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

1999; Elsevier BV; Volume: 274; Issue: 29 Linguagem: Inglês

10.1074/jbc.274.29.20628

1083-351X

Nidhi Sharma, Ana Crane, John P. Clement, Gabriela González, Andrey P. Babenko, Joseph Bryan, Lydia Aguilar‐Bryan,

Hyperglycemia and glycemic control in critically ill and hospitalized patients

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 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 1The abbreviations SUR1high affinity sulfonylurea receptorERendoplasmic reticulumKATPchannelATP-sensitive potassium channelKIRpotassium inward rectifierPBSphosphate-buffered salineEndo Hendoglycosidase HIC50(ATP)IC50 for ATP , and the potassium inward rectifier, KIR6.2, combine to form octameric ATP-sensitive potassium channels, KATP channels, that link glucose metabolism to membrane potential (1Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement IV, J.P. Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1288) Google Scholar, 2Inagaki N. Gonoi T. Clement IV, J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1623) Google Scholar, 3Clement IV, J.P. Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 4Aguilar-Bryan L. Clement IV, J.P. Gonzalez G. Kunjilwar K. Babenko A. Bryan J. Physiol. Rev. 1998; 78: 227-245Crossref PubMed Scopus (516) Google Scholar, 5Aguilar-Bryan L. Bryan J. Endocr. Rev. 1999; 20: 101-135Crossref PubMed Scopus (624) Google Scholar). These channels play a key role in the regulation of insulin secretion, and loss of KATP channel activity has been shown to cause a severe, recessive form of congenital or neonatal hyperinsulinism, designated HI-SUR1 or HI-KIR6.2, depending on which subunit harbors the mutation (5Aguilar-Bryan L. Bryan J. Endocr. Rev. 1999; 20: 101-135Crossref PubMed Scopus (624) Google Scholar). KIR6.2 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. 4Aguilar-Bryan L. Clement IV, J.P. Gonzalez G. Kunjilwar K. Babenko A. Bryan J. Physiol. Rev. 1998; 78: 227-245Crossref PubMed Scopus (516) Google Scholar, 5Aguilar-Bryan L. Bryan J. Endocr. Rev. 1999; 20: 101-135Crossref PubMed Scopus (624) Google Scholar, 6Aguilar-Bryan L. Bryan J. Diabetes Rev. 1996; 4: 336-346Google Scholar, 7Bryan J. Aguilar-Bryan L. Curr. Opin. Cell Biol. 1997; 9: 553-559Crossref PubMed Scopus (107) Google Scholar). C-terminal truncated KIR6.2 subunits generate homomeric K+ channels, (KIR6.2ΔC)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 KATP channels (8Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (683) Google Scholar, 9Drain P. Li L. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13953-13958Crossref PubMed Scopus (174) Google Scholar, 10Babenko A.P. Gonzalez G. Aguilar-Bryan L. Bryan J. FEBS Lett. 1999; 445: 131-136Crossref PubMed Scopus (51) Google Scholar). Zerangueet al. (11Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar) have identified a novel endoplasmic reticulum retention (ER) or retrograde signal in the C terminus of KIR6.1 and in KIR6.2. The same motif is found in SUR1 and SUR2 on the N-terminal side of NBF1. Deletion or mutation of the KIR signal permits surface expression of KIR subunits without SUR1, whereas mutation of the SUR1 signal gives surface expression without a KIR. These signals are proposed to serve as a quality control mechanism that ensures only the surface expression of properly assembled octameric channels (11Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). 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 KATPchannels. The deletion of this anterograde signal can account for the loss of channel activity in some HI-SUR1 mutations. high affinity sulfonylurea receptor endoplasmic reticulum ATP-sensitive potassium channel potassium inward rectifier phosphate-buffered saline endoglycosidase H IC50 for ATP The hamster SUR1 cDNA (1Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement IV, J.P. Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1288) Google Scholar), encoding 1582 amino acids, in the pECE vector (12Ellis L. Clauser E. Morgan D.O. Edery M. Roth R.A. Rutter W.J. Cell. 1986; 45: 721-732Abstract Full Text PDF PubMed Scopus (697) Google Scholar) was truncated by introducing stop codons at positions 1581 (SUR1ΔC2), 1579 (SUR1ΔC4), 1576(SUR1ΔC7), 1570(SUR1ΔC13), 1561(SUR1ΔC22), and 1534(SUR1ΔC49) using conventional polymerase chain reaction. The amino acid substitutions, V1578A, F1574A, L1567A, L1566A, and D1561A, were introduced by double-overlap polymerase chain reaction using overlapping mutagenic primers. PECE-N-6X-hisSUR1 and pECE-N-6X-hisSUR1ΔC49 were generated as described previously (3Clement IV, J.P. Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). Two copies of the myc-epitope were inserted in the first extracellular loop after the first nucleotide binding fold using a unique Bpu1102 site and the following phosphorylated oligonucleotides: forward, 5′-P TCA GCG GTG AGC AGA AAC TAA TTT CTG AGG AGG ACT TAG GGC; and reverse, 5′-P TGA GCC CTA AGT CCT CCT CAG AAA TTA GTT TCT GCT CAC CGC. Multiple clones were picked and sequenced to identify one containing two copies of the c-myc epitope. Mouse KIR6.2 (2Inagaki N. Gonoi T. Clement IV, J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1623) Google Scholar), the kind gift of Dr. Susumu Seino (Chiba University, Chiba Japan), was subcloned into the pECE vector before use. KIR6.2ΔC35 was generated as described (10Babenko A.P. Gonzalez G. Aguilar-Bryan L. Bryan J. FEBS Lett. 1999; 445: 131-136Crossref PubMed Scopus (51) Google Scholar). All constructs were sequenced to confirm the introduction of the desired mutation. COSm6 cells were cultured and transfected as described previously (1Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement IV, J.P. Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1288) Google Scholar,3Clement IV, J.P. Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). Rubidium efflux assays to measure specific glibenclamide-inhibited efflux were done as described (3Clement IV, J.P. Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 13Aguilar-Bryan L. Clement IV, J.P. Nelson D.A. Methods Enzymol. 1998; 292: 732-744Crossref PubMed Scopus (13) Google Scholar). 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 μl of hypotonic buffer (5 mm Tris-HCl, 2 mm EDTA, 0.1m NaCl, pH 7.4) and allowed to swell for 40 min on ice. Cells were homogenized, centrifuged at 1000 × g for 10 min at 4 °C. For further purification, the supernatant was collected and centrifuged at 40,000 × g for 60 min. The pelleted membranes were resuspended in 300 μl of membrane buffer supplemented with protease inhibitors (CompleteTM, Roche Molecular Biochemicals) (50 mm Tris-HCl, 5 mm EDTA; pH 7.4) and stored at −80 °C. The protein concentrations varied from 2–5 mg/ml. Membranes were photolabeled with125I-iodoazidoglibenclamide, kindly provided by Professor Uwe Panten (University of Braunschweig, Braunschweig, Germany), as described (3Clement IV, J.P. Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 14Schwanstecher M. Schwanstecher C. Chudziak F. Panten U. Clement IV, J.P. Gonzalez G. Aguilar-Bryan L. Bryan J. Methods Enzymol. 1999; 294: 445-458Crossref PubMed Scopus (13) Google Scholar). The appearance of SUR1 at the cell surface was quantified using a luminometer-based assay to measure SUR1c-myc. Transfected COSm6 cells were gently washed in Kreb's Ringer PBS and incubated for 1 h at 25 °C with the mouse monoclonal IgG1 c-myc antibody (9E10, Santa Cruz Biotechnology) diluted in Dulbecco's modified Eagles' medium plus 10% fetal bovine serum to a concentration of 0.3 μg/ml. After incubation, the cells were washed 3–4 times with Tris-buffered saline containing 1 mm CaCl2 and 1 mmMgCl2 and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG. The cells were detached using calcium and magnesium-free Tris-buffered saline plus 2 mm EDTA, pelleted, and resuspended in PBS containing 1 mmCaCl2 and 1 mm MgCl2. Chemiluminescence was quantified in aliquots of cells with a Lumat 9507 (EG&G Berthold) using Luminol (Santa Cruz Biotechnology) as the horseradish peroxidase substrate. Immunoprecipitation was done using membranes from transfected COSm6 cells labeled with 125I-azidoglibenclamide and solubilized with 60 mm ofN-dodecyl-β-d-maltoside and 500 mmNaCl for 1 h at 4 °C with continuous rocking. Following centrifugation at 40,000 × g for 30 min, the supernatant was incubated overnight with an agarose-conjugated His-probe (rabbit polyclonal IgG antibodies, H-15, Santa Cruz Biotechnology). The beads were pelleted at 10,000 × gand washed 4–5 times with membrane buffer, and proteins were released from the beads in 30 μl of SDS loading buffer and resolved on 7.5% SDS-polyacrylamide gels. The currents through and surface density of reconstituted KATP channels were measured in the inside-out configuration using the patch-clamp technique at 23–24 °C as described previously (10Babenko A.P. Gonzalez G. Aguilar-Bryan L. Bryan J. FEBS Lett. 1999; 445: 131-136Crossref PubMed Scopus (51) Google Scholar, 15Babenko A.P. Gonzalez G. Aguilar-Bryan L. Bryan J. Circ. Res. 1998; 83: 1132-1143Crossref PubMed Scopus (162) Google Scholar). Deletion of the C terminus of SUR1 affects KATPchannel activity measured here by a 86Rb+efflux assay. The SUR1ΔC2 and ΔC4/KIR6.2 channels show glibenclamide-inhibited 86Rb+ efflux attributable to KATP channels which is comparable with wild-type channels (Fig. 1 A); the SUR1ΔC7/KIR6.2 channels show approximately a 50% decrease in activity, while a complete loss of activity is observed for the SUR1ΔC13, ΔC22, and ΔC49/KIR6.2 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 KIR6.2 (11Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar), we co-expressed the SUR1ΔC constructs with KIR6.2ΔC35 missing 35 residues from its C terminus (10Babenko A.P. Gonzalez G. Aguilar-Bryan L. Bryan J. FEBS Lett. 1999; 445: 131-136Crossref PubMed Scopus (51) Google Scholar). There were no significant differences in the rates of86Rb+ efflux between the SUR1ΔC/KIR6.2 versusSUR1ΔC/KIR6.2ΔC35 channels (Fig. 1 A), 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 KIR6.2. SUR1 is differentially glycosylated, exhibiting a mature or complex glycosylated form (150–170 kDa) and an immature or core glycosylated form (140 kDa) (3Clement IV, J.P. Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 16Aguilar-Bryan L. Nelson D.A. Vu Q.A. Humphrey M.B. Boyd III A.E. J. Biol. Chem. 1990; 265: 8218-8224Abstract Full Text PDF PubMed Google Scholar). Mature SUR1 is present only when the receptor is co-expressed with KIR6.1 or KIR6.2 and has been shown to assemble with KIR6.2 to form active KATP channels in the plasma membrane (3Clement IV, J.P. Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). 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 thecis-Golgi. Co-expression of SUR1ΔC2 or ΔC4 with KIR6.2 yields both the immature and mature glycosylated forms of SUR1 as shown in Fig. 1 B using125I-iodoazidoglibenclamide to identify the receptors. SUR1ΔC7, ΔC13, ΔC22, and ΔC49 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. We used luminometry to quantify the appearance of SUR1c-myc at the cell surface (Fig.1 C). The results confirm the observation that co-expression of SUR1 and KIR6.2 markedly increases their surface expression (11Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). Co-expression of KIR6.2 with full-length SUR1c-myc produced the greatest differential. The SUR1ΔC2c-myc and ΔC4c-myc constructs give ∼ 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.1 B) and KATP channel activity (Fig.1 A). 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 KIR6.2 and the SUR1ΔC subunits were co-assembling, we tested whether the inward rectifier would photolabel with 125I-azidoglibenclamide and co-immunoprecipitate when expressed with the truncated receptors. Fig.2 (left panel) shows that when SUR1ΔC49 or SUR1ΔC221 are co-expressed with KIR6.2, 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 (3Clement IV, J.P. Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). 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 truncated receptor and inward rectifier co-assemble. When N-6X-hisSUR1ΔC49, tagged with six histidine residues at its N terminus, is co-expressed with KIR6.2, the inward rectifier labels with 125I-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 KIR6.2 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.3 A). 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 SUR1c-myc, co-expressed them with KIR6.2, 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 KATP 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 SUR1F1574A/Kir6.2 channels (Fig.4) shows that their ATP sensitivity is similar to wild-type channels (IC50(ATP) = 8.9 ± 03 μm versus 6.9 ± 03 μm for wild type (WT)), but their surface density (N) is ∼5–6 times lower than that of the wild-type, consistent with the86Rb+ efflux and surface expression results. We conclude that amino acids Phe-1574 and Leu-1566 are a part of an anterograde trafficking signal that is required for the surface expression of KATP channels. 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 "−RKR−" endoplasmic reticulum retention, or retrograde, signal in SUR and in the C termini of KIR6.1 and KIR6.2 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, core-glycosylated 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 KIR6.2 as a large octameric complex consistent with its being in active KATP channels on the cell surface (3Clement IV, J.P. Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). 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 KIR6.1 or KIR6.2 produces the immature form of the receptor (3Clement IV, J.P. Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). This is consistent with the observed lack of surface expression of SUR1 in Xenopus oocytes (11Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar) and in COSm6 cells (Fig. 1 C) unless KIR6.1 or KIR6.2 are present and indicates the retrograde signals must be masked before the assembled channels can exit the ER andcis-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 KATP channels when the receptor is expressed with KIR6.2. Co-photolabeling of SUR1 and KIR6.2 with125I-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 SUR1F1574A/KIR6.2 channels indicates they retain normal sensitivity to ATP, but the density of channels is reduced. Expression of KIR6.2ΔC subunits with SUR1ΔC subunits does not lead to surface expression of channels, indicating that the C terminus of SUR does not mask the–RKR− signal in KIR, and our preliminary data indicate it does not mask the –RKR− signal in SUR1. Previous work indicates that SUR1 increases the surface expression of KIR6.2ΔC subunits approximately 8-fold, consistent with the involvement of the anterograde signal in a process that facilitates trafficking to the cell surface (10Babenko A.P. Gonzalez G. Aguilar-Bryan L. Bryan J. FEBS Lett. 1999; 445: 131-136Crossref PubMed Scopus (51) Google Scholar). Substitution of alanines at positions Phe-1574 and Leu-1566 indicate the importance of these residues for anterograde transit of KATP channels. Dileucine motifs have been shown to be important in protein trafficking from the trans-Golgi to a late endosomal/lysosomal compartment (17Letourneur F. Klausner R.D. 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An acidic residue 4–5 residues on the N-terminal side of the dileucine motif can also be important for sorting (24Pond L. Kuhn L.A. Teyton L. Schutze M.P. Tainer J.A. Jackson M.R. Peterson P.A. J. Biol. Chem. 1995; 270: 19989-19997Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 25Geisler C. Dietrich J. Nielsen B.L. Kastrup J. Lauritsen J.P. Odum N. Christensen M.D. J. Biol. Chem. 1998; 273: 21316-21323Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), and a C-terminal glutamate/dileucine motif has been shown to be important for surface expression of the vasopressin V2 receptor (26Schulein R. Hermosilla R. Oksche A. Dehe M. Wiesner B. Krause G. Rosenthal W. Mol. Pharmacol. 1998; 54: 525-535Crossref PubMed Scopus (133) Google Scholar). Interestingly, mutation of the first leucine of the vasopressin V2 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 → Q substitution in the vasopressin V2 receptor largely eliminates surface expression. The vasopressin V2 receptor has no downstream phenylalanine corresponding to SUR1Phe-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. 27Bannykh S.I. Nishimura N. Balch W.E. Trends Cell Biol. 1998; 8: 21-25Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 28Barlowe C. Biochim. Biophys. Acta. 1998; 1404: 67-76Crossref PubMed Scopus (102) Google Scholar, 29Kreis T.E. Lowe M. Pepperkok R. Annu. Rev. Cell Dev. Biol. 1995; 11: 677-706Crossref PubMed Scopus (101) Google Scholar, 30Kuehn M.J. Schekman R. Curr. Opin. Cell Biol. 1997; 9: 477-483Crossref PubMed Scopus (107) Google Scholar, 31Nickel W. Wieland F.T. Histochem. Cell Biol. 1998; 109: 477-486Crossref PubMed Scopus (33) Google Scholar, 32Warren G. Malhotra V. Curr. Opin. Cell Biol. 1998; 10: 493-498Crossref PubMed Scopus (87) Google Scholar). These observations give a molecular insight into the lack of KATP channel activity observed in pancreatic beta cells from patients with persistent hyperinsulinemic hypoglycemia of infancy. Mutations in both SUR1, HI-SUR1, and KIR6.2, HI-KIR6.2, are the cause of a recessive form of this disorder which is characterized by an inappropriate secretion of insulin despite hypoglycemia (5Aguilar-Bryan L. Bryan J. Endocr. Rev. 1999; 20: 101-135Crossref PubMed Scopus (624) Google Scholar, 6Aguilar-Bryan L. Bryan J. Diabetes Rev. 1996; 4: 336-346Google Scholar, 33Permutt M.A. Nestorowicz A. Glaser B. Diabetes Rev. 1996; 4: 347-355Google Scholar). Nonsense and splice site mutations in SUR1 produce C-terminal deletions (5Aguilar-Bryan L. Bryan J. Endocr. Rev. 1999; 20: 101-135Crossref PubMed Scopus (624) Google Scholar) that have been shown to result in a complete loss of KATP channel activity (34Dunne M.J. Kane C. Shepherd R.M. Sanchez J.A. James R.F.L. Johnson P.R.V. Aynsley-Green A. Lu S. Clement IV, J.P. Lindley K.J. Seino S. Aguilar-Bryan L. N. Engl. J. Med. 1997; 336: 703-706Crossref PubMed Scopus (229) Google Scholar). 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 result of deleting the anterograde signal.