either
directly loaded on 8% SDS-polyacrylamide gel or immunoprecipitated with
anti-HA monoclonal antibody. IP,
immunoprecipitates.
Figure 2:
Ba-sensitive
K
currents in X. laevis oocytes injected with
HA-tagged ROMK1 cRNA. A. I-V relationships of
Ba
-sensitive K
(I
) currents in the HA-tagged ROMK1 cRNA
injected oocyte.
, current recordings under two-electrode voltage
clamp in solution of 5 mM K
.
, current
recordings under two-electrode voltage clamp in solution of 5 mM K
and 15 mM tetraethylammonium.
,
current recordings under two-electrode voltage clamp in solution of 5
mM K
and 10 mM Ba
. B, HA-tagged ROMK1 I
currents
exhibited high K
selectivity. External K
was replaced by Na
as indicated in the figure
([K
] +
[Na
]). C, the E
of HA-tagged ROMK1 I
currents was
[K
]
-dependent (slope
= 58 mV/10-fold change in
[K
]).
Figure 3: Expression of the ROMK1 proteins at the plasma membrane of HEK293 cells. A, the HA-tagged ROMK1 cDNA was transiently transfected into HEK293 cells. The crude membranes were isolated from both transfected and untransfected cells and detected by Western blot with anti-HA antibody. B, transfected (and untransfected) HEK293 cells were cell-surface labeled with biotin. Biotinylated cells were immunoprecipitated by anti-HA antibody and analyzed on 10% SDS-polyacrylamide gels. Molecular mass is given in kDa. The position of the ROMK1 bands is indicated by arrows.
Figure 4:
In vivo or in vitro phosphorylation of transfected ROMK1 by PKA. A, ROMK1
cDNA was transfected into HEK293 cells, immunoprecipitated by anti-HA
antibody, and phosphorylated in vitro by PKA in the presence
of [-
P]ATP. B, phosphoamino acid
analysis of the in vitro phosphorylated ROMK1 phosphoprotein
by electrophoresis at pH 3.5. C, ROMK1 cDNA was transfected
into HEK293 cells, metabolically labeled with
P
, and treated with or without forskolin and
IBMX. D, phosphoamino acid analysis of the in vivo phosphorylated ROMK1 phosphoprotein by electrophoresis at pH 3.5.
Molecular mass is given in kDa. The position of the ROMK1 bands is
indicated by arrows. P-Ser, phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine; FSK, forskolin.
Figure 7:
Metabolic labeling of wild-type and mutant
forms of ROMK2 in transfected HEK293 cells with P
. The wild-type and mutant ROMK2 cDNA
constructs were transiently transfected into HEK293 cells,
metabolically labeled with
P
, and treated with
or without forskolin and IBMX. The ROMK2 proteins were
immunoprecipitated with anti-HA antibody and protein A-Sepharose and
then analyzed on 10% SDS-polyacrylamide gels. Molecular mass is given
in kDa. The position of ROMK1 bands are indicated by arrow. FSK, forskolin.
Previous investigations have shown that phosphorylation
by PKA may promote assembly, and insertion, of mature proteins to the
plasma membrane (Green et al., 1991; Ross et al.,
1991; Levin et al., 1995). Thus, we examined whether the
magnitude of surface biotinylation of ROMK1 proteins expressed in
HEK293 cells was altered by forskolin and IBMX treatment. Treatment of
transfected cells with forskolin and IBMX under the same conditions as
in the in vivo phosphorylation experiments did not
significantly increase the amount of biotinylated ROMK1 protein
expression at the plasma membrane of HEK293 cells (data not shown).
Thus, the observed increase in P
incorporation
in response to forskolin is due primarily to the enhancement of
PKA-dependent phosphorylation of the ROMK1 proteins at the plasma
membrane. Phosphoamino acid analyses were performed on the ROMK1
proteins, which were labeled either in vitro or in
vivo. In both cases, we observed that the
P
was exclusively incorporated into serine residues (Fig. 4, B and D). No
P
-phosphothreonine was detected, even when the
TLC plate was exposed to film for up to 30 days. In addition, there
were no detectable counts associated with phosphotyrosine, suggesting
that the basal level of phosphorylation in the absence of forskolin and
IBMX (Fig. 4C) is not due to an endogenous tyrosine
kinase-catalyzed phosphorylation. These data provide strong evidence
that only those putative PKA phosphorylation sites in ROMK containing
serine residues are the potential phosphate receptors.
Figure 5:
In vitro translation of wild-type
and PKA phosphorylation site mutant ROMK2. The wild-type and mutant
ROMK2 cDNA constructs were in vitro translated using
TNT(TM)-coupled reticulocyte lysate and
[S]methionine. Produced proteins were resolved
on 8% SDS-polyacrylamide gel. Control, in vitro translation in
the absence of cDNA. Molecular mass is given in kDa. The position of
the ROMK2 bands are indicated by arrow.
Figure 6: Expression of PKA phosphorylation site mutant ROMK2 at the plasma membrane of HEK293 cells. The wild-type and mutant ROMK2 cDNA constructs were transiently transfected into HEK293 cells and cell-surface labeled with biotin. Biotinylated cells were immunoprecipitated by anti-HA antibody and analyzed on 10% SDS-polyacrylamide gels. Molecular mass is given in kDa. The position of the ROMK2 bands are indicated by arrow.
We next examined the ability of
each mutant to be phosphorylated following exposure to forskolin and
IBMX. Fig. 7A shows that the single mutant ROMK2
channels, R2-S25A and R2-S200A, exhibited 3-fold increases in
P
incorporation in response to cAMP agonists in vivo (a similar result was observed with the R2-S294A
mutant; data not shown). Double mutation of the potential PKA
phosphorylation sites (R2-S25A/S200A, R2-S25A/S294A, and
R2-S200A/S294A; Fig. 7B) resulted in approximately a
50% decrease in the intensity of phosphorylation with forskolin and
IBMX compared with the wild-type ROMK2 protein. The triple mutant
(R2-S25A/S200A/S294A), which lacks all putative PKA phosphorylation
sites, had only a basal level of phosphorylation and failed to show
enhanced phosphorylation intensity after forskolin and IBMX treatment (Fig. 7B, lanes 9 and 10). These
results indicate that in the absence of all three putative
phosphorylation sites, the HA-tagged ROMK2 protein is not a substrate
for PKA. Phosphorylation of the wild-type and mutant HA-tagged ROMK2
proteins are summarized in Table 1.
Figure 8:
Two-dimensional tryptic phosphopeptide
mapping of ROMK2 labeled in vitro. The wild-type or mutant
ROMK2 cDNA constructs were transiently transfected into HEK293 cells,
immunoprecipitated by anti-HA antibody, and phosphorylated in vitro by the catalytic subunit of PKA in the presence of
[-
P]ATP. The ROMK phosphoproteins were
gel-purified and digested with trypsin. The tryptic peptides were then
resolved by electrophoresis and chromatography in two dimensions. The
origins are marked with a circle.
Figure 9:
Ba-sensitive
K
currents in X. laevis oocytes injected with
the wild-type or mutant HA-tagged ROMK2 cRNA. X. laevis oocytes were injected with the wild-type and mutant ROMK2 cRNA and
Ba
-sensitive K
currents (I
). Currents were recorded under
two-electrode voltage clamp in solution of 5 mM K
. Data are expressed as mean ± S.E.
K
currents (µA) are as follows: wild-type, 4.29
± 0.58 (n = 7); S25A, 2.61 ± 0.70 (n = 7, p < 0.02); S200A, 2.30 ± 0.42 (n = 7, p < 0.05); S294A, 2.85 ±
0.39 (n = 7, p < 0.05). p values
were calculated by comparing wild-type with each mutant
form.
It is well established that a variety of voltage-gated and
ligand-gated ion channels are substrates for protein kinases, and
phosphorylation of ion channel proteins on serine, threonine, or
tyrosine residues is considered a ubiquitous mechanism of modulating
ion channel activity (for review, see Catterall(1988) and Levitan
(1988, 1994)). Studies on renal tubules have shown that the low
conductance, ATP-sensitive K channel (K
)
present in apical membranes of rat cortical collecting duct and MTAL
cells is activated by PKA (Wang and Giebisch, 1991a; Kubokawa et
al., 1995a; Wang, 1994a). A recent patch clamp study of ROMK2
expressed in X. laevis oocytes demonstrated that addition of
the catalytic subunit of PKA and MgATP was required to restore channel
activity following phosphatase-induced channel run-down (McNicholas et al., 1994). Thus, these physiological studies suggest that,
similar to the native low conductance K
channel, ROMK
channels are regulated by PKA-mediated phosphorylation and
dephosphorylation processes, and that PKA-dependent phosphorylation is
required for maintaining channel activity. Phosphorylation of K
channels in non-renal cells is also thought to be an important
mechanism for modulation of these metabolically regulated channels
(Ashcroft, 1988; Misler and Giebisch, 1992). However, it is not known
whether ROMK, or indeed any of the cloned inwardly rectifying
K
channels, are substrates for protein kinases. In the
present study, we describe the direct phosphorylation of ROMK K
channel protein in transiently expressed HEK293 cells by PKA.
We epitope-tagged ROMK1 and ROMK2 at the carboxyl termini in order
to immunoprecipitate ROMK with anti-HA antibody. It should be noted
that although ROMK1 and another isoform, ROMK2, differ at the amino
terminus due to alternative splicing, they both form similar functional
K channels when expressed in X. laevis oocytes (Boim et al., 1995) and both isoforms require
PKA-mediated phosphorylation processes for maintenance of channel
activity. (
)Although the HA epitope tag has been used
frequently for verifying the expression of membrane proteins (Attisano et al., 1993; Wrana et al., 1994), the caveat with
epitope tagging of proteins is that the added epitope may disrupt
protein sorting and function. We have shown, however, that the
HA-tagged ROMK1 and ROMK2 channels can be functionally expressed in X. laevis oocytes ( Fig. 2(ROMK1) and Fig. 9(ROMK2)) and that these epitope-tagged constructs are
detected by cell-surface biotinylation in HEK293 cells (Fig. 3B (ROMK1) and Fig. 6(ROMK2)), indicating
that the ROMK-HA proteins are functional and are expressed at plasma
membranes.
The observations that the immunoprecipitated ROMK1
protein from transiently transfected HEK293 cells could be
phosphorylated in vitro by PKA (Fig. 4A) and
that forskolin with IBMX increased P
incorporation into the ROMK1 (Fig. 4C) and ROMK2 (Fig. 7) proteins isolated from transfected HEK293 cells in
vivo demonstrate that ROMK channels are substrates for
PKA-mediated phosphorylation and are consistent with the
electrophysiological studies indicating that ROMK channels are
regulated by PKA-dependent processes (McNicholas et al.,
1994). Phosphoamino acid analyses of both in vitro and in
vivo phosphorylated ROMK1 proteins demonstrated that phosphate is
incorporated only at serine residues Ser-44, Ser-219, and Ser-313 by
PKA (Fig. 4, B and D).
It is quite common
that mutant forms of proteins fail to be transported to the correct
cellular location (Cheng et al., 1990; Welsh and Smith, 1993).
Thus, we tested for plasma membrane expression of ROMK2 in transiently
transfected HEK293 cells by cell-surface biotinylation. The results in Fig. 6clearly show that the serine-to-alanine mutations did not
prevent the ROMK2 mutant proteins from sorting to the plasma membrane
of HEK293 cells. Several lines of evidence from in vitro or in vivo phosphorylation strongly suggest that all three PKA
sites are directly involved in phosphorylation of ROMK. First, the in vitro phosphorylated wild-type ROMK proteins examined by
two-dimensional TLC analysis show three phosphopeptides, which
represent three PKA sites that can be abolished by site-specific
mutagenesis (Fig. 8A). Second, in vivo phosphorylation of the transfected ROMK2 mutant constructs
indicate that the serine-to-alanine substitutions at these three PKA
sites resulted in a decrease in P
incorporation with stimulation with forskolin and IBMX. There was
a 50% decrease in
P
incorporation in the
double-site mutant forms and complete loss of
P
incorporation in the triple mutant form compared with the
wild-type ROMK2 (Fig. 7, A and B). Failure to
detect a significant decline in the
P
incorporation in the single mutant ROMK2 is due possibly to the
high level of phosphorylation. In addition, we were not able to detect
PKA-dependent phosphorylation in vitro when the triple mutated
ROMK2 was examined by phosphopeptide analysis (Fig. 8E). Similarly, the magnitude of
Ba
-sensitive K
currents observed in X. laevis oocytes was dependent on the number of
serine-phosphorylation residues mutated.
From analysis of these
results, which are summarized in Table 1, two major conclusions
can be reached. First, like the CFTR Cl channel and
the insulin receptor (Cheng et al., 1991; Zhang et
al., 1991), phosphorylation of ROMK by PKA is degenerate, meaning
that no one individual site is essential, and yet more than one site is
required for maintaining channel activity. Second, studies on the
triple mutant ROMK2 clearly indicate that there is a direct correlation
between the ROMK phosphorylation and channel activity, and no other
sites are detectable upon phosphorylation of ROMK by PKA. At present,
we have not yet examined the ROMK single-channel properties using patch
clamp techniques. These latter studies may reveal other aspects of
channel function, which are modulated by specific phosphorylated
residues (e.g. the characteristics of MgATP- or pH-mediated
channel inhibition) .
In summary, the present study demonstrates
that ROMK is a phosphoprotein, that the channel can be phosphorylated
by PKA, and that three PKA sites containing serine residues are
essential for ROMK channel activity. Given the critical importance of
this channel for renal K secretion and recycling,
these findings provide important insights into the functional
regulation of ROMK and possibly other K
channels.