A phosphorylation-dependent export structure in ROMK (Kir 1.1) channel overrides an endoplasmic reticulum localization signal.

The cell surface density of functional Kir1.1 (ROMK, KCNJ1) channels in the renal collecting duct is precisely regulated to maintain potassium balance. Here, we explore the mechanism by which phosphorylation of Kir1.1a serine 44 controls plasmalemma expression. Studies in Xenopus oocytes, expressing wild-type, phosphorylation mimic (S44D), or phosphorylation null (S44A) Kir1.1a, revealed that phosphorylation of serine 44 is required to stimulate traffic of newly synthesized channels to the plasma membrane through a brefeldin A-sensitive pathway. ROMK channels were found to acquire mature glycosylation in a serine 44 phosphorylation-dependent manner, consistent with a phosphorylation-dependent trafficking step within the endoplasmic reticulum/Golgi. Serine 44 neighbors a string of three "RXR" motifs, reminiscent of basic trafficking signals involved in directing early transport steps within the secretory pathway. Replacement of the arginine residues with alanine (R35A, R37A, R39A, R41A, or all Arg to Ala) did not restore cell surface expression of the phospho-null S44A channel, making it unlikely that phosphorylation abrogates a nearby RXR-type endoplasmic reticulum (ER) localization signal. Instead, analysis of the compound S44D phospho-mimic mutants revealed that the neighboring arginine residues are also necessary for cell surface expression, identifying a structure that determines export in the biosynthetic pathway. Suppressor mutations in a putative dibasic ER retention signal, located within the cytoplasmic C terminus (K370A, R371A), restored cell surface expression of the phospho-null S44A channel to levels exhibited by the phospho-mimic S44D channel. Taken together, these studies indicate that phosphorylation of Ser44 drives an export step within the secretory pathway to override an independent endoplasmic reticulum localization signal.

The ROMK (aka Kir 1.1 or KCNJ1) subfamily of inward rectifying potassium channels (1) is essential for proper renal potassium excretion and the maintenance of potassium homeostasis. These channels are believed to be a major route for potassium transport into the distal tubule lumen (2,3) and constitute a final regulated component of the potassium secretory machinery in the kidney (4,5). Indeed, aldosterone, vasopressin, and other factors precisely regulate ROMK activity in the renal cortical collecting duct, controlling potassium excretion in accord with the demands of potassium balance. Because ROMK channels normally exhibit a very high open probability (6), physiologic alterations in channel activity are likely to be achieved by regulated changes in the number of active channels at the plasmalemma.
A growing body of data has pointed to the involvement of phosphorylation-dependent trafficking mechanisms in the physiologic control of ROMK cell surface density. For example, Src kinases stimulate ROMK endocytosis and down-regulate channel density in states of potassium deprivation (7)(8)(9)(10). Recent studies have also revealed that mutant forms of the WNK4 kinase can stimulate ROMK channel internalization (11), causing potassium retention in pseudohypoaldosteronism type II (12). By contrast, observations that PKA 2 (13,14) and the aldosterone-induced kinase, SGK1 (14), phosphorylate an N-terminal residue in ROMK, serine 44, to drive cell surface expression (14,15) has suggested a plausible mechanism for the physiological up-regulation of ROMK channel density at the plasmalemma by vasopressin (16) and aldosterone (17).
The mechanism by which phosphorylation of serine 44 controls ROMK cell surface number remains completely unknown, however. In principle, phosphorylation could drive delivery of channels to the cell surface, impair channel internalization or work through a combination of these processes. Observations of ours (14,18) and others (19) that unphosphorylated ROMK channels are inefficiently expressed on the plasmalemma and reside predominately in the endoplasmic reticulum (ER) provide reason to suggest that phosphorylation of the serine 44 residue stimulates anterograde trafficking in the secretory pathway, possibly at the level of the ER/Golgi. Intriguingly, serine 44 juxtaposes putative RXR-type ER localization (20) and Golgi export (21) signals. Here, we test the hypothesis that cell surface expression of ROMK channel is regulated by phosphorylation-dependent export in the secretory pathway through a mechanism involving the phosphoserine 44 and the neighboring arginine motifs.

MATERIALS AND METHODS
Molecular Biology-All studies were performed with modified rat ROMK1 (aka Kir 1.1a) (1), containing an external hemagglutinin (HA) epitope tag as described before (14). Site-directed mutagenesis was performed using a PCR-based strategy with PfuTubo DNA polymerase (QuikChange, Stratagene). The sequence of all modified cDNAs was confirmed by dye termination DNA sequencing (University of Maryland School of Medicine Biopolymer Core). All constructs used for studies in Xenopus oocytes were subcloned between the 5Ј-and 3Ј-untranslated region of the Xenopus ␤-globin gene in the modified pSD64 vector to increase expression efficiency (22). This vector also contains a polyadenylate sequence in the 3Ј-untranslated region (dA23dC30).
cRNA Synthesis-Complementary RNA was transcribed in vitro in the presence of capping analogue G(5Ј)ppp(5Ј) from linearized plasmids containing the cDNA of interest using SP6 RNA polymerase (mMessage Machine, Ambion Inc.). cRNA was purified by spin column chromatog-* This work was supported by National Institutes of Health Grants DK54231 and DK63049 (to P. A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed. E-mail: pwelling@umaryland.edu. raphy (MEGAclear, Ambion Inc.). Yield was quantified spectrophotometrically and confirmed by agarose gel electrophoresis.
Oocyte Isolation and Injection-Oocytes from female Xenopus Laevis (Xenopus Express, Homosassa, FL) were isolated and maintained using the standard procedures as described previously (23). Briefly, frogs were anesthetized with 0.15% 3-aminobenzoate, and a partial oophorectomy was performed through an abdominal incision. Oocyte aggregates were manually dissected from the ovarian lobes and then incubated in OR-2 medium (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , and 5 mM HEPES, pH 7.4) containing collagenase (type 3, Worthington) for 2 h at room temperature to remove the follicular layer. After extensive washing with collagenase-free OR-2, oocytes were stored at 19°C in OR-3 medium (50% Leibovitz's medium, 10 mM HEPES, pH 7.4, containing penicillin/ streptomycin). 12-24 h later, healthy looking, Dumont stage V-VI oocytes were pneumatically injected with 50 nl of diethyl pyrocarbonate-treated water containing 0.75 ng of cRNA (unless noted otherwise) and then stored in OR-3 medium at 19°C.
Electrophysiology-Whole cell currents in Xenopus oocytes were monitored using a two-microelectrode voltage clamp as described previously (6,23 Voltage-sensing and current-injecting microelectrodes had resistances of 0.5-1.5 M⍀ when backfilled with 3 M KCl. Once a stable membrane potential was attained, oocytes were clamped to a holding potential of 0 mV, and currents were recorded during 500-millisecond voltage steps, ranging from Ϫ100 to ϩ40 mV in 20-mV increments. For assessment of cation selectivity (P K /P Na ) reversal potentials were measured as before (23) in 5 mM K and 5 mM Na (85 mM N-methyl-D-glucamine-Cl, 1 mM MgCl 2 . 1 mM CaCl 2 , 5 mM HEPES, pH 7.4). Data were collected using an ITC16 analog-to-digital, digital-to-analogue converter (Instrutech Corp.), filtered at 1 kHz, and digitized on line at 2 kHz using Pulse software (HEKA Electronik) for later analysis. ROMK currents are taken as the Barium-sensitive inward current (2 mM barium acetate) as we have done before (6). Values reported in the text are the barium-sensitive inward currents at Ϫ100 mV.
Surface Expression-Plasmalemma expression of the external HAtagged ROMK1 channel was measured in single oocytes following procedures outlined by Zerangue et al. (20) with slight modifications as before (14). In these studies, oocytes were washed two times in cold OR-2 medium, fixed with 4% formaldehyde in OR-2 for 15 min at 4°C, and washed three times in OR-2. To block spurious antibody binding, oocytes were then incubated for 1 h at 4°C in OR-2 containing 1% bovine serum albumin (BSA). Exposed HA epitopes on the surface of intact oocytes were labeled with a rat monoclonal anti-HA antibody (1 g/ml, Roche Applied Science 3F10, 1% BSA, 4°C overnight), and then oocytes were washed with OR-2 and incubated with horseradish peroxidase-coupled goat anti-rat (1 g/ml, Jackson Laboratories, 1% BSA, 3 h). Cells were washed for 1 h with OR-2 containing 1% BSA and then again for 10 min in OR-2 medium without BSA. Individual oocytes were placed in 50 l of enhanced chemiluminescence substrate (Amersham Biosciences) and incubated for 1 min at room temperature. Luminescence from single oocytes was measured for 10 s in a Sirius luminometer and reported as the average relative light units per second. Average values represent 15-20 oocytes/construct/frog repeated in at least triplicate (three frogs).
Membrane Protein-Oocytes were processed following the protocol described by Kamsteeg and Deen (24) to isolate proteins from total membrane. In brief, oocytes were washed twice in homogenization buffer (80 mM sucrose, 5 mM MgCl 2 , 5 mM NaH 2 PO 4 , 1 mM EDTA, 20 mM Tris, pH 7.4) containing a protease inhibitor mixture (5 g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 5 g/ml pepstatin A) and then broken by trituration with a 25-gauge syringe. To pellet yolk proteins and nuclei, homogenates were spun twice at low speed (100 ϫ g) for 10 min. Supernatants were then spun at high speed (16,100 ϫ g) for 20 min at 4°C to collect the total membrane fraction. Pellets were washed once in the homogenization buffer and spun at top speed again for 15 min and then placed in solubilization buffer (4% sodium deoxycholate, 20 mM Tris, pH 8.0, 5 mM EDTA, 10% glycerol, containing the protease inhibitors) and rocked for 2 h at 37°C. Particulate material was pelleted (16,100 ϫ g for 20 min at 4°C), and the solubilized proteins in the supernatant were resolved by SDS-PAGE electrophoresis and transferred to nitrocellulose membranes. Western blot analysis of total membrane ROMK protein, as described below, was performed to ensure all constructs were translated at relatively equal levels. To study glycosylation, the solubilized membrane protein (20 g) was treated with PNGase F or endoglycoside H (Endo H) (25 units/g of protein) for 37°C according to the recommendations of the manufacturer (New England Biolabs) and then subjected to Western blot analysis.
Cell Surface Immunoprecipitation-Surface delivery of S44A and S44D channels were studied in a qualitative cell surface immunoprecipitation assay. Samples containing the two different mutant channels were processed simultaneously as described below. After recovery from cRNA injection (19°C for 12 h), intact oocytes were placed at room temperature and incubated with an anti-HA antibody (Roche Applied Science rat monoclonal anti-HA; 1:100 in OR-2 media supplemented with 1% BSA) to capture the arrival of channels at the cell surface at specific chase times (2, 4, and 6 h). The different channel mutants were compared using an equal numbers of oocytes (50 oocytes/frog per time point), and the studies were repeated in triplicate (3 frogs). At each time point, oocytes were placed at 4°C in OR-2 medium containing brefeldin A (BFA) (10 M) to stop membrane traffic and then further incubated with the antibody (4 h, 4°C) to optimize labeling of cell surface expressed ROMK channels. Oocytes were washed of unbound antibody with the OR-2 ϩ 1% BSA solution and lysed as above. Surface HA antibody-bound channels were then immunoprecipitated from membrane preparation using Protein G-conjugated Sepharose beads overnight at 4°C, rotating. Beads were washed three times with homogenization buffer ϩ 0.1% Triton. Protein was eluted with SDS sample Buffer, resolved by SDS-PAGE, and transferred onto nitrocellulose for Western blot analysis with anti-rabbit ROMK antibodies. Western blot analysis was then performed to identify surface immunoprecipitated ROMK (1°Ab, anti-ROMK LC35 (25), 1:100; 2°Ab, donkey anti-chicken horseradish peroxidase, 1:5000).
Statistical Analysis-Statistical analysis was performed using GB-Stat TM for Macintosh statistics package (Dynamic Microsystems, Inc). Analysis of variance followed by Fisher's least significant difference method post hoc test was performed to test for significance. Values of at least p Ͻ 0.05 were considered significant. Single exponential curve fits were performed using a Levenberg-Marquardt non-linear least squares optimization in Igor (WaveMetrics, Lake Oswego, OR). All data are given as mean Ϯ S.D. All biochemical studies were repeated in at least triplicate.

RESULTS
To determine whether phosphorylation of serine 44 drives trafficking of ROMK1 channels in the secretory pathway, we first monitored the arrival of newly synthesized ROMK1 channels at the plasmalemma in post-cRNA injection-chase experiments. In these studies, Xenopus oocytes were injected with either wild-type (WT), phosphorylation mimic (S44D), or phosphorylation null (S44A) ROMK cRNA, incubated at 19°C for 12 h, and then "chased" at 22°C in the absence and presence of brefeldin A (10 M) with measurements of ROMK activity (Fig. 1A) and channel surface abundance (Fig. 1C). As shown in Fig. 1A, whole cell K ϩ currents, carried through the WT and the S44D channels but not the S44A channels, appear within the first 2 h of the chase period and then increase further at a quasi-linear rate. The response of WT and S44D was completely inhibited by BFA. Since BFA blocks the transport of newly synthesized membrane proteins at the ER/Golgi without effects on post-endocytotic routing in Xenopus oocytes (26), the response is consistent with the egress of channels through the biosynthetic pathway. The rate of BFA-sensitive traffic to the plasmalemma (Fig. 1B), as inferred from time-dependent increase in the macroscopic potassium conductance, was identical for the wild-type and the phosphorylation mimic (S44D) channel. This latter result agrees with the observations that high levels of cAMP and PKA in the oocyte expression system (27) drive constitutive phosphorylation of Ser 44 (14,28).
Remarkably, the S44A phosphorylation null mutation dramatically inhibited trafficking to the plasmalemma. Since S44A ROMK channels (detected at the cell surface in limited abundance) display the same single channel conductance and open probability as the wild-type channel (28), the absence of significant whole cell channel activity indicates that Ser 44 phosphorylation null channels are inefficiently routed through the biosynthetic trafficking pathway. Indeed, direct measurements of ROMK cell surface expression at steady state by antibody binding and analytical luminometry corroborate the measurements of ROMK activity (Fig. 1C).
To further substantiate the functional studies, we assessed the arrival of externally HA epitope-tagged ROMK channels to the plasmalemma using a dynamic surface labeling and qualitative detection assay (Fig. 2). In these studies, oocytes were incubated in the continued presence of HA antibody during the chase period so that channels that visit the plasmalemma, however transiently, should become bound to antibody. Channels labeled with extracellular antibody were recovered by immunoprecipitation, detected in Western blot analysis, and compared with the total immunodetectable channel in the cell. As shown in Fig. 2, ROMK channels bearing the phosphorylation mimic (S44D) mutation could be readily labeled with HA antibody at the cell surface during the early chase period. In contrast, phosphorylation null (S44A) channels could not be detected at the cell surface. Of note, the total immunodetectable pool of S44A channel consistently migrated as a smaller doublet (ϳ45-48 kb) than the surface expressed S44D channel (ϳ60 kb), presumably reflecting immature processing (see "Glycosidase Analysis"). Collectively, these studies indicate that phosphorylation of serine 44 is required for forward trafficking of channels in the secretory pathway.
Mature Glycosylation Is Dependent on Ser 44 Phosphorylation-ROMK1 channels acquire N-linked glycosylation at a single extracellular asparagine residue, N117 (29), into an Endo H-sensitive core and then are believed to be further processed to an Endo H-resistant form, indicative of maturation beyond the cis-Golgi. We took advantage of this process and examined glycosylation of the Ser 44 mutant ROMK channels to probe for phosphorylation-dependent trafficking from the endoplasmic reticulum through the Golgi (Fig. 3). The WT, S44A, and S44D ROMK proteins migrate on SDS-PAGE gels as a ϳ47and 48.5-kDa doublet. The WT and phospho-mimic, S44D, channel also ran as a higher molecular species, ϳ61 kDa (a broader 61-85-kDa band in longer exposures), that was not seen with the phospho-null S44A channel. PNGase F, an amidase that cleaves all N-glycan chains from asparagine residues, collapsed the 48.5-kDa and the 61-kDa ROMK proteins into a single ϳ47-kDa band. Because 47 kDa is the same size as glycosylationdeficient ROMK1 channels (N117Q) and the predicted molecular mass of unmodified ROMK1, the 48.5-and the 61-kDa proteins represent N-linked glycosolated forms of ROMK1. Endo H, an enzyme that preferentially hydrolyzes high mannose N-glycans on immature membrane proteins, selectively digested the ϳ48.5-kDa form of ROMK. By contrast, the ϳ61-kDa form was resistant to Endo H treatment, indicative of mature glycosylation. Taken together, these studies indicate that ROMK channels acquire mature glycosylation in a manner that is dependent on the conformation of serine 44, consistent with a phosphorylation-dependent trafficking step at the ER/Golgi.
Stability of ROMK at the Plasma Membrane Is Not Dependent on Ser 44 Phosphorylation-In principle, phosphorylation of serine 44 could also influence the stability of ROMK channels at the plasma membrane.
To determine whether this is the case, we assessed the lifetime of wildtype and Ser 44 phosphorylation mutant channels on the plasmalemma. In these studies, we allowed sufficient time after injection (2-3 days) for ROMK to be maximally expressed at the cell surface and then monitored the decay in channel activity (WT, S44D, S44A) under two-microelectrode voltage clamp after the arrival of new channels was blocked with BFA (10 M). Studies of the S44A mutant channel required injecting ten times more cRNA than used with the wild-type and S44D channels to detect measurable activity. Under these conditions, initial whole cell K ϩ current density of the oocytes expressing S44A was well above background and comparable with the other channels (10.83 Ϯ 2 A at Ϫ100 mV in 90 mM K ϩ ). As shown in Fig. 4A, neither of the Ser 44 mutant channels displayed significantly different decay rates than the wild-type channel. Indeed, all were well described by a single exponential time course with half-lives that were statistically indistinguishable (3.2 Ϯ 0.5 h, WT; 4.0 Ϯ 0.7 h, S44D; 3.6 Ϯ 0.5 h, S44A, n ϭ 3 frogs) and similar to previous reports of the wild-type channel (30). Measurements of immunodetectable channel at the cell surface in the presence and absence of brefeldin A confirmed that decay in channel activity was due to channel internalization. With the results above, these studies indicate that phosphorylation of Ser 44 controls surface expression of ROMK through a biosynthetic trafficking mechanism and not through alteration of channel stability at the plasma membrane.
Sequence Requirements for Phosphorylation-dependent Trafficking-The phosphoserine 44 residue juxtaposes a string of three "RXR" motifs ( Fig. 5). Similar basic residue structures have been implicated in the control of membrane protein traffic at early steps in the secretory pathway, acting as signals that drive ER localization (20), release from ER retention (32-34), or Golgi export (21,35). Accordingly, we explored the idea that cell surface expression of ROMK may be controlled by a structure involving phosphoserine 44 and the repeating arginine motifs. Based on present understanding, two different mechanisms can be envisioned. Phosphorylation of Ser 44 could abrogate an RXR-based ER localization signal, reminiscent to the way that phosphorylation is believed to control cell surface expression of N-methyl-D-aspartate receptors (36,37). Alternatively, the structure may encode a phosphorylation-dependent ER release/export signal, which overcomes ER localization. To test the hypothesis and distinguish between these models, we measured plasmalemma expression of external HA epitope-tagged ROMK channels, bearing mutations in the putative trafficking signal. In these studies, we replaced each of the arginine residues with alanine in the context of the phospho-mimic, S44D, or the phospho-null mutation, S44A, so that we could access the role of these residues in trafficking independently from their potential role PKA/SGK1 interaction and phosphorylation. Comparable mutations in the Kir 6.1/6.2 C-terminal "RKR" motif suppress ER retention and increase plasmalemma expression (20). As measured by HA antibody binding and cell surface luminescence, none of the arginine to alanine mutations (R35A, R37A, R39A, R41A, or col- Cell surface immunoprecipitation studies. The arrival of externally HA epitope-tagged ROMK channels to the plasma membrane was measured using a dynamic surface labeling and detection assay. Oocytes were incubated in the continued presence of rat HA antibody during the chase period (2-6 h). Channels labeled with extracellular antibody were recovered by immunoprecipitation, detected in Western blot analysis with rabbit anti-ROMK antibodies (Surface IP), and compared with the total immunodetectable channel in the cell (Whole IP). Exposure time of the S44A surface immunoprecipitation fluorogram is 3ϫ longer than S44D. lective replacement of R35/R37/R39/R41 to A (RallA)) restored cell surface expression of the phospho-null S44A channel, making it unlikely that the N-terminal RXR motifs in the ROMK channel operate as an ER localization signal. Instead, our analysis of the compound S44D phospho-mimic mutants revealed that the neighboring arginine residues are actually necessary for cell surface expression. Collective replacement of the arginine residues with alanine completely blocked cell surface expression of the phospho-mimic channel and S44D channels bearing either R39A or R41A mutations exhibited a significant reduction in plasmalemma expression. Electrophysiological analysis of the phosphomimic channel revealed that the R39A and R41A mutations reduced the macroscopic potassium current density to the same extent as the cell surface density, indicating that the R39A and R41A mutations do not alter the product of the signal channel conductance and the channel open probability. Likewise cation selectivity, as assessed by reversal potential measurements, was not altered by the R39A and R41A mutations (S44D, P K/Na ϭ 15 Ϯ 0.8; S44D/R39A, P K/Na ϭ 13.5 Ϯ 1; S44D/ R41A, P K/Na ϭ 14.7 Ϯ 1, n ϭ 5). Together the results indicate that R39A and R41A mutations reduce surface expression without perturbing the biophysical properties of the channel and, thereby, rule out gross misfolding. Thus, surface expression of ROMK is regulated by an export or release-from-retention structure, involving phosphoserine 44 and at least two of the neighboring arginine residues (Arg 39 and Arg 41 ).
The phosphorylation-dependent release/export determinant appears to override ER/Golgi retention, raising the possibility that an independent structure dictates ER/Golgi localization when the ROMK channel is in a serine 44-unphosphorylated conformation. Analysis of the ROMK primary structure revealed a potential signal (38 -40), consisting of an overlapping RXR and dibasic motif (Fig. 6) at a position within the cytoplasmic C-terminal that is comparable with location of the ER retention signals in Kir 6.X channels (20). Accordingly, we determined the consequence of mutating the critical basic residues in this motif on surface expression of externally HA-tagged ROMK channels. Because the signal is predicted to be the dominant trafficking signal when serine 44 is not phosphorylated, the mutations were studied in the context of the phospho-null, S44A, channel. As measured by HA antibody binding and cell surface luminescence, K370A, R371A, and the double mutants, but not R386A, restored cell surface expression of the phospho-null S44A channel to a level that was not statistically different from the phospho-mimic S44D channel (Fig. 6B). As measured by two-microeletrode voltage clamp as shown in Fig. 6, C and D, the K370A/R371A mutation also restored functional expression of the phospho-null channel. Thus, the C-terminal dibasic motif (K 370 R 371 ), possibly operating with the overlapping RXR-type motif (40) (R 368 MK 370 ), controls intracellular localization of the channel when serine 44 is in the unphosphorylated conformation.

DISCUSSION
Here, we have elucidated the mechanism by which cell surface density of ROMK1 (Kir1.1a) channels is controlled by phosphorylation of a cytoplasmic N-terminal residue, serine 44. Using several independent methods to monitor membrane trafficking of wild-type, phosphorylation mimic (S44D), and phosphorylation null (S44A) Kir1.1a channels in Xenopus oocytes, we found phosphorylation of serine 44 drives efficient transport of newly synthesized channels through the secretory pathway to the plasma membrane. Our observation that ROMK1 channels acquire mature glycosylation in a serine 44 phosphorylation-dependent manner strongly suggests that the regulated trafficking step occurs within the endoplasmic reticulum and/or Golgi apparatus. Mutational analysis revealed that surface expression requires an N-terminal structure containing phosphoserine 44 and a nearby arginine motif, consistent with a structural determinant of forward trafficking in the secretory pathway. Moreover, suppressor mutations within a C-terminal ER localization signal (K 370 R 371 ) rescued cell surface expression of the phosphorylation null (S44A) channel. Taken together, these studies indicate that phosphorylation of serine 44 drives an export step early in the secretory pathway to override an independent endoplasmic reticulum localization signal.
Our studies expand a new and rapidly evolving view that the cell surface density and composition of certain types of channels and receptors can be adjusted by controlling their export from the endoplasmic reticulum and Golgi (41). The current understanding is derived, in part, from well established observations about quality control checkpoints in the secretory pathway. According to current concepts, misfolded or unassembled membrane proteins that escape the endoplasmic reticulum can be retrieved in the cis-Golgi into COP1-coated vesicles for retrograde transport back to endoplasmic reticulum (42) and eventual destruction by the endoplasmic reticulum-associated degradation system (43). A growing body of evidence indicates that the process is dependent on trafficking signals, controlling ER retention, retrieval, and/or export (41). For example, retrograde trafficking of unassembled K ATP channel subunits from the Golgi is specified by RKR signals (20,44), which coordinate interaction with the COP1 complex (34). Likewise, the COP1 complex recognizes a dibasic motif (38) in unassembled nicotinic acetylcholine receptor ␣ subunits (45), identical to the one found here in ROMK. Upon assembly of the complete K ATP channel or Ach receptor complexes, the retention/retrieval signals become masked, providing a quality control mechanism that insures only completely assembled channels and receptors are delivered to the plasma membrane (20).
The work presented here with the ROMK channel, along with exciting recent studies with other membrane proteins, indicate that appropriately folded and assembled membrane proteins might co-opt "quality control" processes in the secretory pathway to regulate biosynthetic trafficking and control cell surface density. Indeed, work with a diverse, albeit very limited, set of receptors and channels has begun to reveal that ER retention signals can be effectively masked by scaffolding protein interaction (36,46) or phosphorylation. Insights into the S44 P -depend-ent trafficking process of the ROMK channel can be gleaned from these other receptors and channels.
Unlike the N-methyl-D-aspartate NR1 receptor, where phosphorylation of residues adjacent to an RXR-type ER retention signal increase surface expression (36,37), our studies with ROMK indicate that the key phosphorylated residue, Ser 44 , is distant in the primary structure from the dibasic ER retention signal, K 370 R 371 . A strategy to translate phosphorylation into ER release, even when phosphorylated residues are not co-linear with the ER retention signal, has recently emerged from work with KCNK-type potassium channels (32,33). In these channels, as well as in the HLA class II-associated invariant chain (47,48) and the nicotinic Ach receptor ␣4 subunit (33), 14-3-3 proteins interact with phosphoserine and arginine containing motifs and effectively suppress ER retention by displacing the COP1 complex. Significantly, this occurs in the KCNK3 channel even though the 14-3-3 binding site is independent from the dibasic ER retention signal (33). Not only does this parallel the separate ER retention and release/export structures in ROMK, the FIGURE 5. Structural requirements of S44 P trafficking. A, mutations in the putative N-terminal trafficking signal were created; B, cell surface expression was measured by HA antibody binding and cell surface luminescence. Each of the arginine residues in the structure was replaced with alanine in the context of the phospho-mimic, S44D, or the phospho-null mutation, S44A. Surface expression is shown relative to the phosphomimic, S44D. The dotted line represents the average background. Bars ϭ mean Ϯ S.D.; *, statistically different from the phospho-mimic channel, S44D. C, functional expression, measured as whole cell potassium current density under two-microelectrode voltage clamp and described under "Materials and Methods," is plotted relative to the phospho mimic, S44D (dark bars). Bars ϭ mean Ϯ S.D., n ϭ 9. Surface expression, as measured in B, is shown by the shaded bars. *, statistically different from the phospho-mimic channel, S44D. FIGURE 6. Suppressor mutations at a C-terminal "KR" site rescue surface expression of the phospho-null S44A channel. A, analysis of the ROMK primary structure revealed a potential ER retention signal at the cytoplasmic C terminus, consisting of an overlapping RXR and dibasic motif. B, mutations in the putative trafficking signal were created in the context of the phospho-null S44A channel, and cell surface expression was measured by HA antibody binding and cell surface luminescence. K370A, R371A, and the double mutants, but not R386A, restored cell surface expression of the phospho-null S44A channel to a level that was not statistically different from the phospho-mimic S44D channel. Surface expression is shown relative to the phospho mimic, S44D. Bars ϭ mean Ϯ S.D. *, statistically different from the phospho-mimic channel, S44D. C, representative whole cell potassium currents and IV relationships of oocytes injected with cRNA encoding the phospho-mimic S44D channel, the phospho-null S44A channel, or the phospho-null, K370A/R371K mutant. D, average whole cell potassium current density is plotted relative to the phospho mimic, S44D (dark bars). Bars ϭ mean Ϯ S.D., n ϭ 10. Surface expression, as measured in B, is shown by the shaded bars. *, statistically different from the phospho-mimic channel, S44D.
14-3-3 binding site in the KCNK3 channel (33) also shares sequence similarity with the release/export structure in ROMK. In fact, both contain an identical motif, RXXS P X, resembling a canonical type II 14-3-3 binding site without the penultimate proline residue (RXXS P XP (49)). Taken together, these observations raise the possibility that a similar 14-3-3-dependent trafficking process is at play with the ROMK channel. Obviously, further studies are required to rigorously explore this hypothesis.
Rather than operate as a release-from-retention signal, it is also conceivable that phosphorylation of serine 44 creates a dominant export signal that simply overrides the C-terminal ER retention signal. Support for this idea is provided by the observation that the phosphorylationdependent trafficking site in ROMK exhibits sequence similarity with a comparable N-terminal structure in the Kir 2.1 channel that has recently been suggested to control forward traffic from the Golgi rather than from the ER (21). The Kir 2.1 structure contains a basic residue cluster, similar to one found in ROMK. Neutralization of several basic residues in this domain, Arg 44 and Arg 46 , causes Kir 2.1 channels to accumulate in the Golgi and dramatically reduce cell surface expression. Remarkably, these two Kir 2.1 residues precisely align with the key arginines (Arg 39 and Arg 41 ) in the ROMK export/release structure. Unlike the N-terminal trafficking site in ROMK, the "Golgi export" determinant in Kir 2.1 does not contain a phosphoacceptor. This may offer a possible explanation for why forward trafficking in the secretory pathway is constitutive in Kir2.1 channel but dependent on phosphorylation in ROMK. In any regard, based on domain homology and common effects of neutralizing mutations on channel trafficking, it seems likely that the related N-terminal structures in ROMK and Kir 2.1 serve related functions. Perhaps, they operate as recognition sites for trafficking machinery, which drive forward transport of cargo in the secretory pathway. Obviously, it will be important to determine whether these N-terminal motifs act as autologus trafficking signals and identify the cellular machineries that decode and act on them.
While this paper was under review, O'Connell et al. (50) provided evidence for a similar Ser 44 phosphorylation-dependent trafficking process, involving a C-terminal ER retention signal. They reached somewhat different conclusions, however, than we and Stockklausner and Klocker (21) did about the requirements of Arg 39 and Arg 41 and homologous residues in other Kir-type channels for efficient forward trafficking. Based on the sole observation that deletion of the N terminus of ROMK through Arg 39 did not modify whole cell current density, O'Connell et al. (50) concluded that the N-terminal arginine track, including Arg 39 and Arg 41 , is not involved in ROMK trafficking. However, it should be pointed out that the group did not study Arg 41 . Furthermore it is unlikely that the N-terminal deletion mutation approach would reveal a specific role of Arg 39 given the different functional roles of the N-terminal domain. Indeed, removal of the N-terminal domain is also predicted to release the ROMK channel from negative regulation by ubiquitination (51) and obscure the precise structural requirements of the phosphorylation-dependent export signal.
The phosphorylation-dependent trafficking process in ROMK is likely to be physiologically important. Factors that influence the activity and cell surface density of ROMK channels in the collecting duct principal cell are believed to have profound effects on renal potassium excretion. For instance, a series of adaptive changes in the collecting duct principal cell take place in response to an increase in dietary potassium, allowing a more effective and enhanced excretion of potassium after an acute potassium load (4). The response, called potassium adaptation, depends on elevated aldosterone levels and other synergistic factors (52), triggering a relatively rapid increase in the number of active ROMK-type channels on the apical plasmalemma (31,53). Because Ser 44 is a substrate for phosphorylation by PKA (13,14) and the aldosterone-induced kinase, SGK-1 (14), our observations provide a potential molecular mechanism for the regulation of ROMK density by dietary potassium, involving a novel phosphorylation-dependent trafficking process.
In summary, we have found that phosphorylation of Ser 44 drives an early export step within the secretory pathway to override an independent endoplasmic reticulum localization signal. Thus, a balance of intracellular retention and phosphorylation-dependent export controls Kir1.1 cell surface density.