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Independent Trafficking of KATP Channel Subunits to the Plasma Membrane

1998; Elsevier BV; Volume: 273; Issue: 6 Linguagem: Inglês

10.1074/jbc.273.6.3369

1083-351X

Elena Makhina, Colin G. Nichols,

Ion channel regulation and function

KATP channels are unique in requiring two distinct subunits (Kir6.2, a potassium channel subunit) and SUR1 (an ABC protein) for generation of functional channels. To examine the cellular trafficking of KATP channel subunits, green fluorescent protein (GFP) was tagged to the cytoplasmic N or C terminus of SUR1 and Kir6.2 subunits and to the C terminus of a dimeric fusion between SUR1 and Kir6.2 (SUR1-Kir6.2). All tagged constructs generated functional channels with essentially normal properties when coexpressed with the relevant other subunit. GFP-tagged Kir6.2 (Kir6.2-GFP) showed perinuclear and plasma membrane fluorescence patterns when expressed alone or with SUR1, and a very similar pattern was observed when channel-forming SUR1-Kir6.2-GFP was expressed on its own. In contrast, whereas SUR1 (SUR1-GFP) also showed a perinuclear and plasma membrane fluorescence pattern when expressed alone, an apparently cytoplasmic fluorescence was observed when coexpressed with Kir6.2 subunits. The results indicate that Kir6.2 subunits traffic to the plasma membrane in the presence or absence of SUR1, in contradiction to the hypothesis that homomeric Kir6.2 channels are not observed because SUR1 is required as a chaperone to guide Kir6.2 subunits through the secretory pathway. KATP channels are unique in requiring two distinct subunits (Kir6.2, a potassium channel subunit) and SUR1 (an ABC protein) for generation of functional channels. To examine the cellular trafficking of KATP channel subunits, green fluorescent protein (GFP) was tagged to the cytoplasmic N or C terminus of SUR1 and Kir6.2 subunits and to the C terminus of a dimeric fusion between SUR1 and Kir6.2 (SUR1-Kir6.2). All tagged constructs generated functional channels with essentially normal properties when coexpressed with the relevant other subunit. GFP-tagged Kir6.2 (Kir6.2-GFP) showed perinuclear and plasma membrane fluorescence patterns when expressed alone or with SUR1, and a very similar pattern was observed when channel-forming SUR1-Kir6.2-GFP was expressed on its own. In contrast, whereas SUR1 (SUR1-GFP) also showed a perinuclear and plasma membrane fluorescence pattern when expressed alone, an apparently cytoplasmic fluorescence was observed when coexpressed with Kir6.2 subunits. The results indicate that Kir6.2 subunits traffic to the plasma membrane in the presence or absence of SUR1, in contradiction to the hypothesis that homomeric Kir6.2 channels are not observed because SUR1 is required as a chaperone to guide Kir6.2 subunits through the secretory pathway. ATP-sensitive potassium (KATP) channels couple cellular metabolism to electrical activity in multiple cell types. Insulin secretion in pancreatic β cells provides one of the best understood examples of such coupling (1Ashcroft F.M. Annu. Rev. Neurosci. 1988; 11: 97-118Crossref PubMed Scopus (768) Google Scholar). KATP channels are generated by coexpression of a potassium channel subunit (Kir6.2) with the sulfonylurea receptor (SUR1) (2Inagaki N. Gonoi T. Clement J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1610) Google Scholar). Neither sulfonylurea receptor nor Kir6.2 subunits are normally capable of channel activity when expressed alone in mammalian cells or in Xenopus oocytes (2Inagaki N. Gonoi T. Clement J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1610) Google Scholar, 3Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P., IV Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1281) Google Scholar, 4Gribble F.M. Ashfield R. Ammala C. Ashcroft F.M. J. Physiol. 1997; 498: 87-98Crossref PubMed Scopus (192) Google Scholar, 5Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (675) Google Scholar). Experiments on functional reconstitution of characteristic β cell currents by coexpression of SUR1 receptor and Kir6.2 suggested their association in a multidomain complex (2Inagaki N. Gonoi T. Clement J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1610) Google Scholar). It has been shown that Kir6.2 subunits form the channel pore (6Shyng S.-L. Ferrigni T. Nichols C.G. J. Gen. Physiol. 1997; 110: 141-153Crossref PubMed Scopus (128) Google Scholar), in a tetrameric complex, with each Kir6.2 subunit associated with 1 SUR1 subunit to form a higher order octamer (7Shyng S.-L. Nichols C.G. J. Gen. Physiol. 1997; 110: 654-655Google Scholar, 8Clement J.P., IV 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 (622) Google Scholar, 9Inagaki N. Gonoi T. Seino S. FEBS Lett. 1997; 409: 232-236Crossref PubMed Scopus (245) Google Scholar), and physical association of the two subunits has been demonstrated biochemically (8Clement J.P., IV 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 (622) Google Scholar). Clement et al. (8Clement J.P., IV 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 (622) Google Scholar) have shown that SUR1 and Kir6.2 protein can be detected in independent expression, but in this case, higher order glycosylation of SUR1 is not seen, and glibenclamide labeling of Kir6.2 is not observed. Fusion proteins in which SUR1 and Kir6.2 are linked in tandem generate functional KATP channels (7Shyng S.-L. Nichols C.G. J. Gen. Physiol. 1997; 110: 654-655Google Scholar, 8Clement J.P., IV 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 (622) Google Scholar). Sedimentation experiments with channel complexes formed either by monomeric SUR1 and Kir6.2 subunits or by fusion proteins (8Clement J.P., IV 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 (622) Google Scholar) further suggest a molecular weight that is consistent with an octameric model of the KATPchannel containing 4 channel and 4 receptor subunits. Nothing is known, however, about the cell biology of complex formation. The inability of Kir6.2 subunits to function as K+ channels in the absence of SUR1 (Inagaki et al. (2Inagaki N. Gonoi T. Clement J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1610) Google Scholar)) may be due to trafficking failure and raises the possibility that one role of SUR1 is to act as a chaperone to permit Kir6.2 subunits to form into a tetramer and/or to traffic to the surface membrane (5Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (675) Google Scholar). Conversely, there is no direct demonstration of the surface expression of SUR1 independent of association with Kir6.2 in the KATP complex. Ozanneet al. (10Ozanne S.E. Guest P.C. Hutton J.C. Hales C.N. Diabetologia. 1995; 38: 277-282Crossref PubMed Scopus (77) Google Scholar) showed that whereas a 170-kDa sulfonylurea receptor (SUR) 1The abbreviations used are: SUR, sulfonylurea receptor; GFP, green fluorescent protein; BHK, baby hamster kidney; ER, endoplasmic reticulum. 1The abbreviations used are: SUR, sulfonylurea receptor; GFP, green fluorescent protein; BHK, baby hamster kidney; ER, endoplasmic reticulum. was present in the plasma membrane, a 140-kDa form was only present on internal membranes. Clement et al. (8Clement J.P., IV 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 (622) Google Scholar) show that SUR1 is seen as a core-glycosylated 140-kDa species seen when expressed in the absence of Kir6.2 but that a higher order (170 kDa) species is seen when coexpressed with Kir6.2. Together, these results might suggest that SUR1 cannot reach the surface membrane in the absence of Kir6.2. In the present study we have utilized green fluorescence protein (GFP) labeling to visualize in vivo Kir6.2 and SUR1 targeting in mammalian cells and to address the question of whether Kir6.2 trafficking to the cell membrane is dependent on association with SUR1 subunits. The results indicate that both SUR1 and Kir6.2 can independently traffic toward the cell membrane. GFP optimized for maximal fluorescence (11Cormack B.P. Valdivia R.H. Falkow S. Gene. 1996; 173: 33-38Crossref PubMed Scopus (2500) Google Scholar) was fused to the N or C terminus of Kir6.2 (GFP-Kir6.2 and Kir6.2-GFP, respectively) or to the C terminus of SUR1 (SUR1-GFP) using an overlapping PCR technique. In the latter case, a linker of six glycine residues was placed in between SUR1 and GFP. A dimeric SUR1-Kir6.2 construct (7Shyng S.-L. Nichols C.G. J. Gen. Physiol. 1997; 110: 654-655Google Scholar, 8Clement J.P., IV 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 (622) Google Scholar) was tagged at the C terminus with GFP by substituting an XhoI-EcoRI fragment containing Kir6.2-GFP. Kir6.2 and its derivatives were expressed under the control of the cytomegalovirus promoter in pCMV6 vector and SUR1-containing constructs in pECE expression vector. GFP-tagged β-1,4-galactosyltransferase gene was a gift from Dr. Jennifer Lippincott-Schwartz (12Cole N.B. Smith C.L. Sciaky N. Terasaki M. Edidin M. Lippincott-Schwartz J. Science. 1996; 273: 797-801Crossref PubMed Scopus (404) Google Scholar). COSm6, HEK293, and BHK cells were transiently transfected using LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturer's suggestions for microscopy and electrophysiology or by a DEAE-dextran/chloroquine method for rubidium flux measurements. In the latter case, cells were plated on glass coverslips at a density of 0.5–2.5 × 105 cells/well (30-mm six-well dishes) and cultured in Dulbecco's modified Eagle's medium plus 10 mm glucose supplemented with fetal calf serum (10%). The following day, 5 μg of total cDNA was transfected into COSm6 cells with diethylaminoethyl dextran (0.5 mg/ml). Cells were incubated for 2 min in HEPES-buffered salt solution containing Me2SO (10%) and then for 4 h in Dulbecco's modified Eagle's medium plus 10 mm glucose plus 2% fetal calf serum and chloroquine (100 μm) and then returned to Dulbecco's modified Eagle's medium plus 10 mm glucose plus 10% fetal calf serum. 86RbCl (1 mCi/ml) was added in fresh growth medium 24 h after transfection. Cells were incubated for 12–24 h before measurement of Rb efflux. For efflux measurements, cells were incubated for 30 min at 25 °C in Krebs-Ringer solution with or without metabolic inhibitors (2.5 mg/ml oligomycin plus 1 mm 2-deoxy-d-glucose). At selected time points, the solution was aspirated from the cells and replaced with fresh solution. The 86Rb+ in the aspirated solution was counted. Cells were cultivated for 24–72 h after transfection and photographed in UV light under 1,000 × magnification in a Zeiss microscope equipped with a 515-nm emission filter. Confocal analysis was performed using an argon-krypton laser (Bio-Rad). Green fluorescence was detected at λ = 515 nm after excitation at λ = 488 nm. Cells were observed on thin coverslips used for their plating or after being treated with 0.1% trypsin for 30 s and resuspended in phosphate-buffered saline. Digitized images from confocal experiments were prepared for presentation using Corel Photopaint (Corel Inc.). Patch-clamp experiments were made at room temperature in a chamber that allowed the solution bathing the exposed surface of the isolated patch to be changed in less than 50 ms. Shards of glass were removed from the culture dishes and placed in the experimental chamber. Micropipettes were pulled from thin-walled glass (WPI Inc., New Haven, CT) on a horizontal puller (Sutter Instrument, Co., Novato, CA). Membrane patches were voltage-clamped with an Axopatch 1B patch-clamp (Axon Inc., Foster City, CA). PClamp software and a Labmaster TL125 D/A converter (Axon Inc.) were used to generate voltage pulses. Data were normally filtered at 0.5–3 kHz, and signals were digitized at 22 kHz (Neurocorder, Neurodata, NY, NY) and stored on video tape. Experiments were replayed onto a chart recorder or digitized into a microcomputer using Axotape software (Axon Inc.). The standard bath (intracellular) and pipette (extracellular) solution had the following composition: 140 mm KCl, 10 mm K-HEPES, 1 mm K-EGTA, pH 7.3, with additions as described. Off-line analysis was performed using ClampFit and Microsoft Excel programs. Wherever possible, data are presented as mean ± S.E. (standard error). Microsoft Solver was used to fit data by a least-square algorithm. To follow KATP channel activity in living cells, GFP was fused to the C terminus of SUR1 (SUR1-GFP) or to the N or C terminus of Kir6.2 (GFP-Kir6.2 or Kir6.2-GFP). To examine functional integrity of chimeric channels, SUR1-GFP was cotransfected with Kir6.2, and Kir6.2-GFP or GFP-Kir6.2 was cotransfected with SUR1 into COSm6 cells. Rubidium efflux measurements performed on the metabolically inhibited transfectants (Fig. 1 A) show that functional KATP channels are formed from each tagged construct when coexpressed with the complementary subunit. Neither GFP-tagged construct of Kir6.2 showed detectable channel activity in the absence of coexpression with SUR1, based on rubidium efflux measurements (not shown). Further characterization of GFP-tagged channels constructs was made using patch-clamp experiments. As shown in Fig. 1, B and C, each of the tagged constructs generated channels that were inhibited by ATP. The tagged SUR1 and Kir6.2 monomers, when coexpressed with the relevant other subunit, generated channels with wild type ATP sensitivity ([ATP] causing half-maximal inhibition (K i) = 13.5 μm and 8.6 μm for SUR1-GFP + Kir6.2 and Kir6.2-GFP + SUR1, respectively, cf. 11.1 μm for wild type channels). SUR1-Kir6.2-GFP triple fusions generated functional channels in the absence of additional subunits. These channels were less sensitive to ATP inhibition, the fitted K i (39.1 μm) being very similar to that seen with expression of untagged SUR1-Kir6.2 dimeric fusion proteins (45.3 μm, Ref. 7Shyng S.-L. Nichols C.G. J. Gen. Physiol. 1997; 110: 654-655Google Scholar; Ref. 8Clement J.P., IV 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 (622) Google Scholar). The functional similarity of tagged channels to the wild type enables us to use them as analogs of the authentic KATP channel in studying channel biogenesis. When GFP is fused to Kir6.2, a strongly fluorescent protein is produced in transfected cells and allows us to follow the fate of the Kir6.2 subunit in vivo. When coexpressed with SUR1 to generate functional KATP channels, the intracellular distribution of GFP-tagged Kir6.2 clearly differs from the distribution of free GFP, as shown in populations of transfected BHK cells (Fig. 2) and in individual COSm6 cells at higher magnification in Fig. 3. A strong signal associated with Kir6.2 is concentrated in the endoplasmic reticulum (ER) and perinuclear space, which contains the Golgi complex (Fig. 3 C). Unfortunately, contrast plasma membrane fluorescence is not obvious in adherent COSm6 cells. To reveal the surface signal, we minimized the cell surface by mild trypsinization immediately before confocal analysis. With visualization in a confocal microscope, edge fluorescence is clearly visible in Kir6.2-GFP + SUR1-transfected COSm6 cells (Fig. 3 D) but is absent in control cells expressing GFP alone (Fig. 3 B) or GFP-tagged β-1,4-galactosyltransferase, an integral membrane protein that resides in the Golgi but not in the plasma membrane (Fig. 3 F, Ref. 12Cole N.B. Smith C.L. Sciaky N. Terasaki M. Edidin M. Lippincott-Schwartz J. Science. 1996; 273: 797-801Crossref PubMed Scopus (404) Google Scholar).Figure 3Fluorescence associated with GFP-tagged Kir6.2 plus SUR1 in COSm6 cells. Fluorescent images of COSm6 cells expressing GFP alone (A), Kir6.2-GFP (C), or GFP-tagged β-1,4-galactosyltransferase (Tgal-GFP) (E) and confocal fluorescent images of trypsinized COSm6 cells expressing GFP alone (B), Kir6.2-GFP and SUR1 (D), or Tgal-GFP (F) are shown. The calibration bar represents 3 μm in A, C, and E and 2 μm in B, D, and F. In this figure and Figs. 4 and 5, white arrows indicate the endoplasmic reticulum, darts indicate the plasma membrane, and asterisksindicate the Golgi apparatus. try, trypsinized.View Large Image Figure ViewerDownload (PPT) In contradiction to the hypothesis that SUR1 might act as a chaperone to guide Kir6.2 through the secretory pathway (4Gribble F.M. Ashfield R. Ammala C. Ashcroft F.M. J. Physiol. 1997; 498: 87-98Crossref PubMed Scopus (192) Google Scholar, 5Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (675) Google Scholar), nearly identical fluorescence patterns were observed when GFP-tagged Kir6.2 constructs were expressed with (Fig. 3, C and D) or without SUR1 (Fig. 4, A and B). Adherent transfected COSm6 cells show the Kir6.2-GFP subunit entering the secretory pathway, but again, a visible plasma membrane signal is not obvious (Fig. 4 A). Again, however, a clear edge fluorescence is apparent after mild trypsinization (Fig. 4 B). Adherent HEK293 cells (Fig. 5) do not spread so thinly on coverslips, and in this case, edge fluorescence is visible in Kir6.2-GFP single transfectants (Fig. 5 A). Since Kir6.2-GFP showed no electrical activity when expressed alone, we could not confirm membrane localization functionally. As a positive control of a homomeric, integral membrane protein with assessable expression, we studied Kir2.3 (HRK1) inward rectifier channel (13Makhina E.N. Kelly A.J. Lopatin A.N. Mercer R.W. Nichols C.G. J. Biol. Chem. 1994; 269: 20468-20474Abstract Full Text PDF PubMed Google Scholar) subunits fused at the C terminus to GFP. Kir2.3-GFP chimeric channels conduct inward rectifier K+ currents with wild type efficiency (14Makhina E.N. Sha Q. Nichols C.G. Biophys. J. 1997; 72 (abstr.): 253Google Scholar) and show essentially identical fluorescence patterns to those seen in Kir6.2-GFP cells (Figs. 4, C and D, and 5 B).Figure 5Fluorescence associated with GFP-tagged inward rectifier subunits in HEK293 cells. Fluorescent images of HEK293 cells expressing Kir6.2-GFP alone (A), Kir2.3-GFP (B), SUR-GFP alone (C), and SUR-Kir6.2-GFP (WTF-GFP) (D) are shown. The calibrationbar represents 3 μm in each case.View Large Image Figure ViewerDownload (PPT) Coexpression of Kir6.2-GFP with SUR1 (in ∼1:1 cDNA ratio), which results in characteristic KATP currents (Fig. 1), does not obviously affect the intracellular distribution of Kir6.2-GFP (Fig. 4, A and B, cf. Fig. 3, C and D). One possible reason is that in coexpression, the predominant Kir6.2 signal that we observe is still actually from non-SUR1-associated subunits. To examine whether the distribution of SUR1-associated Kir6.2 differs from the distribution of Kir6.2 alone, we transfected cells with a SUR1-Kir6.2 tandem (8Clement J.P., IV 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 (622) Google Scholar) tagged at the C-terminal end with GFP (SUR1-Kir6.2-GFP). This triple-fusion construct, lacking free Kir6.2 subunits, shows ER, Golgi, and cell surface fluorescence of a similar intensity to cells expressing Kir6.2-GFP in both COSm6 cells (Fig. 4, E and F) and in HEK293 cells (Fig. 5 D). Since Kir6.2-GFP can traffic toward the plasma membrane independently of SUR1, we addressed the question of whether SUR1 is capable of Kir6.2-independent trafficking to the plasma membrane? COSm6 cells were transfected with SUR1-GFP with or without additional Kir subunits (Figs. 6, 7). In COSm6 cells, SUR1-GFP fluorescence also localizes to the endoplasmic reticulum, perinuclear space (Fig. 6 A), and plasma membrane (Fig. 5 C) in untreated cells, and plasma membrane fluorescence is also clearly visible in trypsinized COSm6 cells (Fig. 6 B). However, a strikingly different fluorescence pattern was observed when SUR1-GFP was cotransfected with Kir6.2 (Fig. 6 C), indicating a physical interaction. The perinuclear and reticular distribution was substantially replaced by a diffuse fluorescence across the cell (Fig. 6 C), although membrane fluorescence was still clearly visible in trypsinized cells (Fig. 6 D). As shown in Fig. 7, this effect of Kir6.2 coexpression is specific. When SUR1-GFP is coexpressed with Kir1.1 (Fig. 7 C) or Kir2.3 (Fig. 7 D), which do not interact with SUR1, the fluorescence is not different from that seen with expression of SUR1-GFP alone (Fig. 7 A).Figure 7Fluorescence associated with GFP-tagged SUR1 subunits indicates a specific interaction with Kir6.2.Fluorescent images of COSm6 cells expressing SUR-GFP alone (A), SUR-GFP and Kir6.2 (B), SUR-GFP and Kir1.1 (C), and SUR-GFP and Kir2.3 (D) are shown. The calibration bar represents 8 μm in each case.View Large Image Figure ViewerDownload (PPT) Neither receptor (SUR1) nor pore-forming (Kir6.2) subunits of the KATP channel normally demonstrate a plasma membrane-associated phenotype, with the exception of sulfonylurea binding to SUR1 in total membranes (3Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P., IV Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1281) Google Scholar), when expressed in the absence of the other subunit. SUR1 appears as a core-glycosylated 140-kDa species in the absence of Kir6.2 (8Clement J.P., IV 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 (622) Google Scholar), and this species does not appear to reach the plasma membrane in various insulin-secreting cell lines (10Ozanne S.E. Guest P.C. Hutton J.C. Hales C.N. Diabetologia. 1995; 38: 277-282Crossref PubMed Scopus (77) Google Scholar). A C-terminal deletion construct (5Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (675) Google Scholar) and even full-length Kir6.2 2S. A. John, J. R. Monck, J. N. Weiss, and B. Ribalet, personal communication. may form some active ATP-sensitive K+ channels in the absence of SUR1, but in both cases a considerably higher level of channel expression is observed when coexpressed with SUR1. Lack of channel function when wild type Kir6.2 is expressed without SUR1 may be due to an inability to express stably or to reach the cell surface and reside there for a physiologically significant period of time. The general concept of multimeric integral membrane protein biogenesis is that subunits assemble in early steps and are co-transported through the Golgi complex and the trans-Golgi network to the plasma membrane (reviewed in Ref. 15Rose J.K. Doms R.W. Annu. Rev. Cell Biol. 1988; 4: 257-288Crossref PubMed Scopus (262) Google Scholar). Unincorporated subunits typically do not reach the plasma membrane because of proteolytic degradation in the endoplasmic reticulum or in lysosomes. The T-cell antigen receptor, consisting of seven transmembrane subunits, provides an example of both degradation pathways. Although the fully assembled, seven-membered complex is transported to the plasma membrane, free α, γ, and δ chains rapidly degrade in the endoplasmic reticulum, and pentamers lacking the ζ chain pass the Golgi complex but are then redirected to lysosomes (reviewed in Ref. 16Klausner R.D. Sitia R. Cell. 1990; 62: 611-614Abstract Full Text PDF PubMed Scopus (455) Google Scholar). To explore the possibility that a similar degradation of free KATP subunits underlies the lack of functional channels in different expression conditions, we labeled them with GFP and followed their fate in living cells. Both SUR1-GFP and Kir6.2-GFP were observed in ER, Golgi, and cell surface compartments, indicating that neither unincorporated subunit contains any specific signal for rapid and effective proteolytic degradation in the ER or rerouting to lysosomes. With a few exceptions (e.g. connexin-43, gap-junction channel, type 2 sodium channel) where oligomerization takes place after exit (17Musil L.S. Goodenough D.A. Cell. 1993; 74: 1065-1077Abstract Full Text PDF PubMed Scopus (399) Google Scholar, 18Green W.N. Millar N.S. Trends Neurosci. 1995; 18: 280-287Abstract Full Text PDF PubMed Scopus (173) Google Scholar), subunit assembly of integral membrane proteins generally occurs in the endoplasmic reticulum, and proteins that fail to assemble into requisite oligomeric complexes are degraded without release from the endoplasmic reticulum (16Klausner R.D. Sitia R. Cell. 1990; 62: 611-614Abstract Full Text PDF PubMed Scopus (455) Google Scholar). In contrast to Kv1.2 or Kv2.2 channels, where auxiliary β subunits promote surface staining with antibodies raised against α-subunits (19Shi G. Nakahira K. Hammond S. Rhodes K.J. Schechter L.E. Trimmer J.S. Neuron. 1996; 16: 843-852Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 20Salinas M. de Weille J. Guillemare E. Lazdunski M. Hugnot J.P. J. Biol. Chem. 1997; 272: 8774-8780Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), coexpression of KATPchannel subunits does not obviously change the expression pattern of individual subunits. The stability of free KATP channel subunits in the secretory pathway indicates that assembly is not necessary for trafficking without necessarily implying that assembly into the functional complex should normally occur after independently reaching the plasma membrane. The formation of functional channels by the SUR1-Kir6.2-GFP triple fusion construct together with the visible intracellular distribution (Fig. 4, E and F) shows that preassembled SUR1 plus Kir6.2 dimer is also not subject to endoplasmic reticular or lysosomal degradation and is as viable outside the plasma membrane as the individual subunits. The effect of coexpression with Kir6.2 on the intracellular distribution of SUR1-GFP (Figs. 6 and 7) indicates a specific physical interaction between SUR1 and Kir6.2, since coexpression with other noninteracting Kir subunits (Kir1.1, Kir2.3) has no effect on the SUR1-GFP fluorescence (Fig. 7). A complex picture is beginning to emerge for KATP channel formation. The demonstration that the channel requires two subunits (2Inagaki N. Gonoi T. Clement J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1610) Google Scholar) and recent indications of an obligate stoichiometry (7Shyng S.-L. Nichols C.G. J. Gen. Physiol. 1997; 110: 654-655Google Scholar, 8Clement J.P., IV 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 (622) Google Scholar, 9Inagaki N. Gonoi T. Seino S. FEBS Lett. 1997; 409: 232-236Crossref PubMed Scopus (245) Google Scholar) might suggest a classical endoplasmic reticular oligomeric assembly (21Hurtley S.M. Helenius A. Annu. Rev. Cell Biol. 1989; 5: 277-307Crossref PubMed Scopus (776) Google Scholar) in which each subunit is necessary for normal trafficking. On the other hand, Ammala et al. (22Ammala C. Moorhouse A. Gribble F. Ashfield R. Proks P. Smith P, A. Sakura H. Coles B. Ashcroft S.J. Ashcroft F.M. Nature. 1996; 379: 545-548Crossref PubMed Scopus (165) Google Scholar) reported that coexpression with SUR1 was capable of conferring sulfonylurea sensitivity on Kir1.1 channels, which can clearly function in the absence of SUR1 (23Ho K. Nichols C.G. Lederer W.J. Lytton J. Vassilev P.M. Kanazirska M.V. Hebert S.C. Nature. 1993; 362: 31-38Crossref PubMed Scopus (832) Google Scholar) and suggested that SUR1 may "promiscuously" couple to different inward rectifier subunits. In the present study, we find that SUR1-GFP fluorescence patterns are affected specifically by expression with Kir6.2 but not by expression with Kir1.1 or Kir2.3. Together with the demonstration that azidoglibenclamide can label Kir6.2, but not Kir1.1, in coexpression with SUR1 (8Clement J.P., IV 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 (622) Google Scholar), these results indicate that SUR1 does not in fact interact promiscuously with other Kir subunits and interacts specifically with Kir6.x subunits. The present results further indicate that independent trafficking of each subunit toward the cell membrane can occur, but altered fluorescence of SUR1-GFP when coexpressed with Kir6.2 indicates a specific interaction and suggests the following as a working hypothesis. Independent synthesis of Kir6.2 tetramers (7Shyng S.-L. Nichols C.G. J. Gen. Physiol. 1997; 110: 654-655Google Scholar) and SUR1, perhaps as monomers, can occur in the endoplasmic reticulum, and these homomeric constructs can traffic to the surface membrane. However, when both subunits are coexpressed, as happens in native cells, a physical interaction between SUR1 and Kir6.2 occurs in the endoplasmic reticulum, possibly involving the C terminus of SUR1, which either facilitates insertion of channel-containing vesicles into the plasma membrane or stabilizes channels once inserted into the plasma membrane. We are grateful to Dr. J. Lippincott-Schwartz for providing us with the β-1,4-galactosyltransferase-GFP construct, to Drs. S. Seino and J. Bryan for providing us with the original Kir6.2 and the SUR1-Kir6.2 fusion, respectively, and to Drs. S. John, J. R. Monck, J. N. Weiss, and B. Ribalet for unpublished data. We are grateful to the Washington University Diabetes Research and Training Center for continued molecular biology support.