From the
Inwardly rectifying K
ROMK1 is an inward rectifier potassium (K
In this
study we found that ROMK1 is N-glycosylated at
Asn
Recombinant baculoviruses were generated by
co-transfection of Sf9 cells with the engineered transfer plasmids and
BaculoGold
The single N-glycosylation consensus sequence of
ROMK1 is located at amino acids 117-119 and has been assigned to
the first extracellular loop between the M1 and H5 segments of the
presently favored topological model (Fig. 1). Total membranes
prepared from Sf9 cells infected with wild-type baculovirus,
recombinant baculovirus encoding the FLAG/ROMK1 channel, or recombinant
baculovirus containing FLAG/ROMK1 that has the N-glycosylation
site removed (N117Q mutant) were analyzed by Western blots. Two bands
were observed, a minor band at 45 kDa and a major band at 43 kDa, in
cells that produced ROMK1, while no bands were detected in wild-type
virus-infected cells (Fig. 1A). Since the 45-kDa band
might constitute glycosylated ROMK1, the cells were incubated with
tunicamycin, an inhibitor of N-glycosylation (Fig. 1B)(19) . Only the 43-kDa band was
observed, suggesting that ROMK1 is N-glycosylated. To further
examine this process, we constructed a form of ROMK1 that had the
consensus sequence removed (N117Q). Similar to tunicamycin-treated
cells, only the lower immunoreactive band was observed (Fig. 1C). There was also high molecular weight
protein(s) observed at the top of the running gels which could be due
to aggregation of the fusion protein. These results show that ROMK1 is
expressed in Sf9 cells and that residue Asn
ROMK1 was
shown to exist in a functional state in the surface membrane of Sf9
cells by patch clamp measurements. Whole cell recordings from cells
expressing ROMK1 showed instantaneous, time-independent currents with
hyperpolarizing pulses (Fig. 2A) similar to the currents
produced by heterologous expression of ROMK1 in Xenopus oocytes(5, 6) . In contrast, currents from cells
that expressed ROMK1, made unglycosylated by either method, were much
smaller and showed clear, time-dependent relaxations (Fig. 2C). The steady-state current versus voltage curves reflect the large reductions in outward and inward
currents for cells expressing unglycosylated ROMK1 (Fig. 2D).
Single channel currents of ROMK1 expressed in Xenopus oocytes are characterized by instantaneous, long lasting openings
interrupted by brief closures(5, 6) . This was also the
case in Sf9 cells (Fig. 3A). In contrast, single channel
currents from the N117Q mutant (Fig. 3B) or
tunicamycin-treated cells (Fig. 3C), have openings at
the beginning of pulses, brief closures, and long closures thereafter.
The summed single channel currents are time-independent for ROMK1,
whereas for unglycosylated ROMK1, they displayed an obvious
time-dependent relaxation. While the kinetic differences were striking,
the single channel conductances were similar for all three types of
ROMK1 channels (Fig. 3D).
The functional studies
(patch clamp), along with the structural studies (Western blots),
demonstrate that both glycosylated and unglycosylated forms of ROMK1
are delivered to the plasma membrane as operational channels.
Tunicamycin treatment and the N117Q mutant produced unglycosylated
ROMK1 monomers, while both glycosylated and unglycosylated forms of the
subunit were generated by wild-type ROMK1 according to structural
analyses. The wild-type ROMK1 channel expresses distinctive single
channel current kinetics when compared to the unglycosylated forms
produced by either the N117Q mutants or treatment with tunicamycin,
suggesting that a glycoform of the channel is on the surface of cells
infected with ROMK1. However, we do not know whether the normally
conducting ROMK1 oligomer contains partially or completely glycosylated
monomers because the Western blot represents ROMK1 from total
membranes, while the functional studies detect the channels in the
plasma membrane. Furthermore, the proportion of glycosylated monomers
may be altered during protein analysis. In any case, the presence of
glycosylated channels seems to prevent the appearance of channel
behavior manifested by unglycosylated channels.
Because the N117Q
mutation and treatment with tunicamycin produced identical structural
and functional effects, glycosylation, not disruption in the primary
sequence, is responsible for the change in P
Membrane channel proteins often contain N-glycosylation
consensus sequences (21, 22, 23, 24, 25) and in
Shaker K
We thank Marjorie Withers for expert technical
assistance with cell culture and Dr. Maurizio Taglialatela for
discussion of single channel analysis.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
channels (IRKs) are
highly K
-selective, integral membrane proteins that
help maintain resting the membrane potential and cell volume. Integral
membrane proteins as a class are frequently N-glycosylated
with the attached carbohydrate being extracellular and perhaps
modulating function. However, dynamic effects of glycosylation have yet
to be demonstrated at the molecular level. ROMK1, a member of the IRK
family is particularly suited to the study of glycosylation because it
has a single N-glycosylation consensus sequence (Ho, K.,
Nichols, C. G., Lederer, W. J., Lytton, J., Vassilev, P. M.,
Kanazirska, M. V., and Herbert, S. C. (1993) Nature 362,
31-38). We show that ROMK1 is expressed in a functional state in
the plasmalemma of an insect cell line (Spodoptera frugiperda,
Sf9) and has two structures, glycosylated and unglycosylated. To test
functionality, glycosylation was abolished by an N117Q mutation or by
treatment with tunicamycin. Whole cell currents were greatly reduced in
both of the unglycosylated forms compared to wild-type. Single channel
currents revealed a dramatic decrease in opening probability, p
, as the causative factor. Thus we have shown
biochemically that the N-glycosylation sequon is
extracellular, a result consistent with present topological models of
IRKs, and we conclude that sequon occupancy by carbohydrate stabilizes
the open state of ROMK1.
)
channel (IRK)
(
)cloned from rat kidney cells (5) and may be very important for potassium exchange in the
kidney. Unlike voltage-dependent K
channels, IRKs
conduct K
current better in the inward rather than
outward direction. There are two reasons for this, block by
intracellular Mg
(5, 6) or
polyamines(7, 8, 9) . This inward rectification
is in part responsible for the physiological role of IRKs in
maintaining the resting membrane potential, excitability, and potassium
exchange. The two-dimensional orientation of IRKs in the plasma
membrane has been predicted from hydropathy plots and
mutagenesis-electrophysiological studies (see Fig. 1)(5, 6, 7, 10, 11) .
There are two transmembrane segments (M1 and M2) and a pore-forming
segment (H5) that links M1 and M2(5, 10) . More
recently, the carboxyl terminus (6) and a negatively charged
residue in M2 (11, 12) have been identified as key pore
determinants.
Figure 1:
ROMK1 expressed in Sf9 cells is N-glycosylated. The left-hand panel shows the
proposed membrane topology of ROMK1 as based on hydropathy plots and
mutagenesis-electrophysiological studies (5, 16-19). A,
Western blots of membrane fractions of Sf9 cells infected with either
FLAG/ROMK1-recombinant (lane 2) or wild-type (lane 1)
baculoviruses. B, immunoblot of total membranes from Sf9 cells
producing ROMK1 in the presence (lane 2) and absence (lane
1) of tunicamycin (25 µg/ml). C, immunoblot of
membranes from Sf9 cells infected with recombinant virus containing the
FLAG/ROMK1 (lane 1) or the N117Q mutant construct (lane
2). Prestained rainbow markers (Amersham Corp.) are indicated by dots: myosin, 220 kDa; phosphorylase B, 97.4 kDa; bovine serum
albumin, 66 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; trypsin
inhibitor, 21.5 kDa; lysozyme, 14.3 kDa (in A). These
molecular mass standards were also used for the immunoblots in Panels B and C (not shown). Arrowheads represent the position of ROMK1.
As integral membrane proteins, many potassium channels
have N-glycosylation consensus
sequences(2, 3, 4, 13) . However, there
is little evidence on whether the channels are glycosylated at these
sites and how glycosylation affects their channel activity. ROMK1 has a N-glycosylation consensus sequence (amino acid residues:
117-119; NRT) in the first extracellular loop (between M1 and H5
segments; Fig. 1). This site has been shown to be utilized in in vitro translation of ROMK1 in the presence of canine
pancreatic microsomes(5) . However, cell-free translation
experiments may not apply in vivo(2) . In addition, the
functional state of the channel was not determined. To investigate the
occurrence of glycosylation and its functional significance, the
baculovirus expression vector system was utilized to express and
characterize ROMK1. This expression system uses Spodoptera
frugiperda (Sf9), a eukaryotic cell, in which N-glycosylation has been demonstrated(1) .
, indicating that this region of the channel is
extracellular. Both forms of ROMK1, glycosylated and unglycosylated,
are present in the plasma membrane in a functional state. From single
channel and whole cell currents it appears that the N-linked
oligosaccharide stabilizes the open state of the channel. Thus,
occupancy of the N-glycosylation sequon of ROMK1 may provide a
means for modulating renal regulation of body potassium and sodium
ions.
Materials
Zwittergent 3-10 was purchased
from Calbiochem; tunicamycin, phenylmethylsulfonyl fluoride, and trypan
blue solution from Sigma; TNM-FH insect medium from JRH Biosciences;
baculovirus agarose and TA cloning kit from Invitrogen;
BaculoGold transfection kit from Pharmingen;
penicillin-streptomycin, pluronic F-68 10% solution, and fetal bovine
serum from Life Technologies, Inc.; and QIAGEN columns from QIAGEN Inc.
Sf9 cells were obtained from American Type Culture Collection.
Anti-FLAG M2 monoclonal antibody was purchased from International
Biotechnologies, Inc.
Construction of Recombinant Baculoviruses
The
FLAG/ROMK1 cDNA in which an oligonucleotide encoding the FLAG epitope
(DYKDDDDK) was fused to the 5` end of ROMK1 was generated by polymerase
chain reaction (PCR). The sense oligonucleotide contained a start
codon, the nucleotide sequence of the FLAG epitope, plus the first 18
nucleotides of the 5` end of ROMK1. The antisense oligonucleotide
consisted of the last 22 nucleotides of the 3` end of ROMK1 and an EcoRI sequence. The template was ROMK1 cDNA(6) . A PCR
overlap extension (14) was used for construction of the
FLAG/ROMK1 mutant (N117Q). Both sense and antisense mutagenesis
complementary oligonucleotides (34 nucleotides) were synthesized with
two mismatches, and FLAG/ROMK1 cDNA was used as a template. The
purified PCR products were ligated into the pCRII vector (Invitrogen)
for sequencing and amplification. The FLAG/ROMK1 and N117Q mutant
constructs were then subcloned into NotI/BamHI
double-digested baculovirus transfer vector (pVL1392). The baculovirus
transfer vector containing FLAG/ROMK1 and the N117Q mutant constructs
were then propagated and purified according to QIAGEN plasmid
purification protocol. Standard methods were used for plasmid DNA
preparation, PCR extensions, PCR site-directed mutagenesis, and DNA
sequencing(15) .
viral DNA (modified AcNPV virus) according to
instructions accompanied with the BaculoGold
transfection
kit. Plaque purification was used to pick a single virus or the
co-transfection viral supernatant was amplified directly as described
below. In either case there did not appear to be a difference in the
quality of the viruses that were used. Plaque assays were performed to
determine viral titers.
Cell Culture and Virus Infections
Sf9 cells were
grown in Hink's TNM-FH insect medium supplemented with 10% (v/v)
fetal bovine serum, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 0.1% pluronic F-68 at 27 °C under natural
atmosphere. The cells were maintained in monolayer cultures and
passaged every 4-6 days. Monolayer Sf9 cell cultures were used
for co-transfections, plaque assays, and initial viral amplifications.
Sf9 cells were grown in suspension cultures for production of high
titer recombinant virus and expression of the fusion proteins at
initial cell densities of 0.5-1 10
cells/ml
and 1.0-1.3
10
cells/ml, respectively.
Recombinant or wild-type viruses were added to cells at a multiplicity
of infection (m.o.i.) of 0.1-0.5 for viral amplification and
about 10 for protein expression. For electrophysiology studies, Sf9
cells were infected in suspension culture and then added about 30 h
postinfection to Petri dishes that contained glass coverslips. Cells in
the Petri dishes were then analyzed 36-70 h postinfection. The
expression of protein from the remainder of the cells in the spinner
flasks were analyzed by Western blot at similar times. All procedures
used for Sf9 cell cultures were performed as described by King and
Posse (16) and Gruenwald and Heitz(17) . Tunicamycin
(25-50 µg/ml) was added to cells about 15 min after
inoculation with recombinant virus.
Membrane Preparation
Sf9 cells were harvested
36-72 h after infection by centrifugation at 1000 g for 10 min. The pellets were washed with phosphate-buffered saline
(50 mM phosphate, pH 7.4, 0.15 M NaCl) three times
and stored at -70 °C until needed or used immediately. Cells
were resuspended at 10
-10
cells/ml in
detergent lysis buffer (50 mM phosphate, pH 7.4, 0.15 M KCl, 1 mM EDTA, 0.5-1 mM dithiothreitol,
0.1% Zwittergent 3-10, 20% glycerol, and 100 µM phenylmethylsulfonyl fluoride) and incubated on ice for 1 h with
gentle agitation every 15 min. The lysed cells were then hand
homogenized (Dounce hand homogenizer, 20-30 strokes) to break up
large cell debris prior to sonication. Aliquots (3-6 ml) of the
lysed cells were sonicated three or four times for 30 s followed by a
30-s rest period using a Heat Systems cell disruptor. The samples were
then spun at 1000
g for 15 min. This low speed
supernatant was centrifuged at 100,000
g for
30-60 min, and the pellet was resuspended in SDS-polyacrylamide
gel electrophoresis sample buffer (2% SDS, 62.5 mM Tris-HCl
(pH 6.8), 10% (v/v) glycerol, 25 mM dithiothreitol, and 0.001%
(w/v) bromphenol blue).
Western Blotting
Proteins were separated on 12.5%
SDS-polyacrylamide gels as described by Laemmli (18) and were
transferred to Immobilon P membranes (Millipore). The nonspecific
protein binding sites of the membranes were blocked with
phosphate-buffered saline plus 5% blocker (Bio-Rad) or 3% bovine serum
albumin (JRH Biosciences) and 0.1% Tween 20 (Sigma) for 2 h. Membranes
were then incubated with M2 antibody at 50 µg/ml (mouse monoclonal
IgG that recognizes the FLAG peptide fused to ROMK1) for 1 h. After
three washes in phosphate-buffered saline containing 0.1% Tween 20, a
goat anti-mouse IgG conjugated to alkaline phosphatase (Organon Teknika
Corp.) was applied at a 1:1000 dilution for 1 h. Membrane blots were
then washed as described above and were developed with nitro blue
tetrazolium (330 µg/ml) and bromo-4-chloro-3-indoyl phosphate (165
µg/ml).
Whole Cell and Single Channel
Recordings
Fire-polished pipette electrodes had tip resistance
of 1-4 megaohms when filled with internal solution for whole cell
recordings and 9-10 megaohms when filled with external solution
for cell-attached single channel recordings. The internal solution had
the following composition (mM): NaCl, 10; potassium aspartate,
10; KCl, 120; MgCl-6H
O, 4; Mes, 135; EGTA, 20;
glucose, 20; sucrose, 20. The external bathing solution contained
(mM): potassium aspartate, 140;
MgCl
-6H
O, 1; HEPES, 10; mannitol, 80. The
osmolarity was about 350 mOsm/kg, and the pH was adjusted to 7.4 with
KOH for both solutions. All voltage-clamp measurements were conducted
at 23-25 °C using an Axopatch 1C (Axon Instruments, Foster
City, CA) amplifier; data was filtered at 1 kHz before digitization via
a Labmaster DMA interface. The pClamp suite of programs was employed
for data acquisition and analysis. Group data are presented as mean
± SE. Student's t test (unpaired) was used to
evaluate the statistical significance of differences between means. A
two-tailed probability of ±5% was taken to indicate statistical
significance.
is
glycosylated. They are consistent with topological models that place
the region between M1 and H5 as extracellular because N-glycosylation only occurs on the luminal side of the
endoplasmic reticulum and Golgi apparatus(20) .
Figure 2:
Whole cell currents of ROMK1 channels
expressed in Sf9 cells. Currents were produced by 100-ms voltage steps
from a holding potential of 0 mV to varying test potentials ranging
from -120 to +80 mV using 10-mV increments at a pulse
interval of 1 s. Typical examples of current traces of ROMK1 (A), N117Q mutant (B), and ROMK1 from
tunicamycin-treated cells (50 µg/ml; C). D,
current-voltage (I-V) relation of ROMK1 (open circles), N117Q
mutant (open squares), and ROMK1 from tunicamycin-treated
cells (filled triangles). Values are mean ± SE obtained
from 12 cells producing ROMK1, 23 cells expressing N117Q mutant, and 25
cells infected with ROMK1-recombinant virus in the presence of
tunicamycin.
As expected for ROMK1, application of
extracellular Ba reduced the currents. For controls,
we found no ROMK1-like currents in cells infected with wild-type virus (Fig. 2B). Nor were endogenous ROMK1-like currents
observed in uninfected cells (data not shown). All cells were of
similar size, and the time course of the infections did not appear to
be a factor in the reduction of whole cell currents. These results
suggest that ROMK1 channels expressed in cells where N-glycosylation is prevented may express at a lower density in
the plasma membrane and/or may have modified channel activity. To
examine these possibilities, single channel currents were measured.
Figure 3:
Single channel ROMK1 currents recorded
from Sf9 cells in cell-attached patches. Currents were produced by
160-ms voltage steps between -130 and +50 mV with 30
consecutive pulses at each potential from a holding potential of 0 mV.
Voltage increments were 20 mV, and the interpulse interval was 1 s. A, B, and C, analog sweeps of single channel
activity from representative experiments. Only traces obtained from
four consecutive pulses at -100 mV are shown for the sake of
clarity. Summation of the single channel activities, expressed as open
probability (%) as a function of pulse duration, are shown under raw
data traces. Base lines are indicated with dash lines and
represent the closed state. D, current-voltage relation of
single channel activity constructed from same cells as shown in A, B, and C. The lines represent a
least-squares linear regression. The resultant slope conductance was
38.4 pS for ROMK1, 38.5 pS for N117Q mutant, and 36.7 pS for ROMK1 from
tunicamycin-treated cells.
P for
the N117Q mutant and tunicamycin-treated cells, was reduced by 73.9%
and 71.5%, respectively (Fig. 4A). The reduction in P
correlates closely with the reduction of 76 and
75% in whole cell currents for these cells. Therefore, the N-linked oligosaccharide moiety appears to have similar
effects on whole cell currents and P
. The large
reduction for the unglycosylated forms does not appear to be due to a
significantly lower density of surface channels because the percentage
of successful patches was the same, about 15% for all three forms of
ROMK1, and the number of channels per patch was the same, about one to
four.
Figure 4:
Characteristics of single channel
activity. A, mean P at pulse potentials
of -100, -130, and -50 mV. Values above the bars indicate the numbers of traces included in analysis, and asterisks indicate statistical significance (p <
0.05, unpaired t tests). B, time constants of channel
closings. Closed times were distributed according to a single
exponential function in ROMK1, and a double exponential function in
both N117Q and ROMK1 plus tunicamycin. The solid bar represents the fast component of channel closings in N117Q and
ROMK1 from tunicamycin-treated cells.
The closed time distributions for both N117Q and
tunicamycin-treated cells were fitted best by a sum of two exponentials (Fig. 4B). The brief is similar to the single
of wild-type ROMK1. The longer
was observed only in the
unglycosylated form and gave rise to the time-dependent relaxation that
was observed in the whole cell currents.
. The
decrease in P
could involve instability of the
ROMK1 oligomer if the oligosaccharide were involved in intra-subunit
interactions that stabilize the open state. Another possibility is that
the carbohydrate moiety participates in binding of a specific ligand
which stabilizes the open state of the channel. It is unlikely that the
mechanism involves changes in surface charge since the P
and closed times are similar, over a wide range from -130
to -50 mV (Fig. 4), and the reduction in whole cell
currents was maintained over an even greater range from -120 to
+80 mV (Fig. 2D). In addition, the N-linked oligosaccharide in Sf9 cells has generally been found
to be of the high mannose (i.e. Man
GlcAc
-Asn) variety (1) rather than complex oligosaccharides as in the case of
Na
channels where surface charge effects due to
glycosylation have been described (21). Western blot analysis also
indicates that the oligosaccharide is simple because the band of the
glycoform is sharp. If complex oligosaccharide synthesis was occurring,
there would be either more than one band or the band would be diffuse.
channels; these sequences are
conserved(13) . Glycosylation of the Shaker H4 (26) and
Kv1.1 channel proteins (27) have been demonstrated, and, as for
ROMK1, the patterns are consistent with proposed topological models.
However, for Shaker and Kv1.1, it was concluded that glycosylation had
no effect on function. In contrast, glycosylation of ROMK1 clearly
alters channel kinetics. In future studies, it will be of interest to
determine whether cells may produce different ratios of unglycosylated
and glycosylated forms of ROMK1 as a novel means of modulating resting
membrane potential and cell volume.
channel; Sf9, Spodoptera frugiderpa cell line; P
, opening probability; PCR,
polymerase chain reaction; Mes,
2-(N-moropholino)ethanesulfonic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.