©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Potassium Channel Structure and Function as Reported by a Single Glycosylation Sequon (*)

Ruth A. Schwalbe , Zhiguo Wang , Barbara A. Wible , Arthur M. Brown (§)

From the (1)Rammelkamp Center for Education and Research, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio 44109-1998

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Inwardly rectifying K 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.


INTRODUCTION

ROMK1 is an inward rectifier potassium (K) 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) .

In this study we found that ROMK1 is N-glycosylated at Asn, 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.


EXPERIMENTAL PROCEDURES

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) .

Recombinant baculoviruses were generated by co-transfection of Sf9 cells with the engineered transfer plasmids and BaculoGold 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-6HO, 4; Mes, 135; EGTA, 20; glucose, 20; sucrose, 20. The external bathing solution contained (mM): potassium aspartate, 140; MgCl-6HO, 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.


RESULTS AND DISCUSSION

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 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) .

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).


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.

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).


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 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. 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. ManGlcAc-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.

Membrane channel proteins often contain N-glycosylation consensus sequences (21, 22, 23, 24, 25) and in Shaker K 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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL36930 and NS23877 (to A. M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Rammelkamp Center for Education and Research, MetroHealth Campus, Case Western Reserve University, 2500 MetroHealth Drive, Cleveland, OH 44109-1998. Tel.: 216-459-5955; Fax: 216-778-8282.

The abbreviations used are: IRK, inwardly rectifying K channel; Sf9, Spodoptera frugiderpa cell line; P, opening probability; PCR, polymerase chain reaction; Mes, 2-(N-moropholino)ethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Marjorie Withers for expert technical assistance with cell culture and Dr. Maurizio Taglialatela for discussion of single channel analysis.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.