| Human Molecular Genetics | Pages |
Functional consequences of ROMK mutants linked to antenatal Bartter's syndrome and implications for treatment
Introduction
Results
Discussion
Materials And Methods
Mutagenesis
Cell culture and recombinant baculoviruses
Immunoaffinity purification
SDS-PAGE and western blotting
Laser confocal microscopy
Patch-clamp recordings
Acknowledgements
References
Functional consequences of ROMK mutants linked to antenatal Bartter's syndrome and implications for treatment
INTRODUCTION
Sodium reabsorption in kidney epithelial cells results from a combination of symport and antiport systems that are driven by the Na+ gradient across the plasma membrane, generated by the Na+-K+ ATPase. A primary mediator of Na+ uptake in the thick ascending loop (TAL) of Henle is the Na-K-2Cl co-transporter (NKCC2) (1). A weak inwardly rectifying ATP-sensitive K+ channel designated as ROMK (9) is critical for K+ secretion and ensures that Na+ uptake via NKCC2 is efficient (2,3). Mutations in NKCC2 recently have been linked to the antenatal form of Bartter's syndrome (ABS), a severe autosomal recessive disease characterized by renal salt wasting, hypotension, hypokalemic metabolic alkalosis and hypercalciuria (4). Elevation of prostaglandin E2 (PGE2) is a secondary phenomenon and has been the basis for treatment of ABS using indomethacin (5,6). However, this treatment does not correct salt wasting. Subsequently, Simon et al. (7) described five kindreds in which ABS did not co-segregate with the NKCC2 locus on chromosome 15 but was linked to the ROMK locus on chromosome 11. Seven ROMK mutations were identified using single-strand conformational polymorphism (SSCP) analysis. A more recent study has also identified ROMK mutations in eight distinct ABS kindreds (8).
ROMK was cloned from rat kidney outer medulla (9) and is a member of the Kir family of K+ channels. Kirs are gated by cytoplasmic blockers rather than membrane potential, as is the case for voltage-dependent K+ channels (Kvs) (9,10-14). Alternative splicing of rat ROMK occurs at the 3[prime] and 5[prime] ends (15,16). Human ROMK shows 93% sequence identity, and alternative splicing produces five distinct transcripts (17,18). All of the isoforms, rat and human, share a highly conserved core to which the Bartter's mutations have been localized (Fig. 1A). Therefore, it is very likely that the function of all isoforms, both rat and human, will be affected in a similar manner. The rat isoforms are expressed differentially along the loop of Henle and distal nephron, and each isoform expresses a functional channel following injection of the cognate cRNA into Xenopus laevis oocytes (15,16,19). Human ROMK and its variants have similar functional properties (17).
Figure 1. Comparison of ABS mutant proteins with WT ROMK1 expressed in Sf9 cells. The ABS mutations are shown in (A). Protein samples were immunopurified and analyzed by western blots. (B) Samples were electrophoresed on 10% SDS gels. The arrowheads denote the glyco- (upper band) and aglyco-forms (lower band) of full-length ROMK1 proteins (left side) and T332-K333, indicated as T332 (right side), respectively. The open circle is the 46 kDa rainbow marker (ovalbumin). (C) Proteins were separated on 12% SDS gels. The two upper arrowheads represent glycosylated (upper) and unglycosylated (lower) WT ROMK1, while the two lower denote the glyco- (upper) and aglyco-forms (lower) of S219R. The 30 (carbonic anhydrase) and 46 kDa markers are indicated by the open circles. Incubation with tunicamycin abolished the glycosylated bands; this is shown for S219R by the + symbol. Based on hydropathy plots, ROMK and other inwardly rectifying K+ channels (Kirs) are topologically similar (9). The absence of a signal sequence in the open reading frame places the N- and C-termini intracellularly. There are two transmembrane segments, M1 and M2, which are assumed to be [alpha]-helical. M1 and M2 are separated by a linker of 17 residues called H5. This segment is homologous to the pore-forming segment found in Kv channels (20,21). Recently, we have shown that N-glycosylation sites inserted in H5 are utilized, indicating that H5 is extracellular (22). Glycosylation enhanced K+ flux by stabilizing the channel open state (23). The functional properties of ROMK and its localization along the loop of Henle and distal nephron are similar to the ATP-regulated channel responsible for K+ secretion in kidney tubules (2,3). The similarity is extended to regulation by the phosphorylation-dephosphorylation cycle (24); cAMP-dependent kinase enhances activity while an Mg2+-dependent phosphatase inhibits activity (25). Three consensus A-kinase sites have been reported, one located at the N-terminus, position 44, and two at the C-terminus, positions 219 and 313 (24). We addressed the functional consequences of four of the seven ROMK mutations reported by Simon et al. (4). We focused on the three missense mutations, A214V, S219R and M357T, and also the C-terminus truncation mutant T332 frameshift, since the three N-terminus mutations that were described should produce C-truncated proteins, lacking the transmembrane segments. We found that M357T expressed normal channel function, indicating polymorphism at this locus. We found that the three remaining mutations had distinctly different consequences. A214V expressed channels that ran down significantly more quickly than the wild-type channels during whole-cell patch-clamp but were otherwise normal. S219R produced proteolytically processed channel subunits which were transported to the plasma membrane but were non-functional. The C-terminus T332 frameshift produced truncated channel subunits that were not transported to the cell membrane.
RESULTS
We constructed the four mutant proteins A214V, S219R, M357T and T332 frameshift using rat ROMK1 cDNA as template. The numbering of the substituted residues is +19 to the numbering in human ROMK2 due to alternative splicing at the N-terminus of ROMK2. Recombinant baculoviruses encoding the ROMK1 variants in insect cells were used for expression and characterization. This system provides sufficient amounts of ROMK1 protein for biochemical characterization (22,23), and the co- and post-translational processing events required for generating functional heterologous proteins have been demonstrated extensively (26). The single-channel currents are virtually identical to those recorded in other cells expressing ROMK (9,11,23). After expression in Sf9 cells, the channels were purified using the M2 Flag antibody directed against the M2 Flag epitope attached to the N-terminus. The epitope did not interfere with wild-type (WT) ROMK1 function (23).
All of the mutants produced both glycosylated (upper band) and unglycosylated (lower band) monomers. The glyco- and aglyco-forms of A214V and M357T were similar to those of WT ROMK1 and ran at ~45 and 43 kDa, respectively (Fig. 1B; cf. ref. 23). Both forms of the T332 frameshift ran faster, at ~43 and 40 kDa, confirming premature termination of the protein. For S219R, the bands ran fastest of all, the minor band running at ~32 kDa and the major one at ~29 kDa (Fig. 1C). The C-terminal fragment was not detected because the M2 Flag was attached to the N-terminus. It is of interest that the unglycosylated band migrated to a position corresponding to the molecular weight calculated for the first 218 amino acid residues of ROMK1. A similar band was not detected in WT ROMK1 or any other ROMK1 mutants that we have studied (22,23).
In all cases, only the unglycosylated band was observed when N-glycosylation was inhibited by tunicamycin (TM) (22,23). Glycosylation and its prevention by TM demonstrate that the Bartter's mutants, just like WT ROMK, were inserted into the endoplasmic reticulum (ER) membrane.
The function of ROMK is to secrete K+, and K+ flux was compared in mutated channels by measuring whole-cell and single-channel currents. A214V and M357T expressed whole Sf9 cell currents similar to WT ROMK1 (23). Inward currents were time independent and the current-voltage relationship showed characteristic weak inward rectification (Fig. 2A). Both A214V and M357T remained selective for K+ as shown by the negative shift in the reversal potential when K+ was replaced with Na+ (Fig. 2B). As in WT, the currents were blocked by Ba2+ at millimolar concentrations and significantly reduced when infected cells were treated with TM. The large reduction in current after TM was shown to result from the absence of N-linked oligosaccharide at position 117 of WT ROMK1 and was due to a decrease in probability of opening, Po (23).
Figure 2. Whole-cell currents of ABS mutants. (A) Whole-cell currents were produced by voltage steps from -120 to +80 in 20 mV increments starting from a holding potential of 0 mV. The cells were perfused with a high K+ solution containing (in mM): K-aspartate 140, MgCl2 1,2-[N-morpholino]ethanesulfonic acid 10, nannitol 60 (pH was adjusted to 6.3 with Tris-OH). The pipette solution contained (in mM): K-aspartate 140, MgCl2 5, HEPES 10, EGTA 10, mannitol 40 (pH adjusted to 7.2 with Tris-OH). (B) Current-voltage (I-V) relationships of A214V (open symbols) and M357T (closed symbols) in a bathing solution containing high K+ (circles) and low K+ (triangles). The low K+ solution contained (in mM): Na-aspartate 135, K-aspartate 5, MgCl2 1, 2-[N-morpholino]ethanesulfonic acid 10, mannitol 60; pH was adjusted to physiological levels, 6.3, with Tris-base. All currents were normalized to currents obtained at -120 mV in the high K+ solution. (C) Inward currents normalized to those obtained at -120 mV are plotted versus time. Currents recorded for WT ROMK1, A214V and M357T are plotted at minute intervals, minus and plus 5 mM K-ATP in the pipette solution, as indicated. Values are given as means ± SEM (n = 3 cells, plus ATP, and n = 8, WT ROMK1; n = 10, A214V; and n = 4, M357T, minus ATP). Whole-cell currents of WT ROMK and M357T ran down slowly, with >75% of the current remaining at 15 min after penetrating the cell with the patch pipette (Fig. 2C). For A214V, current rundown was both faster and greater; at 2 min the current was reduced to 75% and at 12 min to 30%. Position 214 is near the cAMP phosphorylation site at S219, and S219R did not express whole-cell currents (Fig. 2A). In addition, channel rundown due to regulation of ROMK by protein kinase A has been studied extensively (24,25). We therefore wondered if the mutation at 214 hindered channel phosphorylation leading to fast channel rundown. To test this, we added 5 mM ATP to the solution in the patch-clamp pipette and found that rundown was prevented (Fig. 2C). This maneuver also prevented the slow rundown observed in the WT channel. The fast rundown may have arisen in channels having abnormal molecular properties. Moreover, the molecular behaviors of A214V and M357T are unknown. Consequently, we compared single-channel currents in the cell-attached configuration. In this way, we avoided the fast rundown of ROMK1 that occurs in excised patches (9). Open probabilities, open times, close times and single-channel current amplitudes were similar for both A214V and M357T (Fig. 3), and in both cases these values were similar to WT ROMK1 (23). Likewise the slope conductances of the channels were similar. Figure 3. Single-channel currents of ABS mutants recorded in the cell-attached mode. (A) Representative single-channel traces of A214V and M357T at three different test potentials. The currents were examined using voltage steps of 400 ms at a frequency of 1 Hz. The dashed line indicates the closed state. (B) Current-voltage relationships from -140 to -40 mV obtained from three cells expressing A214V and three cells expressing M357T. Single-channel conductance was 38.6 pS for A214V (closed circles) and 38.1 pS for M357T (open circles). The open probabilities, open times and closed times at -100mV for WT ROMK, A214V and M357T were as follows: 87.5 ± 8.2%, 12.1 ± 2.3 ms, 1.75 ± 0.33 ms; 85.4 ± 5.9%, 10.5 ± 2.9 ms, 1.85 ± 0.08 ms; and 84.1 ± 5.7%, 9.8 ± 3.6 ms, 1.58 ± 0.28 ms, respectively. Values are expressed as means ± SD. The pipette and perfusion solutions were high K+ solutions. Data points were fit using linear regression analysis. Unlike A214V and M357T, no currents were observed for either S219R or the T332 frameshift (Fig. 2A). In these circumstances, the lack of current may have been due either to channels that were plasmalemmal but non-functional or to an absence of plasmalemmal channels. To distinguish between these possibilities, ROMK protein was localized in Sf9 cells using the M2 monoclonal antibody and fluorescence immunocytochemistry in combination with laser confocal microscopy. Sf9 cells were examined at 24-28 h post-infection when ROMK immunofluorescence can be assigned to the ER membrane or plasmalemma. The patterns of immunofluorescence were similar for WT ROMK1 (Fig. 4A), M357T (not shown), A214V (Fig. 4B) and S219R (Fig. 4C): a thicker inner ring surrounding the nucleus, a thinner outer ring surrounding the cell and patches of fluorescence in between. The pattern for cells infected with the T332 frameshift (Fig. 4E) was more patchy, and fluorescence was concentrated around the nucleus with no demonstrable outer ring. Similar treatment of uninfected cells and cells infected with cDNA of the human ether-a-go-go gene Herg product, a Kv channel construct lacking the M2 epitope, produced a background signal that was faint to absent. Figure 4. Immunofluorescent localization of WT ROMK (A) and ABS mutants, A214V (B), S219R (C) and T332 (D), in Sf9 cells. The arrows indicate staining at the plasma membrane. Note the absence of the circular pattern of staining at the plasma membrane for T332 (D) and the presence of this pattern for S219R (C). Both of these mutants were non-functional. Proteins were detected using a mouse monoclonal antibody to the M2 Flag epitope and a rabbit anti-mouse secondary antibody conjugated to rhodamine. Images of cells were obtained using a laser scanning confocal microscope.
DISCUSSION
We attribute the outer ring of fluorescence to channels in the cell membrane, the inner ring to channels in the ER membrane and the patches to channels either in the ER membrane or traversing the cytoplasm to destinations such as the plasmalemma. The presence of the outer ring when WT, A214V and M357T were infected is consistent with our recordings of typical whole-cell and single-channel ROMK currents in these cells. S219R channels were also present in the plasmalemma but were non-functional despite being glycosylated. If we recall that unglycosylated S219R migrated as expected for the molecular weight of a protein that was clipped at or near this position, that no full-length form was observed under purification conditions which limit in vitro degradation, that other ROMK1 mutants did not generate this immunoband (22,23), and that plasmalemmal products were present yet membrane currents were absent, it is likely that in vivo proteolysis occurred. Proteolytic enzymes such as trypsin-like endoprotease and carboxypeptidase N are present in Sf9 cells and are capable of identifying and cleaving recombinant proteins (26,27). Thus we infer that mutation of S219 to R219 generates a site at which in vivo proteolysis occurs, resulting in at least two fragments. The presence of trysin-like proteases in Sf9 cells and the introduction of an Arg residue would suggest that the ROMK mutant protein is cleaved by a trypsin-like protease. Non-functional channels may have resulted from one or more of the following: the non-conserved mutation, the absence of phosphorylation or an increase in the degrees of freedom of the broken amide bond.
The T332 frameshift was not present in the plasmalemma, and for this reason ROMK currents were absent. The extracellular regions of the protein did, however, enter the lumen of the ER since glycosylation occurred. Thus, the functional defect in this mutant was improper trafficking to the cell membrane. It remains to be determined if the truncated protein is capable of conducting K+ currents.
A214V was normally distributed to the cell membrane and its significantly faster rundown was prevented by increasing cytoplasmic ATP; thus the fast rundown may reflect defective phosphorylation of the correctly inserted channel. The results are consistent with our hypothesis that residue A214 interacts with the cAMP-dependent A kinase site at S219 to influence phosphorylation. It remains to be determined if A214V reduces phosphorylation or enhances dephosphorylation.
M357T was glycosylated, expressed in the cell membrane, and the single-channel currents and whole-cell current rundown were identical to those of WT ROMK1. This substitution therefore appears to be an allelic polymorphism. Other evidence that supports an allelic polymorphism is that no mutation was found on the other allele and inheritance was fitted with autosomal recessive transmission (7). In addition, no mutation was identified in either ROMK or NKCC2 in one outbred family in the study by Simon et al. (7) and three families in the study by Karolyi et al. (8). The serum potassium levels were also slightly higher in Bartter's patients with ROMK mutations on both alleles than other patients (7). Taken together, these results would suggest the involvement of another gene. However, experimentally, we have not completely eliminated an effect due to regulation by ATP and pH. Regulation of M357T by pH was not addressed since the regulatory regions for internal and external pH have been localized to the N-terminus (29,30) and H5-M131, respectively. Therefore, it is possible but unlikely that these factors will have a large and dramatic effect on the function of M357T.
In this study, we have used rat ROMK1, and all ROMK isoforms, in both rat and human, share the highly conserved core sequence in which all the mutations have been identified, and consequently the activity of all the isoforms is expected to be affected in a similar manner. The present experiments demonstrate that the mechanisms producing defective K+ flux in the patients studied by Simon et al. (7) differ for each mutation. Each mutation implies a different therapeutic modality which might be used in conjunction with indomethacin. Synthetic chaperones might successfully direct the T332 frameshift to the cell membrane; protease inhibitors might interrupt proteolytic processing of S219R and phosphodiesterase, or phosphatase inhibitors might normalize phosphorylation of A214V.
MATERIALS AND METHODS
Mutagenesis
All ROMK1 Bartter's mutants were constructed by PCR overlap extension (28) using Flag-ROMK1 cDNA as template (23). The M2 Flag epitope was fused to the 5[prime] end of ROMK1 for immunoaffinity purification, western blots and immunocytochemistry. PCR products were subcloned into pCRII (Invitrogen) for amplification and sequencing, and subsequently subcloned into NotI-BamHI double-digested baculovirus transfer vector (pVL 1392).
Cell culture and recombinant baculoviruses
Sf9 cells were grown in Hink's TNM-FH insect medium supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.1% pluronic F-68 at 27°C under natural atmosphere. Monolayer cultures were passaged every 4-6 days for maintaining cells. Suspension cultures were utilized for infection at a cell density of 1-1.2×106 cells/ml. The recombinant baculoviruses were generated by co-transfection of Sf9 cells with baculovirus vectors containing the ROMK1 construct and Baculogold viral DNA (modified AcNPV) according to the manufacturer's instructions (Pharminogen). Viral seed stocks were prepared by amplification using monolayer cultures. Suspension culture at a cell density of 0.5-0.8×106 cells/ml was used for generating high titer virus. The multiplicity of infection (m.o.i.) for amplifications of recombinant virus was <1; for production of recombinant protein, the m.o.i. was 5-20. The viral supernatant and/or the infected cells were collected 1-4 days after the infection. TM (25 µg/ml) was added to cells 5-15 min after inoculation with viral supernatant. Standard procedures for routine sub-culturing and co-transfection were followed (26).
Immunoaffinity purification
Sf9 cells (4×106-5×106) were harvested 22-28 h post-infection by centrifugation at 1000 g for 10 min. Pellets were washed with cold phosphate-buffered saline (PBS) and then resuspended in cell lysis buffer (50 mM phosphate, pH 7.4, 0.3 M KCl, 2% Zwittergent 3-10) in the absence and presence of 100 µM phenylmethylsulfonyl fluoride and 200 µM aminobenzamidine, plus 125 µl of the M2 antibody affinity gel resuspended in PBS which recognizes the Flag epitope. The sample was rocked gently overnight, the resin allowed to settle and the supernatant discarded. Resin was washed with the solubilization buffer without the detergent (8 ml) and then with PBS. All purifications steps were done at ~4°C. Beads were resuspended in 100-150 µl of SDS sample buffer and heated at 90°C for 4 min.
SDS-PAGE and western blotting
Reducing 10 and 12% SDS-polyacrylamide gels were used to separate the proteins. Electrophoresed proteins were transferred to Immobilon P membranes and incubated with the M2 antibody (mouse monoclonal IgG) at a concentration of 50 µg/ml and then with goat anti-mouse IgG conjugated to alkaline phosphatase (1:1000) (22,23). The gels were run until the carbonic anhydrase ran off the gel and ~1 cm from the bottom of the gel for 10 and 12%, respectively to separate the glyco- and aglyco-forms. Only the portion of gels that contained the ROMK1 proteins are shown.
Laser confocal microscopy
Sf9 cells infected for 20-28 h in suspension culture were plated in cell wells containing glass coverlips, allowed to adhere for at least 15-20 min and then washed with PBS. Cells were then fixed with paraformaldehyde (3%) for 5-15 min and washed three times with PBS. Cells were permeabilized and non-specific binding was blocked using PBS plus 3% bovine serum albumin (BSA), 0.1% Tween-20 and 0.03% Triton X-100. Fixed cells were incubated with the primary antibody M2 (1:100) overnight at 4°C, washed three times with blocking buffer without Triton X-100, and incubated with a rhodamine-conjugated sheep anti-mouse antibody (1:100) for 1 h at room temperature. Cells were washed three times with PBS. The glass coverslips were mounted with mounting medium (Vectashield). Cells were examined with a Noran Instruments ODYSSEY XL confocal laser scanning microscope equipped with a Nikon inverted microscope using a ×60 objective lens. Antibodies were suspended in PBS, plus 3% BSA and 0.1% Triton X-100.
Patch-clamp recordings
Details have been provided in Schwalbe et al. (23). Briefly, cells were added 20-24 h post-infection to Petri dishes containing glass coverslips. Currents were measured at 23-25°C using an Axoclamp 1B amplifier (Axon Instruments, Foster City, CA) with membrane capacitance and resistance compensation. Fire-polished patch pipettes had tip resistances of 2-5 and 9-10 M[sigmav] for whole-cell and single-channel measurements, respectively. Voltage pulse protocols and data acquisition and analysis were carried out using the pClamp suite of programs (Axon Instruments). Data were filtered at 1 kHz and subsequently digitized at 5 kHz. Cells were perfused for no more than 20 min.
ACKNOWLEDGEMENTS
We thank Tom Carrol for assistance with DNA sequencing. This work was supported by Kidney Foundation of Ohio and National Institute of Health Grants DK52990 (to R.A.S.), HL36930 and HL55404 (to A.M.B.).
REFERENCES
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