The C Terminus of SUR1 Is Required for Trafficking of KATP Channels*

In beta cells from the pancreas, ATP-sensitive potassium channels, or KATP channels, are composed of two subunits, SUR1 and KIR6.2, assembled in a (SUR1/KIR6.2)4 stoichiometry. The correct stoichiometry of channels at the cell surface is tightly regulated by the presence of novel endoplasmic reticulum (ER) retention signals in SUR1 and KIR6.2; incompletely assembled KATPchannels fail to exit the ER/cis-Golgi compartments. In addition to these retrograde signals, we show that the C terminus of SUR1 has an anterograde signal, composed in part of a dileucine motif and downstream phenylalanine, which is required for KATPchannels to exit the ER/cis-Golgi compartments and transit to the cell surface. Deletion of as few as seven amino acids, including the phenylalanine, from SUR1 markedly reduces surface expression of KATP channels. Mutations leading to truncation of the C terminus of SUR1 are one cause of a severe, recessive form of persistent hyperinsulinemic hypoglycemia of infancy. We propose that the complete loss of beta cell KATP channel activity seen in this form of hyperinsulinism is a failure of KATPchannels to traffic to the plasma membrane.

In pancreatic beta cells, the high affinity sulfonylurea receptor, SUR1 1 , and the potassium inward rectifier, K IR 6.2, combine to form octameric ATP-sensitive potassium channels, K ATP channels, that link glucose metabolism to membrane potential (1)(2)(3)(4)(5). These channels play a key role in the regulation of insulin secretion, and loss of K ATP channel activity has been shown to cause a severe, recessive form of congenital or neonatal hyperinsulinism, designated HI-SUR1 or HI-K IR 6.2, depending on which subunit harbors the mutation (5). K IR 6.2 forms the channel pore that is regulated by SUR1, and both subunits are required to form a fully functional channel sensitive to ATP, MgADP, sulfonylureas, and potassium channel openers (for reviews, see Refs. 4 -7). C-terminal truncated K IR 6.2 subunits generate homomeric K ϩ channels, (K IR 6.2⌬C) 4 , in the absence of SUR1 that have the correct conductance and are weakly inhibited by ATP but have altered kinetics and lack the other properties of wild-type K ATP channels (8 -10). Zerangue et al. (11) have identified a novel endoplasmic reticulum retention (ER) or retrograde signal in the C terminus of K IR 6.1 and in K IR 6.2. The same motif is found in SUR1 and SUR2 on the N-terminal side of NBF1. Deletion or mutation of the K IR signal permits surface expression of K IR subunits without SUR1, whereas mutation of the SUR1 signal gives surface expression without a K IR . These signals are proposed to serve as a quality control mechanism that ensures only the surface expression of properly assembled octameric channels (11). We show here that there is an additional level of regulation of trafficking; the C terminus of SUR1 has an anterograde signal that is required for surface expression of K ATP channels. The deletion of this anterograde signal can account for the loss of channel activity in some HI-SUR1 mutations.
Membranes were prepared from transfected COSm6 cells grown on 150-mm plates. Cells were washed twice with PBS, pH 7.4, and collected by incubating at 4°C with PBS supplemented with 2 mM EDTA. The cells were pelleted, resuspended in 200 -1000 l of hypotonic buffer (5 mM Tris-HCl, 2 mM EDTA, 0.1 M NaCl, pH 7.4) and allowed to swell for 40 min on ice. Cells were homogenized, centrifuged at 1000 ϫ g for 10 min at 4°C. For further purification, the supernatant was collected and centrifuged at 40,000 ϫ g for 60 min. The pelleted membranes were resuspended in 300 l of membrane buffer supplemented with protease inhibitors (Complete TM , Roche Molecular Biochemicals) (50 mM Tris-HCl, 5 mM EDTA; pH 7.4) and stored at Ϫ80°C. The protein concentrations varied from 2-5 mg/ml. Membranes were photolabeled with 125 I-iodoazidoglibenclamide, kindly provided by Professor Uwe Panten (University of Braunschweig, Braunschweig, Germany), as described (3,14).
The appearance of SUR1 at the cell surface was quantified using a luminometer-based assay to measure SUR1 c-myc . Transfected COSm6 cells were gently washed in Kreb's Ringer PBS and incubated for 1 h at 25°C with the mouse monoclonal IgG 1 c-myc antibody (9E10, Santa Cruz Biotechnology) diluted in Dulbecco's modified Eagles' medium * This work was supported by Juvenile Diabetes Foundation International Grant 397003 (to A. P. B.), by National Institutes of Health Grants DK44311 and DK52771 (to J. B.), and by grants from the American Diabetes Association and the Houston Endowment (to L. A.-B.). 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.
Immunoprecipitation was done using membranes from transfected COSm6 cells labeled with 125 I-azidoglibenclamide and solubilized with 60 mM of N-dodecyl-␤-D-maltoside and 500 mM NaCl for 1 h at 4°C with continuous rocking. Following centrifugation at 40,000 ϫ g for 30 min, the supernatant was incubated overnight with an agarose-conjugated His-probe (rabbit polyclonal IgG antibodies, H-15, Santa Cruz Biotech-nology). The beads were pelleted at 10,000 ϫ g and washed 4 -5 times with membrane buffer, and proteins were released from the beads in 30 l of SDS loading buffer and resolved on 7.5% SDS-polyacrylamide gels.
The currents through and surface density of reconstituted K ATP channels were measured in the inside-out configuration using the patch-clamp technique at 23-24°C as described previously (10,15).

RESULTS
Deletion of the C terminus of SUR1 affects K ATP channel activity measured here by a 86 Rb ϩ efflux assay. The SUR1⌬C2 and ⌬C4/K IR 6.2 channels show glibenclamide-inhibited 86 Rb ϩ efflux attributable to K ATP channels which is comparable with wild-type channels (Fig. 1A); the SUR1⌬C7/K IR 6.2 channels show approximately a 50% decrease in activity, while a complete loss of activity is observed for the SUR1⌬C13, ⌬C22, and ⌬C49/K IR 6.2 channels. The results suggest that the last 5 to 13 amino acids of the C terminus of SUR1 are essential for obtaining channel activity. To determine whether the SUR1 C terminus was masking the ER retention signal on K IR 6.2 (11), we co-expressed the SUR1⌬C constructs with K IR 6.2⌬C35 missing 35 residues from its C terminus (10). There were no significant differences in the rates of 86 Rb ϩ efflux between the SUR1⌬C/ K IR 6.2 versus SUR1⌬C/K IR 6.2⌬C35 channels (Fig. 1A), indicating that the loss of channel activity resulting from deletion of the C terminus of SUR1 does not depend on the presence of the ER retention signal on K IR 6.2.
SUR1 is differentially glycosylated, exhibiting a mature or complex glycosylated form (150 -170 kDa) and an immature or core glycosylated form (140 kDa) (3,16). Mature SUR1 is present only when the receptor is co-expressed with K IR 6.1 or K IR 6.2 and has been shown to assemble with K IR 6.2 to form active K ATP channels in the plasma membrane (3). The mature receptor is resistant to Endo H, whereas the immature, core glycosylated species is deglycosylated to a 137-kDa species (data not shown). Endo H removes high mannose oligosaccharide chains that are added in the ER, thus the appearance of an Endo H-resistant form indicates processing of the oligosaccharides beyond the cis-Golgi. Co-expression of SUR1⌬C2 or ⌬C4 with K IR 6.2 yields both the immature and mature glycosylated forms of SUR1 as shown in Fig. 1B using 125 I-iodoazidoglibenclamide to identify the receptors. SUR1⌬C7, ⌬C13, ⌬C22, and ⌬C49 show essentially a complete lack of complex glycosylation. The results are consistent with either a defect in the processing of oligosaccharides on the C-terminal truncated receptors, with a failure of the receptors to traffic to the medial Golgi apparatus and thus the cell surface, or with a failure of subunits to co-assemble.
with full-length SUR1 c-myc produced the greatest differential. The SUR1⌬C2 c-myc and ⌬C4 c-myc constructs give ϳ 60% of this increase, whereas further deletions reduced surface expression to background values. The loss of surface expression observed with C-terminal deletion parallels the loss of mature SUR1 (Fig. 1B) and K ATP channel activity (Fig. 1A). The results are consistent with a failure of the C-terminal deleted receptors to traffic beyond the cis-Golgi to the plasma membrane.
To determine whether K IR 6.2 and the SUR1⌬C subunits were co-assembling, we tested whether the inward rectifier would photolabel with 125 I-azidoglibenclamide and co-immunoprecipitate when expressed with the truncated receptors. Fig. 2 (left panel) shows that when SUR1⌬C49 or SUR1⌬C221 are co-expressed with K IR 6.2, both the receptor and inward rectifier are photolabeled consistent with their physical association with SUR1. We have previously shown that the inward rectifier alone does not label (3). Comparison with the wild-type control indicates that the mature form of the receptor is absent in both cases. The right panel in Fig. 2 demonstrates that the trun-cated receptor and inward rectifier co-assemble. When N-6X-his SUR1⌬C49, tagged with six histidine residues at its N terminus, is co-expressed with K IR 6.2, the inward rectifier labels with 125 I-iodoazidoglibenclamide and can be co-immunoprecipitated using anti-histidine tag antibodies (Fig. 2, Co-IP).
The results indicate the C-terminal truncated receptors can assemble with K IR 6.2 but then fail to traffic into the Golgi and plasma membrane.
To determine which amino acids in the C terminus of SUR are important, we compared the distal 25 amino acids of SUR1, SUR2A, and SUR2B (Fig. 3A). The amino acids 24, 22, 17, 9, and 5 residues from the C-terminal were conserved, whereas amino acid 16 was one of a dileucine pair. We engineered alanine substitutions at positions Val-1578, Phe-1574, Leu-1567, Leu-1566, and Asp-1561 in SUR1 c-myc , co-expressed them with K IR 6.2, and analyzed their channel activity and surface expression. As shown in Fig. 3, B and C, substitution of alanine at positions Phe-1574 and Leu-1566 gave parallel reductions in K ATP channel activity measured as glibenclamide-inhibited efflux and in surface expression measured by the appearance of the myc tag. Companion experiments indicate there is a parallel decrease in maturation of the receptor (data not shown).
Single channel recordings of the SUR1 F1574A /K ir 6.2 channels (Fig. 4) shows that their ATP sensitivity is similar to wild-type channels (IC 50(ATP) ϭ 8.9 Ϯ 03 M versus 6.9 Ϯ 03 M for wild type (WT)), but their surface density (N) is ϳ5-6 times lower than that of the wild-type, consistent with the 86 Rb ϩ efflux and surface expression results. We conclude that amino acids Phe-1574 and Leu-1566 are a part of an anterograde trafficking  4. Analysis of currents through wild-type and SUR1 F1574A / K IR 6.2 channels. Potassium currents through excised patches from COSm6 cells transfected with K IR 6.2 plus wild-type SUR1, top trace, or SUR1 F1574A , bottom trace, were recorded as described (15). The quasisteady state sensitivity to inhibitory ATP was estimated as described (15). The relative NPo values versus [ATP] for the wild-type channels (filled circles) and SUR1 F1574A /K IR 6.2 channels were fit to a pseudo-Hill equation. The values obtained were: IC 50(ATP) ϭ 6.9 Ϯ 0.3 (h ϭ 1.05) versus 9.1 Ϯ 0.3 (h ϭ 1.1) for the wild-type versus SUR1 F1574A /K IR 6.2 channels, respectively. signal that is required for the surface expression of K ATP channels.

DISCUSSION
These results identify an export or anterograde signal on the C terminus of the sulfonylurea receptor, SUR1, which is required for surface expression of ATP-sensitive potassium channels. The anterograde signal and the recently described "ϪRKRϪ" endoplasmic reticulum retention, or retrograde, signal in SUR and in the C termini of K IR 6.1 and K IR 6.2 are summarized schematically in Fig. 5. These signals act as a quality control mechanism to ensure that only fully assembled, octameric ATP-sensitive potassium channels reach the plasma membrane.
The results are consistent with our previous observations on glycosylation of the receptor. The mature, complex glycosylated receptor is Endo H-resistant, whereas the immature, coreglycosylated receptor is Endo H-sensitive. The Endo H resistance of mature SUR1 indicates it has been transported from the ER and cis-Golgi to the medial and trans-Golgi where further processing takes place. The mature receptor has been shown to be associated with K IR 6.2 as a large octameric complex consistent with its being in active K ATP channels on the cell surface (3). Their Endo H sensitivity indicates the immature, core glycosylated receptors are retained in the ER and cis-Golgi and do not undergo further processing. Expression of SUR1 without K IR 6.1 or K IR 6.2 produces the immature form of the receptor (3). This is consistent with the observed lack of surface expression of SUR1 in Xenopus oocytes (11) and in COSm6 cells (Fig. 1C) unless K IR 6.1 or K IR 6.2 are present and indicates the retrograde signals must be masked before the assembled channels can exit the ER and cis-Golgi. Deletion of as few as seven residues from the C terminus of SUR1 reduces the amount of mature receptor, the level of surface expression, and the density of K ATP channels when the receptor is expressed with K IR 6.2. Co-photolabeling of SUR1 and K IR 6.2 with 125 I-iodoazidoglibenclamide and co-immunoprecipitation of the two subunits indicates they can assemble, which suggests the C terminus of SUR is not required for subunit association. Analysis of the currents from SUR1 F1574A /K IR 6.2 channels indicates they retain normal sensitivity to ATP, but the density of channels is reduced. Expression of K IR 6.2⌬C subunits with SUR1⌬C subunits does not lead to surface expression of channels, indicating that the C terminus of SUR does not mask the -RKRϪ signal in K IR , and our preliminary data indicate it does not mask the -RKRϪ signal in SUR1. Previous work indicates that SUR1 increases the surface expression of K IR 6.2⌬C subunits approximately 8-fold, consistent with the involvement of the anterograde signal in a process that facilitates trafficking to the cell surface (10).
Substitution of alanines at positions Phe-1574 and Leu-1566 indicate the importance of these residues for anterograde transit of K ATP channels. Dileucine motifs have been shown to be important in protein trafficking from the trans-Golgi to a late endosomal/lysosomal compartment (17)(18)(19)(20) and for internalization of receptors following protein kinase activation and phosphorylation (21)(22)(23). An acidic residue 4 -5 residues on the N-terminal side of the dileucine motif can also be important for sorting (24,25), and a C-terminal glutamate/dileucine motif has been shown to be important for surface expression of the vasopressin V 2 receptor (26). Interestingly, mutation of the first leucine of the vasopressin V 2 receptor has a much larger inhibitory effect than substitution of the second, the same pattern as we observe for SUR1. However, mutation of Asp-1561, five residues upstream of the dileucine motif in SUR1 had no significant effect, whereas an E 3 Q substitution in the vasopressin V 2 receptor largely eliminates surface expression. The vasopressin V 2 receptor has no downstream phenylalanine corresponding to SUR1 Phe-1574 . The cellular receptors for these signaling motifs have not been identified, and it is unclear whether the C terminus of SUR interacts with COPI or COPII proteins or with COP-associated adaptor proteins (for reviews, see Refs. [27][28][29][30][31][32]. These observations give a molecular insight into the lack of K ATP channel activity observed in pancreatic beta cells from patients with persistent hyperinsulinemic hypoglycemia of infancy. Mutations in both SUR1, HI-SUR1, and K IR 6.2, HI-K IR 6.2, are the cause of a recessive form of this disorder which is characterized by an inappropriate secretion of insulin despite hypoglycemia (5,6,33). Nonsense and splice site mutations in SUR1 produce C-terminal deletions (5) that have been shown to result in a complete loss of K ATP channel activity (34). Although many of the truncated receptors may be incapable of producing functional channels for other reasons, our results indicate that even if channels do assemble they will not reach the cell surface and that the "primary" defect associated with mutations in SUR1 that truncate the receptor will be a failure to traffic correctly as a FIG. 5. Summary model for assembly and maturation of K ATP channels. The scheme for masking of the retrograde ER retention signals is taken from Zerangue et al. (11). The anterograde signal is required for the channels to exit the ER/cis-Golgi compartment as indicated by surface expression and the appearance of complex glycosylated SUR1. Deletion of the C terminus of SUR1 inhibits transit from the ER/cis-Golgi compartment. result of deleting the anterograde signal.