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• W1977946638 abstract "8-Cyclopentyl-1,3-dipropylxanthine (CPX) and 1,3-diallyl-8-cyclohexylxanthine (DAX) are xanthine adenosine antagonists which activate chloride efflux from cells expressing either wild-type or mutant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR). These drugs are active in extremely low concentrations, suggesting their possible therapeutic uses in treating cystic fibrosis. However, knowledge of the mechanism of action of these compounds is lacking. We report here that the same low concentrations of both CPX and DAX which activate chloride currents from cells also generate a profound activation of CFTR channels incorporated into planar lipid bilayers. The process of activation involves a pronounced increase in the total conductive time of the incorporated CFTR channels. The mechanism involves an increase in the frequency and duration of channel opening events. Thus, activation by these drugs of chloride efflux in cells very likely involves direct interaction of the drugs with the CFTR protein. We anticipate that this new information will contribute fundamentally to the rational development of these and related compounds for cystic fibrosis therapy. 8-Cyclopentyl-1,3-dipropylxanthine (CPX) and 1,3-diallyl-8-cyclohexylxanthine (DAX) are xanthine adenosine antagonists which activate chloride efflux from cells expressing either wild-type or mutant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR). These drugs are active in extremely low concentrations, suggesting their possible therapeutic uses in treating cystic fibrosis. However, knowledge of the mechanism of action of these compounds is lacking. We report here that the same low concentrations of both CPX and DAX which activate chloride currents from cells also generate a profound activation of CFTR channels incorporated into planar lipid bilayers. The process of activation involves a pronounced increase in the total conductive time of the incorporated CFTR channels. The mechanism involves an increase in the frequency and duration of channel opening events. Thus, activation by these drugs of chloride efflux in cells very likely involves direct interaction of the drugs with the CFTR protein. We anticipate that this new information will contribute fundamentally to the rational development of these and related compounds for cystic fibrosis therapy. Cystic fibrosis (CF) 1The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; CPX, 8-cyclopentyl-1,3-dipropylxanthine; DAX, 1,3-diallyl-8-cyclohexylxanthine; PKA, protein kinase A; DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; NBF, nucleotide-binding fold. 1The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; CPX, 8-cyclopentyl-1,3-dipropylxanthine; DAX, 1,3-diallyl-8-cyclohexylxanthine; PKA, protein kinase A; DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; NBF, nucleotide-binding fold. is the most common, fatal, autosomal recessive disease in the United States, affecting nearly one birth in 2000 (1Boat T.F. Welsh M.J. Beaudet A.L. Scrivier C.L. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. 6th Ed. McGraw-Hill, New York1989: 2649-2680Google Scholar, 2Welsh M.J. Tsui L.C. Boat T.F. Beaudet A.L. Scriver C.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Diseases. 7th Ed. McGraw-Hill, NY1995: 3799-3876Google Scholar). The responsible gene has been identified as CFTR (cystic fibrosistransmembrane conductance regulator), and the vast majority of CF patients carry a deletion of phenylalanine from position 508 (ΔF508; Refs. 3Riordan J.R. Rommens J.M. Karem 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. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5935) Google Scholar, 4Rommens J.M. Iannuzzi M.C. Karem B-S. Drumm M.L. Melmer G. Dean M. Rozmahel J.M. Cole J.L. Kennedy D. Hidaka N. Zsiga M. Buchwald M. Riordan J.R. Tsui L-C. Collins F.S. Science. 1989; 245: 1059-1065Crossref PubMed Scopus (2533) Google Scholar, 5Kerem B. Rommens J.M. Buchanan J.A. Markiewicz D. Cox T.K. Chakravarti A. Buchwald M. Tsui L-C. Science. 1989; 245: 1073-1080Crossref PubMed Scopus (3220) Google Scholar). The physiological function of CFTR includes cAMP-activated chloride channel activity (6Anderson M.P. Rich D.P. Gregory R.J. Smith A.C. Welsh M.J. Science. 1991; 251: 679-682Crossref PubMed Scopus (432) Google Scholar, 7Rommens J.M. Dho S. Bear C.E. Kartner N. Kennedy D. Riordan J.R. Tsui L-C. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7500-7504Crossref PubMed Scopus (80) Google Scholar, 8Li M. McCann J.D. Liedke C.M. Nairn A.C. Geeengard P. Welsh M.J. Nature. 1988; 331: 358-360Crossref PubMed Scopus (261) Google Scholar, 9Hwang T-C. Lu L. Zeitlin P.L. Gruenert D.C. Haganir R. Guggino W.B. Science. 1989; 244: 1351-1353Crossref PubMed Scopus (162) Google Scholar), and the mutation compromises the ability of CFTR to traffic out of the endoplasmic reticulum to its appropriate location on the apical plasma membrane (10Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. O'Riordan G.A. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1423) Google Scholar). Repair of the mutation can be effected by transferring wild-type CFTR into the mutant cell (11Drumm M. Pope H.A. Cliff W.H. Rommens J.M. Marvin S.A. Tsui L-C. Collins F.S. Wilson J.M. Cell. 1990; 62: 1227-1233Abstract Full Text PDF PubMed Scopus (490) Google Scholar), and the long term strategy for treatment of cystic fibrosis has thus become focused on gene therapy at the level of the whole organism. However, it has also been observed that the ΔF508 CFTR does have intrinsic channel activity (12Drumm M.L. Wilkinson D.J. Smit L.S. Worrell R.T. Strong T.V. Frizzell R.A. Dawson D.C. Collins F.S. Science. 1991; 254: 1797-1799Crossref PubMed Scopus (421) Google Scholar, 13Pasyk E.A. Foskett J.K. J. Biol. Chem. 1995; 270: 12347-12350Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Furthermore, incubation of some mutant cells at low temperature (14Denning G.M. Anderson M.P. Amara J.F. Marshall J. Smith A.E. Welsh M.J. Nature. 1992; 358: 761-764Crossref PubMed Scopus (1061) Google Scholar), or in chemical chaperones such as 1 mglycerol (15Sato S. Ward C.L. Krouse M.E. Wine J.J. Kopito R.R. J. Biol. Chem. 1996; 271: 635-638Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar), do permit the mutant CFTR to traffic out of the endoplasmic reticulum to the plasma membrane. Once at the membrane, the mutant CFTR exhibits cAMP-activated chloride channel activity, thereby permitting functional repair. These data have thus been interpreted to indicate that the ΔF508-CFTR is intrinsically active, and more recent data have shown that in some cells a small but measurable portion of the mutant CFTR actually spontaneously traffics to the vicinity of the plasma membrane (16Dalemanns W. Barby P. Champigny G. Jallet S. Dott K. Dreyer S.D. Crystal R.G. Pavirani A. Lecocq J-P. Lazdunski M.J. Nature. 1991; 354: 526-528Crossref PubMed Scopus (569) Google Scholar, 17Haws C.M. Nepomuceno I. Krouse M.E. Wakelee H. Law T. Xia Y. Nguyen H. Wine J.J. Am. J. Physiol. 1996; 270: 1544-1555Crossref PubMed Google Scholar). These data therefore suggest that an alternative way to overcome the reduced chloride transport in CF cells might be to find compounds which further activate the small portion of mutant CFTR chloride channels which have trafficked to the cell membrane. The adenosine A1-receptor antagonist CPX (8-cyclopentyl-1,3- dipropylxanthine) has been shown to stimulate36[Cl−] efflux from pancreatic CFPAC-1 cells (18Eidelman O. Guay-Broder C. van Galen P.J.M. Jacobson K.A. Fox C. Turner R.J. Cabantchik Z.I. Pollard H.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5562-5566Crossref PubMed Scopus (91) Google Scholar) in the concentration range of 20–100 nm. These cells are homozygous for the ΔF508 genotype common to most cases of cystic fibrosis. Similar results were obtained with the CF tracheal epithelial cell line IB3–1 expressing the ΔF508 allele, and with recombinant mouse fibroblast NIH 3T3 cells (19Guay-Broder C. Jacobson K.A. BarNoy S. Cabantchik Z.I. Guggino W.B. Zeitlin P.L. Turner R.J. Vergara L. Eidelman O. Pollard H.B. Biochemistry. 1995; 34: 9079-9087Crossref PubMed Scopus (54) Google Scholar). Schweibert et al. (20Schweibert E. Gruenert D. Stanton B. Pediatric Pulm. 1992; S8: 257Google Scholar) have also shown that CPX (50 nm) activates outward chloride currents in whole cell patch studies of primary explants of nasal epithelial cells from homozygous ΔF508 CF patients as well as wild-type CFTR control cells. More recently, Haws et al.(17Haws C.M. Nepomuceno I. Krouse M.E. Wakelee H. Law T. Xia Y. Nguyen H. Wine J.J. Am. J. Physiol. 1996; 270: 1544-1555Crossref PubMed Google Scholar) showed that CPX could activate iodide efflux from recombinant cells expressing ΔF508 CFTR. Thus wherever the CFTR mutant has been expressed, or in some favorable cases the wild-type CFTR, an effect of CPX on chloride efflux can be demonstrated. In considering how CPX activates wild-type CFTR or repairs mutant CFTR, it is possible that CPX action might either be directly on the CFTR molecule, or be secondary to binding of CPX to another protein (21Casavola V. Turner R.J. Guay-Broder C. Jacobson K.A. Eidelman O. Pollard H.B. Am. J. Physiol. 1995; 269: C226-C233Crossref PubMed Google Scholar). As indicated above, CPX is best known as an adenosine A1-receptor antagonist (22Jacobson K.A. van Galen P.J.M. Williams M. J. Med. Chem. 1992; 35: 407-422Crossref PubMed Scopus (504) Google Scholar). However, Northern blot analysis and structure-activity relationship (“SAR”) studies with 26 different compounds indicate that the A1-receptor is not responsible for CPX action in CFPAC cells (23Jacobson K.A. Guay-Broder C. van Galen P.J.M. Gallo-Rodriguez C. Melman N.K.A. Jacobson M.A. Eidelman O. Pollard H.B. Biochemistry. 1995; 34: 9088-9094Crossref PubMed Scopus (37) Google Scholar). As one useful example, 1,3-diallyl-8-cyclohexylxanthine (DAX), a poor A1antagonist, is also highly potent and efficacious in stimulating chloride efflux from CFPAC-1 cells (23Jacobson K.A. Guay-Broder C. van Galen P.J.M. Gallo-Rodriguez C. Melman N.K.A. Jacobson M.A. Eidelman O. Pollard H.B. Biochemistry. 1995; 34: 9088-9094Crossref PubMed Scopus (37) Google Scholar). Thus the site of action of these xanthines in stimulating chloride efflux appears to represent a novel site of action, possibly involving direct interaction with the CFTR molecule. Additional information consistent with this latter possibility are data indicating that radiolabeled CPX binds with high affinity to the recombinant first nucleotide-binding fold (NBF-1) of CFTR (24Cohen B.E. Lee G. Jacobson K.A. Kim Y-C. Huang Z. Sorscher E.J. Pollard H.B. Biochemistry. 1997; 36: 6455-6461Crossref PubMed Scopus (66) Google Scholar) and to a subdomain within NBF-1 (25Lee G. Cohen B.E. BarNoy S. Eidelman O. Jaobson K.A. Pollard H.B. Pediatr. Pulm. 1995; S12: 185Google Scholar). To test this hypothesis directly at the single channel level, we have incorporated recombinant wild-type CFTR channels from HEK293 cell microsomal membranes into planar lipid bilayers, and tested whether CPX could activate chloride currents through CFTR channels. In addition, we tested whether DAX, another active CPX analogue, could also activate chloride currents through CFTR channels. We report here that both CPX and DAX potently activate cAMP-dependent CFTR chloride channels. Furthermore, both compounds affect CFTR channel kinetics differently, and in a manner predictable from their respective actions on chloride efflux from different cell types. Furthermore both CPX and DAX also bind with high affinity to the recombinant first nucleotide-binding fold domain (NBF-1) of CFTR (24Cohen B.E. Lee G. Jacobson K.A. Kim Y-C. Huang Z. Sorscher E.J. Pollard H.B. Biochemistry. 1997; 36: 6455-6461Crossref PubMed Scopus (66) Google Scholar). These data together indicate that the mechanism of CFTR channel activation very likely involves direct interaction of the drugs with the CFTR protein. We anticipate that this new information will contribute fundamentally to the rational development of these and related compounds for cystic fibrosis therapy. Wild-type CFTR cDNA was subcloned into the eukaryotic expression vector pCEP4 (Invitrogen) between the NheI and XhoI restriction sites to create the recombinant vector, pCEP4(CFTR) (11Drumm M. Pope H.A. Cliff W.H. Rommens J.M. Marvin S.A. Tsui L-C. Collins F.S. Wilson J.M. Cell. 1990; 62: 1227-1233Abstract Full Text PDF PubMed Scopus (490) Google Scholar). A human embryonic kidney cell line (HEK293-EBNA: Invitrogen) was used for the transfection and expression of CFTR protein (26Xie J. Drumm M.L. Ma J. Davis P.B. J. Biol. Chem. 1995; 270: 28084-28091Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 27Xie J. Drumm M.L. Zhao J. Ma J. Davis P.B. Biophysical J. 1996; 71: 3148-3156Abstract Full Text PDF PubMed Scopus (25) Google Scholar, 28Ma J. Tasch J.E. Tao T. Zhao J. Xie J. Drumm M.L. Davis P.B. J. Biol. Chem. 1996; 271: 7351-7356Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). This cell line contains the vector pCMV-EBNA which constitutively expresses the Epstein-Barr virus EBNA-1 gene product which increases the transfection efficiency of Epstein-Barr virus-based vectors. The parent cell line was maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% glutamine. Geneticin (G418, 250 μg/ml) was added to the cell culture media to maintain selection of the cells containing pCMV-EBNA vector until after CFTR gene transfer. pCEP4(CFTR) was then introduced to the cell using Lipofectin reagent (Life Technologies), and 2 days after transfection, the cells were passaged and selected for hygromycin resistance (hygromycin B, 260 μg/ml). Three weeks after transfection, microsomal vesicles were isolated from transfected cells. The expression of CFTR protein was conformed by Western blot using an antibody against the R domain of CFTR (mAb 13-1, Genzyme; Ref. 28Ma J. Tasch J.E. Tao T. Zhao J. Xie J. Drumm M.L. Davis P.B. J. Biol. Chem. 1996; 271: 7351-7356Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Microsomal vesicles were isolated from HEK 293 cells expressing wild-type CFTR protein using a modified protocol of Gunderson and Kopito (29Gunderson K.L. Kopito R.R. J. Biol. Chem. 1994; 269: 19349-19353Abstract Full Text PDF PubMed Google Scholar), as described previously (26Xie J. Drumm M.L. Ma J. Davis P.B. J. Biol. Chem. 1995; 270: 28084-28091Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 30Tao T. Xie J. Drumm M.L. Zhao J. Davis P.B. Ma J. Biophys. J. 1996; 70: 743-753Abstract Full Text PDF PubMed Scopus (33) Google Scholar). Briefly, 12 × 75-cm2 flasks of HEK 293 cells transfected with pCEP4(CFTR) vectors were harvested. The cell pellet was resuspended in ice-cold hypotonic lysis buffer (10 mm HEPES/NaOH, pH 7.2, 1 mm EDTA, 5 μm diisopropyl fluorophosphate, 10 μg/ml pepstatin A, 10 μg/ml aprotinin, and 10 mg/ml benzamidine) before lysis by 10 strokes in a tight-fitting Dounce glass homogenizer, followed by 15 strokes after the addition of an equal volume of sucrose buffer (500 mm sucrose, 10 mm HEPES/NaOH, pH 7.2). Microsomes were collected by centrifugation of a postnuclear supernatant (600 × g for 15 min) at 100,000 ×g for 45 min, and resuspended in 1 ml of prephosphorylation buffer (250 mm sucrose, 10 mm HEPES/NaOH, pH 7.2, 5 mm Mg-ATP, and 100 units/ml PKA catalytic subunit). The membrane vesicles were stored at a protein concentration of 2–6 mg/ml at −75 °C until use. CPX was synthesized according to GMP (“Good Manufacturing Practice”) as part of our program for preparing CPX for clinical trials on cystic fibrosis patients. The CPX was solubilized at a concentration of 10 mm in dimethyl sulfoxide, and further diluted in dimethyl sulfoxide prior to dilution into the chamber solution. Prior to recording the effects of the drug, the contents of the chamber were mixed for 30 s using an internal mixing apparatus. The final concentration of dimethyl sulfoxide never exceeded 1%, and this amount of dimethyl sulfoxide alone was found to be entirely inactive, either on the naked bilayer or on incorporated CFTR channels. DAX was synthesized as described previously (23Jacobson K.A. Guay-Broder C. van Galen P.J.M. Gallo-Rodriguez C. Melman N.K.A. Jacobson M.A. Eidelman O. Pollard H.B. Biochemistry. 1995; 34: 9088-9094Crossref PubMed Scopus (37) Google Scholar). Planar bilayers were formed by applying a suspension of palmitoyloleoyl phosphatidylethanolamine and palmitoyloleoyl phosphatidylserine, 1:1, 50 mg/ml, each in n-decane, to a hole of about 100–120 μm in diameter in a thin TeflonTM film separating two compartments that contained defined salt solutions (31Arispe N. Rojas E. Hartman J. Sorscher E.J. Pollard H.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1539-1543Crossref PubMed Scopus (48) Google Scholar). Channels were incorporated from a suspension of microsomes prepared from HEK293 cells expressing wild-type CFTR. The microsomes were added to the cis chamber in small aliquots, and incorporation occurred directly from the experimental solutions. Currents associated with CFTR channels were observed shortly after the bilayer system was exposed to protein kinase A (100 units/ml). The specific conditions include a KCl gradient (cis = 200 mm; trans = 50 mm), (1 mm) MgCl2, and ATP (2 mm) in cis, and 10 mm Tris/HEPES in cis and trans, adjusted to a final pH of 7.0. Single channel currents were recorded using a patch-clamp amplifier (AXOPATCH-1D equipped with a CV-4B 0.1–100 Bilayer headstage, Axon Instruments, Foster City, CA), and data were stored on magnetic tape using a pulse-code modulation/video cassette recorder digital system (Toshiba) with a frequency response in the range from direct current to 25,000 Hz. Off-line analysis of the recorded CFTR channel activity was carried out using the software package pClamp 5.51 and 6. (Axon Instruments, Foster City, CA). Data base files were obtained from playbacks of the experimental records digitized using a 12-bit analog to digital converter (TL-1 DMA interface, Axon Instruments) using the fetchex subroutine. The channel current signal from the PCM-VCR was fed through a low pass filter (eight-pole Bessel 902 LPF; Frequency Devices Inc., Haverhill, MA) in series with the ADC module. The filtering level was set between 50 and 100 Hz. As a rule the data base used for open time and close time distribution corresponded to filtered recordings with a signal to noise ratio of ≥4:1. HEK293 cells expressing a permanently transfected CFTR gene were grown and microsomes were prepared by differential centrifugation (30Tao T. Xie J. Drumm M.L. Zhao J. Davis P.B. Ma J. Biophys. J. 1996; 70: 743-753Abstract Full Text PDF PubMed Scopus (33) Google Scholar). Microsomal membrane vesicles carrying the expressed protein were then incorporated into a planar lipid bilayer, and ionic currents measured in the presence of a chemical and potential gradient. Currents associated with CFTR channels were observed shortly after the bilayer system was exposed to protein kinase A (100 units/ml). The specific ionic conditions, as described under “Materials and Methods,” were maintained throughout all the experiments described in this work. As previously reported, these channels are insensitive to DIDS (50 μm), blocked by diphenylamine-2-carboxylate (300 μm), and are selective for chloride (26Xie J. Drumm M.L. Ma J. Davis P.B. J. Biol. Chem. 1995; 270: 28084-28091Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 27Xie J. Drumm M.L. Zhao J. Ma J. Davis P.B. Biophysical J. 1996; 71: 3148-3156Abstract Full Text PDF PubMed Scopus (25) Google Scholar, 30Tao T. Xie J. Drumm M.L. Zhao J. Davis P.B. Ma J. Biophys. J. 1996; 70: 743-753Abstract Full Text PDF PubMed Scopus (33) Google Scholar). Fig. 1 A, illustrates typical current events observed upon application of electrical potentials (−30, −50, or −80 mV) to the cis compartment. These events correspond to conductances of 7–10 pS, and additional current levels, double that of the first, are occasionally observed. These data indicate that more than one channel of the same type has been incorporated in the bilayer. In addition, we observe subconductances of about 2.5 pS, as described previously for CFTR channels (30Tao T. Xie J. Drumm M.L. Zhao J. Davis P.B. Ma J. Biophys. J. 1996; 70: 743-753Abstract Full Text PDF PubMed Scopus (33) Google Scholar). Fig. 1 B shows the expression of several combinations of these large and small CFTR conductances. To better visualize the relationships a more expanded amplitude scale is employed for records taken at −80 mV. In Fig. 1 B, part a) multiple larger current events are shown. In Fig. 1 B, part b, isolated smaller current events (smaller conductance) are shown, and in Fig. 1 B, part c, mixtures of the larger and smaller current events are observed. The relationship between amplitude and voltage for this family of current events is shown in the appended I-V curve (Fig. 1 C). The values of current in the I-V curves are calculated as the arithmetic mean ± S.E. The larger slope conductance (solid circles) is 6.7 pS, with an equilibrium potential of −21.5 mV. The smaller slope conductance (solid squares) is 2.6 pS, with an equilibrium potential of −22 mV. Thus on the basis of blocker sensitivity, chloride selectivity, and conductance states these channels correspond to CFTR activity. CFTR was incorporated into the planar lipid bilayer, and the channel modestly activated by addition of PKA and ATP (see “Materials and Methods”), Fig. 2 A. These data represent the control condition, in which the dominant motif is relatively low CFTR channel activity at the conductance level of 8.3 pS, and generated by a −50 mV driving force potential. Fig. 2 B shows continuous recordings of CFTR channel activity, generated by the same driving force 5 min after 500 nm CPX was added to the cis side of the planar lipid bilayer system. The figure represents 2 min of continuous recording. The overall general activity is considerably increased, and the system exhibits multilevels of current that were not observed under control conditions. Upon elevation of the CPX concentration to 2 μm (see Fig. 2 C), the general pattern of activity is reduced relative to the maximum at 500 nm CPX. However, it remains higher than control. This type of “bell-shaped response” is precisely what would have been predicted from our previously published results with the effect of CPX on chloride efflux from CF cells (19Guay-Broder C. Jacobson K.A. BarNoy S. Cabantchik Z.I. Guggino W.B. Zeitlin P.L. Turner R.J. Vergara L. Eidelman O. Pollard H.B. Biochemistry. 1995; 34: 9079-9087Crossref PubMed Scopus (54) Google Scholar, 23Jacobson K.A. Guay-Broder C. van Galen P.J.M. Gallo-Rodriguez C. Melman N.K.A. Jacobson M.A. Eidelman O. Pollard H.B. Biochemistry. 1995; 34: 9088-9094Crossref PubMed Scopus (37) Google Scholar). A kinetic analysis of these data, shown below, indicates that the effect of CPX is to increase the number and duration of open events of CFTR channels without modifying their conductance and selectivity. The I-V relationship for CFTR channels under control and 500 nm CPX conditions are shown in Fig. 3 A. To prepare these data we plotted the arithmetic mean current amplitude of unitary events recorded at different electrical potentials (≥59 events in control conditions and ≥257 events in the presence of CPX, for each potential). Regression lines fit for the two conditions are statistically identical, indicating that the slope conductances (8.2 pS) and equilibrium potentials (12 mV) for ion fluxes are the same for both conditions. This indicates that the ionic selectivity and the unitary conductance of CFTR channels are not affected by the interaction with the drug. Rather, the increased current activity of the system must be due to a direct effect of CPX on the kinetics of the CFTR channel behavior. The action of CPX on the CFTR conductance can be most clearly visualized by construction of amplitude histograms from current records. Fig. 3 B, left panel, shows such an amplitude histogram from a control record summarizing the distribution of amplitudes at −50 mV. The Gaussian distribution of the mean amplitudes of these events gives an average amplitude of 0.48 pA (59 unitary events). In the presence of 500 nm CPX (Fig. 3 B, right panel) the amplitude histogram of all events occurring during 2 min recording reveals that the events are distributed in three levels. The principal Gaussian peak (number 1)(749 events) is very similar to that of the control in the absence of CPX. The additional peaks (number 2 (257 events) and number 3 (9 events)) are exact multiples of the principal peak. These data indicate that the CPX-induced increase in CFTR activity are likely to be additional CFTR channels. A second approach to the analysis of CPX action on the CFTR channel is to create a scatter plot of amplitudes as a function of duration of the events. The large solid dot symbols in Fig. 3 Care the values of current amplitudes at −50 mV under control conditions. The cross symbols are the values of current amplitude after the application of 500 nm CPX. These data indicate that whatever the duration of the event the mean amplitudes are the same, regardless of the presence or absence of CPX. Furthermore, the control condition and the lower level measured in the presence of CPX appear to be quite coincident. However, in the presence of CPX, a very well defined second level occurs at an amplitude which is twice the control. Furthermore, this level is constant regardless of the duration of the event. The conclusions from both analytic approaches similarly indicate that CPX increases the number of active CFTR channels. Data shown later (see Fig. 7) indicate that CPX also increases the open time probability. As mentioned above, the smaller 2.5 pS conductance associated with CFTR expression can, on occasion, be incorporated as a single channel in isolation from the larger 7–10 pS CFTR conductance. We therefore took advantage of several such instances to study the influence of CPX on the smaller conductance. Addition of CPX also increased the overall activity of the 2.5 pS conductance. For a better signal to noise ratio, we analyzed this increased activity at a large potential. As shown in Fig. 4 A, control conditions at a membrane potential of −100 mV (upper panel) are characterized predominantly by brief spikes, which under the 50 Hz filtering conditions give an apparent average open time of 8 ms and an apparent arithmetic mean amplitude of 0.08 pA. After the addition of 500 nm CPX the activity of the channel increases profoundly, with the apparent arithmetic mean amplitude increasing to 0.14 pA (Fig. 4 A, lower panel). The effect of CPX is to increase the duration of single events. Quantitatively, the open time probability increases from about 8% in control conditions to 35–38% in the presence of CPX. Since the control amplitude of 0.08 pA is the average spike amplitude observed under high filtering conditions (50 Hz), the apparent increase in current amplitude mediated by CPX may not be the real effect of the drug. This is because the amplitudes of longer events are less attenuated by the filtering condition. Thus, alternatively, the apparent increase in current amplitude could also be a consequence of the ability of CPX to prolong the open time of individual events. To test the latter hypothesis we plotted open time histograms of the 2.5 pS CFTR conductance for both control and 500 nm CPX conditions. Channel activity of the 2.5 pS conductance under control conditions is characterized by predominantly brief spikes with an average duration of 8 ms (see upper panel of Fig. 4 B). A minor peak is observed at approximately 24 ms. In the presence of CPX (see lower panel, Fig. 4 B) the increase in open time duration is not monotonic but is distributed into three main Gaussian populations, with peak durations of 9, 27, and 44 ms, respectively. However, we observe that the 27 ms population in the CPX treated condition is actually present to a very minor extent in the drug-free condition (viz. the 24 ms population in the control histogram in the upper panel of Fig. 4 B). These data therefore suggest that the CPX may activate or modify existing 2.5 pS CFTR channels, rather than merely recruit cryptic or new CFTR channels. We conclude that these data support the concept that addition of CPX not only increases the number of events, but also their duration. DAX is also a potent activator of chloride conductance in CF cells, much like CPX, which we hypothesized might also activate CFTR channels. To study this possibility we followed the same protocol used to study CPX. Therefore, prior to the addition of DAX we established an active CFTR channel using PKA/ATP (see “Materials and Methods”). We then added DAX at various concentrations and studied the channel activity at various membrane potentials. In all conditions, addition of DAX produced a profound increase in basal channel activity. Fig. 5 A summarizes one of those results for a driving force potential of −50 mV. The data representing the control condition show a dominant motif of relatively low activity at the level of 7–10 pS. The 2.5 pS conductance is absent from this example. DAX (500 nm) is then added to the cisside of the planar lipid bilayer system. As shown in Fig. 5 B, the number of channel events is considerably increased. By inspection, at least four different levels c" @default.
• W1977946638 created "2016-06-24" @default.
• W1977946638 creator A5003537359 @default.
• W1977946638 creator A5015264004 @default.
• W1977946638 creator A5034755455 @default.
• W1977946638 creator A5091587615 @default.
• W1977946638 date "1998-03-01" @default.
• W1977946638 modified "2023-10-04" @default.
• W1977946638 title "Direct Activation of Cystic Fibrosis Transmembrane Conductance Regulator Channels by 8-Cyclopentyl-1,3-dipropylxanthine (CPX) and 1,3-Diallyl-8-cyclohexylxanthine (DAX)" @default.
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