In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer
2006; Springer Nature; Volume: 25; Issue: 20 Linguagem: Inglês
10.1038/sj.emboj.7601373
ISSN1460-2075
AutoresMartin Mense, Paola Vergani, D. Mccall White, Gal Altberg, Angus C. Nairn, David C. Gadsby,
Tópico(s)Adenosine and Purinergic Signaling
ResumoArticle12 October 2006free access In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer Martin Mense Martin Mense Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, NY, USA Search for more papers by this author Paola Vergani Paola Vergani Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, NY, USAPresent address: Department of Pharmacology, University College London, London WC1E 6BT, UK Search for more papers by this author Dennis M White Dennis M White Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, NY, USA Search for more papers by this author Gal Altberg Gal Altberg Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, NY, USA Search for more papers by this author Angus C Nairn Angus C Nairn Department of Psychiatry, Yale University, New Haven, CT, USA Search for more papers by this author David C Gadsby Corresponding Author David C Gadsby Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, NY, USA Search for more papers by this author Martin Mense Martin Mense Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, NY, USA Search for more papers by this author Paola Vergani Paola Vergani Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, NY, USAPresent address: Department of Pharmacology, University College London, London WC1E 6BT, UK Search for more papers by this author Dennis M White Dennis M White Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, NY, USA Search for more papers by this author Gal Altberg Gal Altberg Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, NY, USA Search for more papers by this author Angus C Nairn Angus C Nairn Department of Psychiatry, Yale University, New Haven, CT, USA Search for more papers by this author David C Gadsby Corresponding Author David C Gadsby Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, NY, USA Search for more papers by this author Author Information Martin Mense1, Paola Vergani1, Dennis M White1, Gal Altberg1, Angus C Nairn2 and David C Gadsby 1 1Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, NY, USA 2Department of Psychiatry, Yale University, New Haven, CT, USA *Corresponding author. Laboratory of Cardiac/Membrane Physiology, Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, USA. Tel.: +1 212 327 8680; Fax: +1 212 327 7589; E-mail: [email protected] The EMBO Journal (2006)25:4728-4739https://doi.org/10.1038/sj.emboj.7601373 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The human ATP-binding cassette (ABC) protein CFTR (cystic fibrosis transmembrane conductance regulator) is a chloride channel, whose dysfunction causes cystic fibrosis. To gain structural insight into the dynamic interaction between CFTR's nucleotide-binding domains (NBDs) proposed to underlie channel gating, we introduced target cysteines into the NBDs, expressed the channels in Xenopus oocytes, and used in vivo sulfhydryl-specific crosslinking to directly examine the cysteines' proximity. We tested five cysteine pairs, each comprising one introduced cysteine in the NH2-terminal NBD1 and another in the COOH-terminal NBD2. Identification of crosslinked product was facilitated by co-expression of NH2-terminal and COOH-terminal CFTR half channels each containing one NBD. The COOH-terminal half channel lacked all native cysteines. None of CFTR's 18 native cysteines was found essential for wild type-like, phosphorylation- and ATP-dependent, channel gating. The observed crosslinks demonstrate that NBD1 and NBD2 interact in a head-to-tail configuration analogous to that in homodimeric crystal structures of nucleotide-bound prokaryotic NBDs. CFTR phosphorylation by PKA strongly promoted both crosslinking and opening of the split channels, firmly linking head-to-tail NBD1–NBD2 association to channel opening. Introduction The cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the gene found mutated in cystic fibrosis patients (Riordan et al, 1989), is a member of the superfamily of ATP-binding cassette (ABC) transporter ATPases. Like most eukaryotic ABC proteins, CFTR comprises two transmembrane domains each linked to a cytoplasmic nucleotide-binding domain (NBD1, NBD2) (Figure 1). Unlike any other ABC protein, however, CFTR functions as a Cl−- ion channel. Opening and closing of a CFTR channel, like the transport function of other ABC proteins, is regulated by ATP binding and hydrolysis at the NBDs. But, channel activity requires prior cAMP-dependent protein kinase (PKA)-mediated phosphorylation of the regulatory (R) domain that connects CFTR's two homologous halves (e.g., Gadsby and Nairn, 1999; Sheppard and Welsh, 1999). Figure 1.Native cysteines of human epithelial CFTR. Topological cartoon of CFTR showing the 18 cysteines indicated by their position numbers, the transmembrane domains (gray), the R domain (green), and the relative locations of Walker A (red) and ABC signature (purple) motifs in the nucleotide-binding domains, NBD1 (yellow) and NBD2 (cyan). Download figure Download PowerPoint Much is known about CFTR's role in epithelial function and about how CFTR channel activity is regulated (Kirk and Dawson, 2003), but little is known about CFTR structure. The sole exceptions are a low-resolution map from cryo-electron microscopy of 2D crystals of CFTR (Rosenberg et al, 2004), and high-resolution structures of NBD1 from mouse and human CFTR (Lewis et al, 2004, 2005; Thibodeau et al, 2005) that display the overall architecture, and arrangement of ATP-contacting 'Walker A and B' motifs and of the 'ABC signature' (LSGGQ) sequence, found in all other isolated NBD structures (e.g., Armstrong et al, 1998; Hung et al, 1998; Karpowich et al, 2001; Yuan et al, 2001). Evidence from crystal structures of prokaryotic NBD homodimers (Hopfner et al, 2000; Smith et al, 2002; Chen et al, 2003; Zaitseva et al, 2005) and full transporters (Locher et al, 2002; Reyes and Chang, 2005), and from biochemical tests on intact prokaryotic (Fetsch and Davidson, 2002) or eukaryotic ABC transporters (Loo et al, 2002) suggests that NBD pairs may, at least transiently, form homodimeric complexes with two ATP binding sites within the dimer interface (for a review, see Higgins and Linton, 2004). In the homodimeric crystal structures, each ATP links the Walker motifs of one NBD to the signature sequence of the opposing NBD, in a rotationally symmetric head-to-tail dimer (Hopfner et al, 2000; Smith et al, 2002; Chen et al, 2003; Zaitseva et al, 2005). We have proposed that in CFTR a corresponding association between ATP-bound NBD1 and NBD2 accompanies opening of the ion channel (Vergani et al, 2003, 2005). We present here direct evidence, from sulfhydryl-specific crosslinking studies in living cells, that activation of human CFTR channels by phosphorylation promotes formation of a head-to-tail NBD1–NBD2 complex with an interface that mimics that found in homodimeric prokaryotic NBD crystals. Furthermore, by crosslinking during electrophysiological recordings, we demonstrate that the crosslinked NBD1–NBD2 complex characterizes an open-channel conformation of CFTR. Results Removal of native cysteines from CFTR Before target cysteines for crosslinking could be introduced into CFTR, it was necessary to remove native cysteines to preclude their reaction with sulfhydryl reagents. We used PCR-based mutagenesis and/or gene synthesis techniques to replace all 18 native cysteines in human CFTR (Figure 1) with serines, one at a time or in groups. Functional expression of wild type (WT; Figure 2A, middle) and of mutant (e.g., Figure 2A, bottom) CFTR channels was assessed using two-microelectrode voltage-clamp recordings of forskolin-activated Cl− currents in cRNA-injected Xenopus oocytes (e.g., Chan et al, 2000). Forskolin (the adenylyl cyclase agonist and hence stimulator of PKA activity) elicited robust responses in most mutant channels lacking one or more cysteines, including channels in which all of the cysteines except two (Cys 590 and 592 in NBD1) had been replaced by serines (16CS; Figure 2B and C). Functional expression was markedly diminished when C590 and C592 were both mutated to serine, regardless of whether the other 16 cysteines remained (data not shown) or had all been replaced by serines (Figure 2B, 16CS+C590S/C592S). Nor could C590 and C592 be replaced by alanine, threonine or phenylalanine (Figure 2B), but function was similar to that of the 16CS background when they were replaced by leucines (16CS+C590L/C592L; Figure 2A and B) or valines (16CS+C590V/C592V; Figure 2B). In excised patches, where [ATP] and PKA activity could be controlled independently, Cys-free (16CS+C590V/C592V) CFTR channels, like WT, required phosphorylation by PKA before they could be opened by ATP, closed upon ATP removal, and were activated half-maximally by ∼50 μM [ATP] (Supplementary Figure S1); their single-channel conductance was very slightly larger than that of WT CFTR channels. Figure 2.Expression and function of cysteine-deficient CFTR channels in Xenopus oocytes. (A) Two-microelectrode voltage-clamp current recordings from uninjected oocyte and oocytes expressing WT CFTR (2.5 ng cRNA) or HA-tagged Cys-free CFTR 16CS+C590L/C592L (20 ng cRNA); vertical current deflections monitor conductance, which was transiently increased by brief exposure to 40 μM forskolin (between arrows). (B) Summary of mean±s.d. whole-oocyte conductances determined as in (A), before ('resting', white bars) and at maximal forskolin effect ('stimulated', gray bars), 3 days after injection of 20 ng cRNA encoding HA-tagged CFTR 16CS constructs containing native C590 and C592 or substitutions at those positions as indicated; forskolin elicited significant conductance only with C590 and C592 unchanged (131±6 μS, n=3), or replaced by valines (119±6 μS, n=15) or leucines (155±9 μS, n=3). (C) Conductances from oocytes injected with 2.5 ng cRNA, and measured 1 day later for WT (153±17 μS, n=3), or 3 days later for HA-tagged 16CS mutants with C590/C592 (42±10 μS, n=6) or C590V/C592 V (51±3 μS, n=6). This under-represents the functional difference between WT and mutants, as conductance is enhanced by injecting more cRNA or allowing more time for its expression. (D) WT CFTR and Cys-free CFTR (16CS+C590V/C592V) were immunoprecipitated from membranes of oocytes injected with cRNA amounts indicated, and subjected to SDS-PAGE and Western blot analysis; arrows mark core-glycosylated and mature fully glycosylated CFTR. Download figure Download PowerPoint Functional expression of CFTR correlates with the appearance of fully glycosylated CFTR protein (Gregory et al, 1991; Denning et al, 1992), and so we tested whether the smaller activated whole-oocyte conductance in intact oocytes expressing the mutants (Figure 2C) might reflect their impaired maturation. Western blotting of immunoprecipitated channels (Figure 2D) supported this interpretation. Oocytes injected with 20 ng WT CFTR cRNA yielded dense bands (Figure 2D, lane 2) of both core-glycosylated and fully glycosylated WT CFTR. But maturation of Cys-free CFTR was less efficient, and after injection of 20 ng Cys-free CFTR cRNA only a weak band corresponding to mature fully glycosylated CFTR could be detected (Figure 2D, lane 4) whereas a stronger signal reflected core-glycosylated protein (lower arrow). Introduction of target cysteines for crosslinking studies On the basis of crystal structures of nucleotide-bound prokaryotic NBD homodimers and of monomeric NBD1 F508A from human CFTR, we made a homology model of the anticipated CFTR NBD1–NBD2 complex (Figure 3; see Materials and methods). We then selected candidate pairs of positions for crosslinking across three different regions of the heterodimeric interface: the 'NBD2' composite catalytic site (comprising NBD2 Walker A and B motifs and NBD1 signature sequence), a central region, and the 'NBD1' composite site (including NBD1 Walker motifs and NBD2 signature sequence). Figure 3.Homology model of a head-to-tail CFTR NBD1–NBD2 heterodimer, based on crystal structures of human CFTR NBD1 F508A and of ATP- or AMPPNP-bound NBDs of other ABC proteins (Materials and methods). Two ATP molecules (CPK-colored stick structures) are sandwiched between the Walker A and ABC signature sequences of opposing NBDs, NBD1 (yellow) and NBD2 (blue). To test this model by sulfhydryl-specific crosslinking, the residues in spacefill were mutated to cysteines in pairs, chosen so that an interfacial crosslink would span the 'NBD1' composite site (cyan, orange, and purple residue pairs), or a central region (green residues), or the 'NBD2' composite site (red residues). Due to sequence and structural differences between CFTR's NBD1 and other NBDs, the position of S434 is uncertain. Download figure Download PowerPoint Introducing target cysteines into the NBDs of full-length Cys-free CFTR further impaired protein maturation (data not shown). We were nevertheless able to retain the benefit of a cysteine-free background by carrying out crosslinking experiments on split CFTR channels (Chan et al, 2000; Csanády et al, 2000), comprising a native NH2-terminal half (amino acids 1–633, including all nine native cysteines) and a Cys-free COOH-terminal half (amino acids 634–1480, with nine Cys-to-Ser mutations), both of which expressed well and were readily detected in Westen blots (see below). After introduction of a target cysteine in each half, the COOH-terminal half channel contained only a single cysteine. Any observed sulfhydryl-specific crosslinking of NH2- and COOH-terminal half channels could therefore be unequivocally ascribed to linkage via that target cysteine. In addition, the use of split channels enhanced the relative mobility shift upon crosslinking, thereby rendering the identification of crosslinked product unambiguous. Target cysteines were introduced one at a time into the half channels in place of the following residues: S1248 in the NBD2 Walker A sequence and S549 in the NBD1 LSGGQ signature sequence; S605 in NBD1 and A1374 in NBD2, both in central positions of the putative dimer interface; A462 in the NBD1 Walker A and S1347 in LSHGH of NBD2. Comparatively inefficient crosslinking across the latter NBD1 composite site in early tests prompted introduction of additional cysteine pairs in that region, replacing S459 in NBD1 Walker A and V1379 in NBD2, and S434 in NBD1 and D1336 in NBD2. We confirmed that after introduction of each individual target cysteine, into either NH2-terminal or Cys-free COOH-terminal half, co-expression in oocytes with the complementary half channel lacking introduced cysteines yielded mature fully glycosylated COOH-terminal CFTR halves in Western blots (broad band at ∼130 kDa in Figure 4B, arrow; Figure 5, top row); some core-glycosylated COOH-terminal half-channel protein is also evident (sharp bands near 85 kDa in Figure 4B). The full glycosylation implies that the protein was correctly folded and trafficked to the surface membrane (Gregory et al, 1991; Denning et al, 1992; Chan et al, 2000). Accordingly, application of forskolin to raise PKA activity in oocytes expressing these split CFTR channels containing either one, or both, introduced target cysteines elicited robust increases in conductance (see Figures 4A and C); CFTR channels show low basal activity in resting oocytes, reflecting basally active PKA, but channel activity is enhanced several-fold upon stimulation of PKA by forskolin (Chan et al, 2000; Csanády et al, 2005). Similar split CFTR channels, though incorporating all native cysteines, were previously found to display basal and stimulated activity essentially like WT CFTR (Chan et al, 2000; Csanády et al, 2000). Comparable levels of expression (Figures 6, 7, 8 and 9 and Supplementary Figure S2) and function (e.g., Figure 4A and C) were obtained when both co-expressed half channels contained a target cysteine. Figure 4.Expression (A, B) and function (A, C) of split CFTR channels containing introduced cysteines. Oocytes were injected with 5+5 ng cRNA encoding NH2-terminal (1–633), and COOH-terminal (634–1480) 9CS (Ser replacing Cys at positions 647, 832, 866, 1344, 1355, 1395, 1400, 1410 and 1458), half channels containing either no introduced cysteine in NBD1 or NBD2 ('no C' label in A, B), or a single introduced cysteine at the residue position indicated beside each point in (A), above each lane in (B), and above each recording in (C). (A) Summary of resting and stimulated whole-oocyte conductances (μS) normalized to relative expression level, plotted against levels of expression (normalized to the highest measured value). (B) Expression levels were measured from a Western blot (anti-R-domain antibody) of ∼25 μg of membrane proteins from 30 to 32 resting oocytes as gray level intensities of the broad bands of fully glycosylated COOH-terminal half channels (arrow); the lower sharp bands near 85 kDa are core-glycosylated COOH-terminal half channels. (C) Three examples of conductance measurements, at rest and after stimulation with 40 μM forskolin, in oocytes containing an introduced cysteine in NBD1 (top), or in NBD2 (middle), or in both NBDs (bottom). Download figure Download PowerPoint Figure 5.The absence of efficient crosslinking when no, or only one, engineered cysteine is present. Oocytes injected with 5+5 ng cRNA encoding NH2-terminal (1–633), and COOH-terminal (634–1480) 9CS, half channels containing no (background), or just one, introduced cysteine in either NBD1 or NBD2, at the position indicated above each panel. Oocytes were untreated, or pretreated with 40 μM forskolin plus 1 mM IBMX and then incubated with 300 μM BMOE or 600 μM BMH at room temperature, as indicated. Membrane proteins were analyzed by SDS-PAGE and blotted with antibody against the R domain (COOH-terminal half-channel, upper blots) or antibody against the NH2-terminus (lower blots). Note that upon forskolin treatment, phosphorylation of the R domain slows mobility of the COOH-terminal half channel (Csanády et al, 2005) resulting in the appearance of a sharp band (at ∼90 kDa) just above the ∼85 kDa core-glycosylated band. No strong high-molecular-mass band, reflecting crosslinked product, is evident in any lane, but arrows mark weak, dispersed, unidentified BMOE- and/or BMH-induced bands discernible in some blots with antibody against the NH2-terminus. Download figure Download PowerPoint Figure 6.Crosslinking across the 'NBD2' composite catalytic site, between position 1248 in NBD2 Walker A and position 549 in NBD1 LSGGQ. Western blots identify the NH2-terminal half channel (1–633), S549C (left panel; lower arrow), the COOH-terminal half channel (634–1480) 9CS+S1248C (right panel; core-glycosylated, ∼85–90-kDa, bands; fully glycosylated, lower arrow), and crosslinked product (both panels; arrows labeled X-link). Incubation temperature, presence or absence of 300 μM BMOE or 600 μM BMH, and/or treatment with 40 μM forskolin plus 1 mM IBMX ('fsk') are indicated below each lane. Forskolin increased the yield of crosslinked product four-fold for BMOE and two-fold for BMH. Samples in lanes 8 and 16 are from uninjected control oocytes. Download figure Download PowerPoint Figure 7.Crosslinking between central region residues, 605 of NBD1 and 1374 of NBD2. Western blots show CFTR half channels (1–633) S605C (left panel; lower arrow), (634–1480) 9CS+A1374C (right panel; core-glycosylated, ∼85–90 kDa, bands; fully glycosylated, lower arrow), and crosslinked product (both panels; arrows labeled X-link). Incubation temperature, presence or absence of 300 μM BMOE or 600 μM BMH, and/or treatment with 40 μM forskolin plus 1 mM IBMX ('fsk') are indicated below each lane. Forskolin increased the yield of crosslink product 870-fold for BMOE and four-fold for BMH. Download figure Download PowerPoint Figure 8.Crosslinking across the 'NBD1' composite site. Incubation temperature, presence or absence of 300 μM BMOE or 600 μM BMH, and/or treatment with 40 μM forskolin plus 1 mM IBMX ('fsk') are indicated below each lane in all panels. Left panels show NH2-terminal half channels (lower arrows), right panels show COOH-terminal half channels (core-glycosylated, ∼85–90-kDa bands; fully glycosylated, lower arrows), and all panels show crosslinked product (upper arrows labeled X-link); some other BMOE- and/or BMH-induced bands, mostly in the blots with anti-NH2-terminal antibody, likely reflect crosslinking of NH2-terminal half channels (with nine native Cys) to unknown proteins. (A) CFTR half channels (1–633) A462C (left panel) and (634–1480) 9CS+S1347C (right panel). Forskolin increased the yield of crosslinked product >1000-fold for both BMOE and BMH; lanes 8 and 16 are samples from uninjected oocytes. (B) CFTR half channels (1–633) S459C (left panel) and (634–1480) 9CS+V1379C (right panel). Forskolin increased crosslink product yield nine-fold for BMOE and 5-fold for BMH. (C) CFTR half channels (1–633) S434C (left panel) and (634–1480) 9CS+D1336C (right panel), as well as crosslinked product (arrow labeled X-link, both panels). Forskolin increased crosslink product yield 9-fold for BMH. Crosslink yield for BMOE was negligibly small. Download figure Download PowerPoint Figure 9.Tests of crosslinking between NBD1 and NBD2 using other combinations of the target cysteines. The presence or absence of 300 μM BMOE or 600 μM BMH and/or treatment with 40 μM forskolin plus 1 mM IBMX ('fsk') are indicated for each lane in all panels. (A) Oocytes were injected with cRNAs encoding NH2-terminal (1–633) and COOH-terminal (634–1480) 9CS half channels, each containing one introduced cysteine, in the combinations indicated above each panel. Incubations were at room temperature. Western blots identify NH2-terminal half channels (lower rows) and core- and fully glycosylated COOH-terminal half channels (upper row). (B) Western blots show CFTR half channels (1–633) S549C (left panel, lower arrow) and (634–1480) 9CS+A1374C (right panel, core-glycosylated, ∼85–90-kDa bands; fully glycosylated, lower arrow), as well as crosslinked product (both panels, arrows labeled X-link). Incubation temperature is indicated below each lane. Forskolin increased the yield of crosslink product eight-fold for BMOE and two-fold for BMH. Download figure Download PowerPoint Crosslinking tests We assessed crosslinking of target cysteines by exposing intact oocytes, both at rest and after PKA stimulation by forskolin plus IBMX, to a membrane-permeant bismaleimide-based crosslinking reagent, bismaleimidoethane (BMOE) or bismaleimidohexane (BMH), and then analyzing membrane proteins by SDS-PAGE. In control tests, oocytes expressing split CFTR channels containing only a single target cysteine were exposed to BMOE or BMH under optimal conditions (see below). Western blots with an anti-R-domain antibody identified both core-glycosylated (∼85 kDa, poorly phosphorylated; ∼90 kDa after strong phosphorylation by PKA) and fully glycosylated (∼130 kDa) COOH-terminal half channels (Figure 5, top row), and blots of the same membranes with anti-NH2-terminal antibody showed the NH2-terminal half channels (∼55 kDa; Figure 5, bottom row). However, the blots show no BMOE- or BMH-induced bands that are detected with both antibodies and so provide no evidence for crosslinking between any introduced cysteine in NBD2 and a native cysteine in the NH2-terminal half channel. The lack of strong bands of higher molecular mass in either blot indicates that there was no efficient crosslinking of introduced target cysteines to each other, offering no convincing evidence for the presence of homodimeric half-channel complexes. Nonetheless, occasional faint BMOE- and/or BMH-induced bands are discernible in Figure 5 (and to varying extents also in Figures 6, 7, 8 and 9), usually confined to the blots with the anti-NH2-terminal antibody. These largely indistinct unidentified bands (arrows in Figure 5) likely reflect crosslinking of the more reactive NH2-terminal half channels (due to its nine native cysteines) to other proteins, possibly in oligomeric complexes. 'NBD2' composite site, with S549C and S1248C In contrast to these results with single introduced cysteines, BMOE (flexible spacer, reactive groups ⩽8 Å apart) or BMH (flexible spacer length, 16 Å) application to oocytes co-expressing CFTR half channels (1–633) S549C and (634–1480) 9CS+S1248C, with both target cysteines in the NBD2 composite catalytic site (Figure 3), yielded a clear crosslinked product (Figure 6, arrows labeled X-link) not seen without crosslinking reagent (lanes 1 and 9). The product band detected with antibody against the NH2-terminal half channel was of identical molecular mass to that identified with antibody against the COOH-terminal half channel, strongly suggesting that the two half channels were crosslinked. The crosslinked product migrated with an apparent molecular mass (∼250 kDa) slightly higher than that of linear, full-length, mature CFTR protein (∼200 kDa; Figure 2D). We estimated crosslink yield as the fractional intensity of crosslinked product signal relative to the sum of signal intensities of crosslinked product plus fully glycosylated, but non-crosslinked, monomer; strengthening of the crosslinked product signal was generally associated with a corresponding loss of signal in the fully glycosylated monomer band (Figures 6, 7, 8 and 9). The efficiency of crosslinking appeared similar for BMOE and BMH, and was somewhat higher when carried out at 23°C (lanes 2–5, 10–14) than on ice (lanes 6, 7, 14 and 15). Importantly, although crosslinking was observed in resting oocytes (lanes 2, 4, 10 and 12), after stimulation of PKA activity by preincubation of oocytes with 40 μM forskolin plus 1 mM IBMX (phosphodiesterase inhibitor, to sustain elevated [cAMP]), the yield of crosslinked product was increased several-fold (e.g., compare lane 10 with 11, 12 with 13). Preincubation of oocytes with forskolin and IBMX alone (without crosslinking reagent) yielded no crosslinked product bands (Supplementary Figure S3). Central region, with S605C and A1374C Similar results were obtained with the pair of cysteines introduced at positions 605 and 1374, predicted to lie near the center of the proposed NBD1–NBD2 interface. A crosslinked product with apparent molecular mass of ∼220 kDa was evident (Figure 7, arrows labeled X-link) in lanes from oocytes incubated with BMOE or BMH at room temperature or on ice (lanes 2–7 and 9–14), but not from untreated oocytes (lanes 1 and 8). The same molecular mass product band was detected with antibodies against epitopes in the NH2-terminal or the COOH-terminal half channel. PKA stimulation again resulted in a strong, several-fold, increase in crosslinking yield (compare lane 2 with 3, 4 with 5, 9 with 10, and 11 with 12). 'NBD1' composite site, with A462C and S1347C, S459C and V1379C, and S434C and D1336C At the NBD1 composite site, we first examined crosslinking between positions homologous to those tested successfully at the NBD2 composite site. However, crosslinking between position 462 in NBD1 Walker A and position 1347 in NBD2 LSHGH sequence was weak, although still apparently enhanced upon PKA stimulation (Figure 8A; compare lane 10 with 11, 12 with 13). Concerned that this weak crosslink signal might reflect steric hindrance by the ATP believed to remain bound at the NBD1 composite site for prolonged periods (Basso et al, 2003), we introduced two additional pairs of target cysteines near this site. One pair, at positions 459 in NBD1 and 1379 in NBD2, showed robust crosslinking with either BMOE or BMH (Figure 8B) and, once again, crosslinking yield was increased several-fold after stimulation of PKA with forskolin and IBMX (e.g., compare lane 2 with 3, or 9 with 10). Crosslinking was weaker, but still evident, when carried out on ice (lanes 6, 7, 13 and 14). The other cysteine pair, at positions 434 in NBD1 and 1336 in NBD2, yielded crosslinked product only with the longer crosslinker BMH (Figure 8C, lanes 5, 7, 12 and 14) but not with BMOE (lanes 3, 6, 10 and 13), in contrast to all other pairs described so far. That crosslink was obtained both at room temperature and on ice and, like other crosslinks, was enhanced upon stimulation of PKA activity. In addition to these specific crosslink products recognized by antibodies to both half channels, Figure 8 shows other BMOE- and/or BMH-induced bands, predominantly in the blots with anti-NH2-terminal antibody. Because some migrate at a molecular mass overlapping that of fully glycosylated COOH-terminal half channels, we cannot discern whether these reflect crosslinked complexes of NH2-terminal half channels with COOH-terminal halves (complexes somehow truncated, or core-glycosylated) or with unidentified proteins. Tests with other combinations of NBD1 and NBD2 introduced cysteines To test the specificity of the interfacial crosslinks established in Figures 6, 7 and 9, we co-expressed six different pairs of the same target cysteines, again one in NBD1 and one in NBD2 in split CFTR channels, but now in new combinations. For five of these new pairs, C434 with C1374, C459 with C1248, C605 with C1336, C549 with C1336, and C549 with C1379, no crosslinked p
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