May 18, 1994 - FREDERIC BECQ*, TIMOTHY J. JENSENt, XIu-BAO CHANGt, ... LAP-CHEE Tsuit§, MANUEL BUCHWALD*§, JOHN R. RIORDANt¶, AND JOHN W. HANRAHAN*II ...... Farley, J. R., Ivey, J. L. & Baylink, D. J. (1980)J. Biol.
Proc. Natl. Acad. Sci. USA Vol. 91, pp. 9160-9164, September 1994
Physiology
Phosphatase inhibitors activate normal and defective CFTR chloride channels (rundown/cystic fibrosis transmembrane conductance regulator)
FREDERIC BECQ*, TIMOTHY J. JENSENt, XIu-BAO CHANGt, ANNA SAVOIAt, JOHANNA M. ROMMENSt§, LAP-CHEE Tsuit§, MANUEL BUCHWALD*§, JOHN R. RIORDANt¶, AND JOHN W. HANRAHAN*II *Department of Physiology, McGill University, 3655 Drummond Street, Montr6al, PQ Canada H3G 1Y6; Departments of tBiochemistry and *Genetics, Research Institute, Hospital for Sick Children, and Departments of WMolecular and Medical Genetics and Clinical Biochemistry, University of Toronto, Toronto, ON Canada M5G 1X8
Communicated by Robert Berliner, May 18, 1994
using membrane-associated phosphatase activity as a pharmacologic target for the treatment of CF (19).
The cystic fibrosis transmembrane conducABSTRACT tance regulator (CFTR) chloride channel is regulated by phosphorylation and dephosphorylation at multiple sites. Although activation by protein kinases has been studied in some detail, the dephosphorylation step has received little attention. This report examines the ehanisms responsible for the dephosphorylation and spontes deactivation ("rundown") of CFTR chloride channels excised from transfected Chinese hamster ovary (CHO) and human airway epithelial cells. We report that the alkali phosphatase inhibitors bromotetramisole, 3-isobutyl-1-methylxanthine, theophyfline, and vanadate slow the rundown of CFTR channel activity in excised membrane patches and reduce dephosphorylation of CFTR protein in isolated membranes. It was also found that in unstimulated cells, CFTR chanels can be activated by exposure to phosphatase inhibitors alone. Most importantly, exposure of mammalian cells to phosphatase inhibitors alone activates CFTR channels that have disease-causing mutations, provided the mutant channels are present in the plasma membrane (R117H, G551D, and AF508 after cooling). These results suggest that CFTR dephosphorylation is dynamic and that membraeassociated phosphatase activity may be a potential therapeutic target for the treatment of cystic fibrosis.
MATERIALS AND METHODS Patch-Clamp Experiments. CHO cells (6, 16) and human airway epithelial cells stably expressing wild-type CFTR were used. Simian virus 40-transformed human airway epithelial cells (NP34) were infected with supernatant from PA317 cells to produce an amphotrophic retrovirus containing the full-length CFTR cDNA. After selection in medium with G418, individual colonies were isolated and CFTR expression was confirmed by Northern blotting (A.S. and M.B., unpublished work). Single-channel currents were recorded from both cell-attached and excised patches, as described (6). The pipette solution contained 150 mM NaCl, 2mM MgCl2, and 10 mM Tes (pH 7.4); the bath contained 145 mM NaCl, 4 mM KCl, 2 mM MgCl2, and 10 mM Tes (pH 7.4). Throughout the study the pipette puller was set at similar parameters to minimize variations in patch area. Cells were stimulated with 15 ,uM forskolin [dissolved at 15 mM in dimethyl sulfoxide (DMSO); final DMSO concentration, 0.1%] during cell-attached experiments. Channels were activated in inside-out patches by exposure to 1 mM MgATP and 180 nM PKA catalytic subunit. Channels that had been activated on-cell were rapidly excised for studies of rundown. The duration of channel activity was measured as the time between excision and the initial loss of channel activity-i.e., when the patch became quiescent for >10 sec. Open probability (PO) was calculated for 10-sec intervals during recordings that lasted .600 sec, as described (6). Experiments were carried out at room temperature. Data are presented as the mean ± SEM. Statistical significance was assessed at the 95% confidence level with Student's t test. Pharmacological Agents. 1,3-Dimethylxanthine (theophylline), 3-isobutyl-1-methylxanthine (IBMX), 1,3,7-trimethylxanthine (caffeine), sodium metavanadate (NaVO3), sodium fluoride, and dipyridamole were obtained from Sigma and used at 1 mM final concentration; forskolin (15 ,uM) was from Calbiochem; 9,10-deepithio-9,10-didehydroascanthifolocin (okadaic acid, 10 p;M) was from Research Biochemicals; 6-(4-bromophenyl)-2,3,5,6-tetrahydroimidazo[2,1-b]thiazole ethanedioate [(-)-p-bromotetramisole, active isomer; (+)-pbromotetramisole, inactive isomer) were from Aldrich Chem (Metuchen, NJ) and were used at 1 mM final concentration. Type II bovine cardiac PKA catalytic subunit (prepared in the laboratory of M. P. Walsh) was described previously (6).
Cystic fibrosis (CF) is a recessive disorder characterized by defective electrolyte transport in epithelial cells of the lungs, pancreas, and other organs. It is caused by alterations in the cystic fibrosis transmembrane conductance regulator (CFTR; refs. 1-3), a low-conductance chloride channel regulated by phosphorylation (4-12) and ATP (4, 13). Much attention has been focused on the control of CFTR by protein kinases (14-16), but there is some evidence that its activity also depends on membrane-associated phosphatases (6, 17). Activity of CFTR channels usually disappears within 2 min following excision of membrane patches from cAMPstimulated cells (6, 8, 10-12). This spontaneous loss of activity, or "rundown," is not strongly affected by MgATP alone (11) but is prevented when protein kinase A (PKA) and MgATP are provided (6). Rundown is probably not due to an irreversible loss of agents or enzymes critical for channel function, because phosphorylation restores channel activity (6, 11, 12). The rundown of CFTR is probably due to a membrane-bound phosphatase activity, which is not counteracted by cytosolic kinases in cell-free patches, as proposed previously for calcium channel rundown (18). We have used transfected Chinese hamster ovary (CHO) and human airway epithelial cells expressing CFTR to determine the molecular mechanisms underlying rundown of CFTR chloride channels and to investigate the potential of
Abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; IBMX, 3-isobutyl-1-methylxanthine; PKA, protein kinase A; PO, open probability. IlTo whom reprint requests should be addressed.
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1 A and B show the rundown of CFTR channels and PO after membrane patches were excised from forskolin-stimulated CHO cells. Rundown was independent of voltage. The mean duration of CFTlR activity in excised patches was 89 ± 10 sec for CHO cells (n = 13) and 84 ± 13 sec for airway cells (n = 8). The rundown reflected dephosphorylation, as it could be prevented by addition of PKA and MgATP (Fig. 1C). In the presence of MgATP (1 mM), the rundown was slowed to 131 + 11 sec (n = 9; Fig. 1F) and 150 ± 35 sec (n = 3, Fig. 1F) in CHO and airway cells, respectively, and was accelerated by addition of alkaline phosphatase plus ATP when compared with ATP alone. It was reversed by the addition of PKA in the presence of ATP (Fig. 1D). These results suggest that dephosphorylation by endogenous phosphatase is an important mechanism for controlling CFTR activity in both cell types. Phosphatase Inhibitors Slow the Rundown. Using the rundown of channel activity in excised patches as an assay of membrane-associated phosphatase activity, we examined the effects of several putative phosphatase inhibitors. Vanadate (1 mM), which inhibits many enzymes that catalyze phosphotransfer reactions, including phosphatases (22, 23), strongly suppressed rundown (Fig. 1F) in patches excised from CHO cells (237 ± 9 sec, n = 5) and airway cells (310 ± 33 sec, n = 3). Similarly, the potent alkaline phosphatase inhibitor (-)-p-bromotetramisole (1 mM) (24-27) slowed rundown to 361 ± 46 sec (n = 11) and 295 ± 52 (n = 3) in CHO and airway epithelial cells, respectively (Fig. 1F), whereas the inactive isomer (+)-p-bromotetramisole did not (data not shown). Surprisingly, the strongest inhibitor of rundown was IBMX (Fig. 1E), a widely used phosphodiesterase inhibitor which has many actions, including inhibition of some phosphatases (28-30). Channel activity was recorded routinely for >400 sec after excision with IBMX in the bath solution (CHO, 400 ± 35 sec, n = 7; airway, 460 ± 41 sec, n = 4; Fig. 1F), and this persistence of activity may have been due, in part, to phosphatase inhibition. Similar inhibition of rundown
Alkaline phosphatase from bovine intestinal mucosa was obtained from Sigma. In Vitro Phosphorylation and Dephosphorylation. Membrane vesicles were prepared by a procedure modified from Bell et al. (20). Briefly, cells were washed and lysed on ice with a hypotonic buffer, LB (10 mM KCl/1.5 mM MgCl2/10 mM Tris HCl, pH 7.4), scraped into 1 ml of LB plus protease inhibitors, and ruptured with a homogenizer. Cell nuclei and organelles were spun down at 4000 x g for 5 min at 40C; the supernatant was collected and spun again at high speed for 20 min to pellet the membranes. Membranes were suspended in phosphorylation buffer and a 10-gl sample was assayed for protein. For in vitro phosphorylation, 50 pg of the fresh membrane sample was exposed to 10 gCi (370 kBq) of [y-32P]ATP (Amersham) and 50 units of PKA (Promega) for 15 min at 20°C. The final reaction volume (100 A4) also contained 10 pg of bovine serum albumin and 20 /uM nonradioactive ATP, with or without putative phosphatase inhibitors. The reaction was stopped by adding 0.9 ml of RIPA buffer [1% (wt/vol) deoxycholic acid/1% (vol/vol) Triton X-100/0.1% (wt/vol) SDS/150 mM NaCl/20 mM Tris'HCl, pH 7.4] with continued incubation for 15 min at 40C. Phosphorylated CFTR was immunoprecipitated by adding 1 pg of the monoclonal antibody M3A7 (21) with overnight incubation at 4°C. Prewashed protein G-Sepharose beads (25 jld) were added to the sample tubes for a 1-hr incubation at 4°C with shaking. Protein was eluted from the beads with Laemmli sample buffer at 20°C for 10 min. Sample eluates were run in Tris/glycine/7.5% polyacrylamide gels at 150 V. The gels were dried onto filter paper and autoradiographs were exposed for 18 hr.
RESULTS Rundown of CFTR Channel Activity in Excised Patches. We began by examining the properties of the rundown of CFTR chloride channels from CHO and airway epithelial cells. Fig. B +60n-1\,B
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FIG. 1. Effect of phosphatase inhibitors on the rundown of CFT'R channel activity in CHO and airway epithelial cells. (A) Channels were activated on a CHO cell by 15 ,uM forskolin and then excised at the arrow. (B) Time dependence of P. estimated for consecutive 10-sec sweeps. Excision was at time 0. (C and D) Channel activity immediately after patch excision in the presence of 1 mM ATP plus 180 nM PKA and ATP plus alkaline phosphatase (AP), respectively. In Fig. 1D, 180 nM PKA was added after rundown. (E) Effect of 1 mM IBMX on rundown. (F) Histogram summarizing effects of 1 mM ATP and phosphatase inhibitors [1 mM vanadate (VO43-), 1 mM IBMX, 1 mM (-)-p-bromotetramisole (Br-t) and 10 AM okadaic acid (okad)] on the duration of channel activity in patches excised from CHO and airway epithelial cells. Bars show means and SEM of 3-13 experiments.
9162
Physiology: Becq et A
was observed with theophylline (1 mM, n = 6), another xanthine derivative having phosphatase inhibitory properties (28-30), but not with caffeine (1 mM, n = 5) or dipyridamole (1 mM, n = 3), two structurally unrelated phosphodiesterase inhibitors that do not affect phosphatases in other systems (28-30). Okadaic acid (10 ,uM), which inhibits types 1 and 2A protein phosphatases (31), had no effect on rundown (Fig. IF). Although the rundown parameters observed for CHO and airway epithelial cells were very similar, we cannot exclude the possibility that there may be subtle differences in phosphatase regulation of CFTR expressed in these cells. Dynamic Control of Channel Activity by Phosphatase Inhibitors. If (-)-p-bromotetramisole, IBMX, and theophylline inhibit phosphatases as suggested from the rundown experiments, they should also shift the equilibrium between phosphorylation and dephosphorylation when excised patches are bathed with a phosphorylation mixture, and alter PO accordingly. This was indeed observed. (-)-p-bromotetramisole increased PO 2-fold and increased the apparent number of channels per patch (N) from 2.9 ± 0.04 (n = 12) to 7.5 ± 0.72 (n = 6) in the presence of PKA (180 nM) and MgATP (1 mM) (N was assessed as the maximum number of levels observed during long recordings). Similar changes were obtained with IBMX and theophylline, but not with okadaic acid (data not shown). Direct Measurement of CFTR Phosphorylation. To confirm that (-)-p-bromotetramisole and other inhibitors of rundown alter the phosphorylation of CFTR protein, we examined their effects on phosphorylation of CFTR in isolated CHO membranes under conditions that approximated those during the excised-patch recordings. Consistent with their inhibitory effects on channel rundown in membrane patches, IBMX, (-)-p-bromotetramisole, and vanadate all led to increases in CFTR labeling when isolated membranes were incubated in the presence of exogenous PKA catalytic subunit and ['y.32P]ATP, presumably by inhibition of endogenous phosphatase activity (Fig. 2 A and B). Okadaic acid (10 ILM), which did not affect rundown (Fig. iF), nevertheless led to increased CFTR labeling under these conditions (Fig. 2 A and B), implying that okadaic acid-sensitive dephosphorylation occurs at sites that are not essential for rundown. Fig. 2 C and D indicate that exogenous alkaline phosphatase removes a large fraction of the [32P]phosphate from immunoprecipitated CFTR. This is consistent with our previous studies showing that exogenous alkaline phosphatase could reverse PKAmediated activation of CFTR (6, 16). Dephosphorylation by exogenous alkaline phosphatase was at least partially blocked by the same inhibitors that reduced endogenous phosphatase activity (Fig. 2 C and D). As expected, okadaic acid did not influence the action of exogenous alkaline phosphatase, although some radiolabeled phosphate was removed by exogenous protein phosphatase 2A (Fig. 2 C and D), as reported previously (32). Phosphatase Inhibitors Activate CFTR Channels On Cells. The action of phosphatase inhibitors on CFTR channels was also investigated in resting (unstimulated) cells, because some phosphorylation of CFTR has been reported under these conditions (14-16), presumably due to basal activity of PKA or some other kinase. Addition of 1 mM (-)-pbromotetramisole activated CFTlR channel activity on airway epithelial cells (Fig. 3A). The mean number of active channels in each patch increased from 0 to 4.0 ± 0.7 (n = 11) and 3.2 + 1 (n = 5) on CHO and airway epithelial cells, respectively (Fig. 3E). By comparison, the number of CFTR channels on CHO and airway epithelial cells activated by 15 AM forskolin was 9.3 ± 1.6 (n = 13) and 5.5 ± 1.1 (n = 9), respectively (Fig. 3E). The intracellular concentration of cAMP in CHO cells (evaluated with a cAMP enzyme immunoassay kit from Sigma) did not increase in the presence of 1 mM (-)-pbromotetramisole (5.3 ± 0.8 pmol per well, n = 3) compared
Proc. Natl. Acad. Sci. USA 91 (1994)
with control (6.4 + 0.1 pmol per well, n = 3) or forskolin (66.6 ± 10 pmol per well, n = 3). During parallel studies of phosphorylation in cultured cells, exposure to (-)-pbromotetramisole, IBMX, or vanadate increased CFTR phosphorylation to levels that were somewhat lower than those observed during forskolin stimulation, consistent with the functional response. The (+)-p-bromotetramisole isomer, which does not inhibit alkaline phosphatase (24-26), did not activate CFTR channels under these conditions. Phosphatase Inhibitors Activate Mutated CFTR Channels On Cells. The suitability of using phosphatase inhibitors as therapeutic targets was examined by using CFTR channels that have disease-causing mutations. We characterized the responses of two CFTR mutants that are processed similarly to wild type and are delivered to the plasma membrane; R117H, which causes a relatively mild (pancreatic-sufficient) form of CF (33, 34), and G551D, which does not respond to cAMP and causes severe disease (35, 36). Both R117H and G551D channels were activated on CHO cells by exposure to (-)-p-bromotetramisole (PO = 0.14 ± 0.1, n = 5, and 0.35 ± 0.07, n = 4, respectively) in the absence of cAMP agonists (Fig. 3 B-E), even though forskolin alone did not open G551D channels in 14 of 14 trials (Fig. 3 C and E). (-)-p-Bromotetramisole also slowed the rundown of R117H and G551D in excised patches, as was observed with wild-type channels. The mean number of mutant channels per patch that were activated in the presence of (-)-p-bromotetramisole (R117H, 2.0 ± 0.37, n = 7; G551D, 3.0 + 0.27, n = 31) was comparable to that determined for cells expressing wild-type CFTR (CHO, 4.0 ± 0.7, n = 11; airway; 3.2 ± 1.0, n = 5). Single-channel conductances of R117H (5.7 ± 0.15 pS, n = 7) and GS51D (5.3 ± 0.37 pS, n = 10) channels were decreased slightly (Fig. 3D) compared with wild-type channels (6.8 ± 0.21 pS, n = 6, P < 0.05). IBMX (1 mM) stimulated these mutants (Fig. 3E) and also activated AFS08 channels on CHO cells (unitary conductance, 6.8 ± 0.14 pS, n = 3) when the cells had been maintained at 28°C for 48 hr to allow some channels to reach the plasma membrane (37).
DISCUSSION An Endogenous Phosphatase Activity Contributes to the Rundown of CFTR Channels. Our working hypothesis was
that rundown mainly reflects the tonic activity of a membrane-bound phosphatase that normally dephosphorylates CFTR channels on cells. In support of this, rundown of CFTR channel activity sometimes develops slowly on cells (8, 10) but occurs consistently and rapidly when membrane patches are excised from most cell types into bath solution lacking PKA (6, 8-12). The data show that rundown of CFTR channel activity is significantly slowed when phosphatase inhibitors are present in the bath, consistent with regulation by an endogenous, membrane-associated phosphatase activity. It is conceivable that this phosphatase activity, which is functionally linked to CFTR, may also be physically associated with the channel protein in situ. Phosphatase Control of the Phosphorylation State and Channel Activity of CFIR. The 32p labeling data in Fig. 2 provide direct evidence that inhibitors of rundown increase the phosphorylation of CFTR. Previous studies have yielded differing views concerning phosphatase regulation of CFTR. Anderson et al. (4) did not observe rundown in mouse NIH 3T3 fibroblasts and human HeLa cells, and Berger et al. (32) have suggested that the main phosphatase acting on CFTR could be protein phosphatase 2A but not alkaline phosphatase (32). Our results on CHO and airway cells clearly show rundown in excised patches and an effect of bromotetramisole, IBMX, theophylline, and vanadate on CFTR channel activity, but no effect of okadaic acid, suggesting that endogenous protein phosphatase 2A does not mediate rundown. The reason for the discrepancy between laboratories is not known; it may
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FIG. 2. Phosphorylation and dephosphorylation of CFTR in CHO membranes in the presence or absence of agents that inhibit rundown. (A) Effect of phosphatase inhibitors on incorporation of [32P]phosphate into CFTR in the presence of PKA. (B) Densitometry histogram corresponding to lanes shown in A. (C) Effect of phosphatase inhibitors on dephosphorylation of immunoprecipitated CFTR by purified protein phosphatases. The lanes (left -s right) show radioactivity in the immunoprecipitate after (i) incubation with [y-32P]ATP (1 mM; 15 min) without PKA, (ii) 30-min incubation with [y-32P]ATP (1 mM; 15 min) and PKA catalytic subunit (175 nM), (iii) control 30-min incubation at 200C, (iv) 30-min exposure to calfintestinal alkaline phosphatase (AP, 100 units/ml), (v) 30 min in the presence of AP and 2 mM IBMX, (vi) 30-min exposure to AP and 20 mM NaF, (vii) 30-min exposure to AP and 2 mM ATP, (viii) 30-min exposure to AP plus 10 pM okadaic acid (Okad), (ix) 30-min exposure to protein phosphatase 2A (PP2A, 2.8 units/ml), and (x) 30-min exposure to both PP2A and Okad. (D) Densitometry analysis of lanes shown in C.
reflect the lower density of channels in the present study or the different cell type used. A phosphatase inhibitor profile similar to the one reported here has also been found in human pancreatic duct epithelial cells (17). Moreover, okadaic acid does not affect the downregulation of short-circuit current after T84 human colon cancer cells are stimulated with cAMP (unpublished observations). Contrary to a previous study (32), we have demonstrated that alkaline phosphatase indeed dephosphorylates CFrR, consistent with its effects on channel activity (6, 17). The ability of phosphatase inhibitors alone to activate CFTR channels was unanticipated, although there is at least one precedent for channel activation by phosphatase inhibition: exposure to okadaic acid alone activates potassium channels in tracheal smooth muscle (38). IBMX inhibition of membrane-associated phosphatases may also be partly responsible for the surprising ability of IBMX to activate CFTR mutants expressed in Xenopus oocytes (39), although IBMX has many other actions, including a direct stimulatory effect on CFTR (unpublished observations). Another xanthine derivative, 8-cyclopentyl-1,3-dipropylxanthine, has also been
reported to activate AF508 CFTR channels (40), but whether this involves a phosphatase is unknown. Kinase activation alone is not sufficient to induce maximal phosphorylation of CFTR or maximal channel activity; these require simultaneous inhibition of the endogenous CFTR-associated phosphatase activity. This has implications for patch-clamp studies and for phosphorylation experiments with cultured cells, since critical sites could escape detection if they are dephosphorylated rapidly during membrane isolation. If the membrane-associated phosphatase activity were regulated, it would provide more precise control over CFTR channel function in situ. Phosphatase Inhibitors and Disease. This study reports strong activation of the G551D mutant in mammalian cells; the inability of forskolin to open G551D channels was apparently bypassed by inhibition of endogenous phosphatase activity. Enhanced secretion may help explain the ability of levamisole, a compound related to bromotetramisole, to reduce the frequency, severity, and duration of upper respiratory tract infections in children (41-43). The activation of defective R117H and G551D channels by phosphatase inhib-
Physiology: Becq et al.
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FIG. 3. Activation of wild-type and mutated CFTR channels in cell-attached patches by phosphatase inhibitors alone. (A and B) Wild-type or R117H channel activity on cells incubated with (-)-pbromotetramisole (Br-t, 1 mM) or IBMX (1 mM), respectively. (C) Activation of G551D channels by 1 mM Br-t; note that forskolin alone did not induce channel activity. (D) Current-voltage relationships for wild-type (WT) and mutant channels activated by Br-t on the cells. (E) Number of CF1TR channels activated on cells by forskolin (15
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itors also suggests that these phosphatases may serve as a useful target for drugs designed to alleviate symptoms in individuals with mutations that permit channels to reach the plasma membrane ("class II" mutations; ref. 33). Phosphatase inhibitors could also be used to improve the efficacy of future gene and other therapies for the treatment of CF. We thank A. Boucher, J. Eng, and J. A. Tabcharani for technical assistance and Dr. M. Gola for helpful discussions. This work was supported by a Canadian Cystic Fibrosis Foundation Studentship to F.B., Canadian Cystic Fibrosis Foundation postdoctoral fellowships to X.-B.C. and A.S., a research scholar award from the Fonds de la Recherche en Sante du Quebec to J.W.H., and grants from the Canadian Cystic Fibrosis Foundation, the U.S. Cystic Fibrosis Foundation, Association Frangaise de Lutte Contre la Mucoviscidose, and the Respiratory Health Network of Centres of Excellence. Rommens, J. M., lannuzzi, M. C., Kerem, B.-S., Drumm, M. L., Melmer, G., Dean, M., Rozmahel, R., Cole, J. L., Kennedy, D., Hidaka, M., Zsiga, M., Buchwald, M., Riordan, J. R., Tsui, L.-C. & Collins, F. S. (1989) Science 245, 1059-1065. 2. Riordan, J. R., Rommens, J. M., Kerem, B.-S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M. L., Iannuzzi, M. C., Collins, F. S. & Tsui, L.-C. (1989) Science 245, 1066-1073. 3. Kerem, B.-S., Rommens, J. M., Buchanan, J. A., Markiewicz, D.,
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40. 41. 42.
43.
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