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J W Hanrahan

Publications and source records attributed to J W Hanrahan.

At least 19 recordsLinked to original sources
PubMed2003

Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA.

Activation of the cystic fibrosis transmembrane conductance regulator (CFTR) channel by protein kinase A (PKA) is enhanced by protein kinase C (PKC). However, the mechanism of modulation is not known and it remains uncertain whether PKC acts directly on CFTR or through phosphorylation of an ancillary protein. Using excised patches that had been pre-treated with phosphatases, we found that PKC exposure results in much larger PKA-activated currents and shifts the PKA concentration dependence. To examine if these effects are mediated by direct PKC phosphorylation of CFTR, a mutant was constructed in which serines or threonines at nine PKC consensus sequences on CFTR were replaced by alanines (i.e. the '9CA' mutant T582A/T604A/S641A/T682A/S686A/S707A/S790A/T791A/S809A). In excised patches, 9CA channels had greatly reduced responses to PKA (i.e. 5-10 % that of wild-type), which were not enhanced by PKC pre-treatment, although the mutant channels were still functional according to iodide efflux assays. Stimulation of iodide efflux by chlorophenylthio-cAMP (cpt-cAMP) was delayed in cells expressing 9CA channels, and a similar delay was observed when cells expressing wild-type CFTR were treated with the PKC inhibitor chelerythrine. This suggests that weak activation by PKA in excised patches and slow stimulation of iodide efflux from intact cells are specifically due to the loss of PKC phosphorylation. Finally, PKC caused a slight activation of wild-type channels when added to excised patches after phosphatase pre-treatment but had no effect on the mutant. We conclude that direct phosphorylation of CFTR at one or more of the nine sites mutated in 9CA is required for both the partial activation by PKC and for its modulation of CFTR responses to PKA.

Alkaloids↗
PubMed2002

Altered channel properties of porins from Haemophilus influenzae: isolates from cystic fibrosis patients.

Changes in amino-acid sequence of the unique pore-forming protein of H. influenzae (OmpP2; porin) have been associated with increased antimicrobial resistance in H. influenzae strains isolated from cystic fibrosis patients. From patients who were subjected to long-term antimicrobial therapy, H. influenzae strains 67d and 69a (patient 27) and strains 77a and 77f (patient 30) were isolated. Strains 67d and 77a were previously shown to have elevated values for minimal inhibitory concentrations of antibiotics compared to strains 69a and 77f. Porins were extracted from all four H. influenzae strains by detergent treatment and purified to homogeneity by ion exchange chromatography. By reconstitution of the clinical Hi porins into planar lipid bilayers, single-channel conductance, ionic selectivity, and voltage-gating characteristics were assessed. Porins 77a and 77f displayed similar single-channel conductance and ionic selectivity. Current-voltage relationships were determined for the different porins: porin 77f displayed substantial voltage gating at both positive and negative polarity; porin 77a gated at negative polarity only. Porins 67d and 69a showed substantial differences in their pore-forming properties: the single-channel conductance of porin 69a was significantly increased (1.05 nS) relative to porin 67d (0.73 nS). Porin 67d was twice as permeable to cations as porin 69a, and at both positive and negative polarities the extent of voltage gating was greater for porin 67d relative to porin 69a. Expression of the porins in an isogenic, porin-deleted H. influenzae background allowed for assessment of the contribution of each porin to the minimum inhibitory concentrations of various antimicrobial compounds. Porin 67d was found to have a decreased susceptibility to the antimicrobials novobiocin and streptomycin. This decreased susceptibility of porin 67d to novobiocin and streptomycin correlates with its decrease in single-channel conductance.

Amino Acid Sequence↗
PubMed2001

Mutagenesis identifies amino acid residues in extracellular loops and within the barrel lumen that determine voltage gating of porin from Haemophilus influenzae type b.

Porin (341 amino acids; M(r) 37 782) of Haemophilus influenzae type b mediates exchange of solutes between the external environment and the periplasm of this Gram-negative bacterium. Positively charged residues in the extracellular loops have been shown to be involved in the voltage gating of this protein. To further elucidate our observations on the functional properties of this channel, we mutated seven lysines (Lys(48), Lys(161), Lys(165), Lys(170), Lys(248), Lys(250), and Lys(253)) to glutamic acid. The selected residues were previously shown to be accessible to chemical modification, and they map to three locations: loop 4 and loop 6, and within the barrel lumen. The seven mutant proteins were purified, and each was reconstituted into planar lipid bilayers to characterize its channel forming properties. The single substitution mutant porins displayed increased single channel conductances in 1 M KCl ranging between 134 and 178% of the single channel conductance for wild-type Hib porin. Six of the seven mutant porins also displayed altered current-voltage relationships when compared to wild-type Hib porin. Whereas Lys(170)Glu had activity similar to wild-type Hib porin, Lys(48)Glu, Lys(248)Glu, and Lys(253)Glu showed substantial voltage gating at both positive and negative polarities. Lys(161)Glu and Lys(250)Glu gated only at negative potentials, and Lys(165)Glu gated only at positive potentials. Rather than ascribing one specific loop in gating, our analyses of these mutant Hib porins suggest that voltage gating can be attributed to contributions from loops 4 and 6 and a residue within the barrel lumen.

Amino Acid Sequence↗
PubMed2001

Regulation of the CFTR channel by phosphorylation.

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are regulated tightly by protein kinases and phosphatases. The regulatory domain of CFTR has about 20 potential sites for phosphorylation by protein kinases A (PKA) and C (PKC). The reason for this large number of sites is not known, however their conservation from fish to humans implies that they play important roles in vivo. PKA is an important activator, and its stimulation of CFTR is enhanced by PKC via mechanisms which are not fully understood. The physiological stimuli of CFTR are not known for some epithelia, and it appears likely that other serine/threonine and even tyrosine kinases also regulate CFTR in particular tissues. Phosphatases that deactivate CFTR have yet to be identified definitively at the molecular level, however CFTR is regulated by a membrane-bound form of protein phosphatase-2C (PP2C) in several cell types. Patch-clamp studies of channel rundown, co-immunoprecipitation, chemical cross-linking studies, and pull-down assays all indicate that CFTR and PP2C are closely associated within a stable regulatory complex. Understanding the regulation of CFTR by PP2C is a priority due to its potential as a target for pharmacotherapies in the treatment of cystic fibrosis.

Cystic Fibrosis↗
PubMed2001

Asymmetric structure of the cystic fibrosis transmembrane conductance regulator chloride channel pore suggested by mutagenesis of the twelfth transmembrane region.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel contains 12 membrane-spanning regions which are presumed to form the transmembrane pore. Although a number of findings have suggested that the sixth transmembrane region plays a key role in forming the pore and determining its functional properties, the role of other transmembrane regions is currently not well established. Here we assess the functional importance of the twelfth transmembrane region, which occupies a homologous position in the carboxy terminal half of the CFTR molecule to that of the sixth transmembrane region in the amino terminal half. Five residues in potentially important regions of the twelfth transmembrane region were mutated individually to alanines, and the function of the mutant channels was examined using patch clamp recording following expression in mammalian cell lines. Three of the five mutations significantly weakened block of unitary Cl(-) currents by SCN(-), implying a partial disruption of anion binding within the pore. Two of these mutations also caused a large reduction in the steady-state channel mean open probability, suggesting a role for the twelfth transmembrane region in channel gating. However, in direct contrast to analogous mutations in the sixth transmembrane region, all mutants studied here had negligible effects on the anion selectivity and unitary Cl(-) conductance of the channel. The relatively minor effects of these five mutations on channel permeation properties suggests that, despite their symmetrical positions within the CFTR protein, the sixth and twelfth transmembrane regions make highly asymmetric contributions to the functional properties of the pore.

Amino Acid Sequence↗
PubMed2000

Charged residues in surface-located loops influence voltage gating of porin from Haemophilus influenzae type b.

Porin of Haemophilus influenzae type b (341 amino acids; M(r) 37782) determines the permeability of the outer membrane to low molecular mass compounds. Purified Hib porin was subjected to chemical modification of lysine residues by succinic anhydride. Electrospray ionization mass spectrometry identified up to 12 modifications per porin molecule. Tryptic digestion of modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight mass spectrometry mapped the succinylation sites. Most modified lysines are positioned in surface-located loops, numbers 1 and 4 to 7. Succinylated porin was reconstituted into planar lipid bilayers, and biophysical properties were analyzed and compared to Hib porin: there was an increased average single channel conductance compared to Hib porin (1.24 +/- 0.41 vs. 0.85 +/- 0.40 nanosiemens). The voltage-gating activity of succinylated porin differed considerably from that of Hib porin. The threshold voltage for gating was decreased from 75 to 40 mV. At 80 mV, steady-state conductance for succinylated porin was 50-55% of the instantaneous conductance. Hib porin at 80 mV showed a decrease to 89-91% of the instantaneous current levels. We propose that surface-located lysine residues are determinants of voltage gating for porin of Haemophilus influenzae type b.

Amino Acid Sequence↗
PubMed2000

Molecular determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel pore.

Ionic selectivity in many cation channels is achieved over a short region of the pore known as the selectivity filter, the molecular determinants of which have been identified in Ca(2+), Na(+), and K(+) channels. However, a filter controlling selectivity among different anions has not previously been identified in any Cl(-) channel. In fact, because Cl(-) channels are only weakly selective among small anions, and because their selectivity has proved so resistant to site-directed mutagenesis, the very existence of a discrete anion selectivity filter has been called into question. Here we show that mutation of a putative pore-lining phenylalanine residue, F337, in the sixth membrane-spanning region of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel, dramatically alters the relative permeabilities of different anions in the channel. Specifically, mutations that reduce the size of the amino acid side chain present at this position virtually abolish the relationship between anion permeability and hydration energy, a relationship that characterizes the anion selectivity not only of wild-type CFTR, but of most classes of Cl(-) channels. These results suggest that the pore of CFTR may indeed contain a specialized region, analogous to the selectivity filter of cation channels, at which discrimination between different permeant anions takes place. Because F337 is adjacent to another amino acid residue, T338, which also affects anion selectivity in CFTR, we suggest that selectivity is predominantly determined over a physically discrete region of the pore located near these important residues.

Amino Acid Substitution↗
PubMed2000

Role of protein phosphatases in the activation of CFTR (ABCC7) by genistein and bromotetramisole.

Genistein and bromotetramisole (Br-t) strongly activate cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) chloride channels on Chinese hamster ovary cells and human airway epithelial cells. We have examined the possible role of phosphatases in stimulation by these drugs using patch-clamp and biochemical methods. Genistein inhibited the spontaneous rundown of channel activity that occurs after membrane patches are excised from cAMP-stimulated cells but had no effect on purified protein phosphatase type 1 (PP1), PP2A, PP2B, PP2C, or endogenous phosphatases when assayed as [(32)P]PO(4) release from prelabeled casein, recombinant GST-R domain fusion protein, or immunoprecipitated full-length CFTR. Br-t also slowed rundown of CFTR channels, but, in marked contrast to genistein, it did inhibit all four protein phosphatases tested. Half-maximal inhibition of PP2A and PP2C was observed with 0.5 and 1.5 mM Br-t, respectively. Protein phosphatases were also sensitive to (+)-p-Br-t, a stereoisomer of Br-t that does not inhibit alkaline phosphatases. Br-t appeared to act exclusively through phosphatases since it did not affect CFTR channels in patches that had low apparent endogenous phosphatase activity (i.e., those lacking spontaneous rundown). We conclude that genistein and Br-t act through different mechanisms. Genistein stimulates CFTR without inhibiting phosphatases, whereas Br-t acts by inhibiting a membrane-associated protein phosphatase (probably PP2C) that presumably allows basal phosphorylation to accumulate.

Animals↗
PubMed2000

Airway plumbing.

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Animals↗
PubMed1999

Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum.

CFTR possesses a large cluster of strict dibasic consensus sites for phosphorylation by protein kinase A (PKA) in the R-domain and an obligatory dependence on phosphorylation is a hallmark of CFTR Cl(-) channel function. Removal of as many as 11 of these sites reduces the conformational change in the R-domain and the degree of channel activation in response to PKA. However, until recently a completely PKA-unresponsive CFTR variant has not been reported, leaving open the possibility that the residual response may be mediated by associating ancillary phosphoproteins. We traced the residual PKA-catalyzed (32)P-labelling of the variant with 11 sites mutagenized (11SA) to distinct CNBr phosphopeptides within the R-domain. Mutagenesis of 4 additional monobasic sites in these segments produced a 15SA variant in which Cl(-) channel response to PKA was abolished. Therefore, it can be concluded that ancillary phosphoproteins do not contribute to CFTR activation by PKA. Notably, however, the 15SA protein did exhibit a low level of constitutive channel activity not dependent on PKA, which might have reflected a down-regulating effect of phosphorylation of one or two of the 15 sites as suggested by others. However, this did not prove to be the case.Since immature CFTR has been claimed to be active in the endoplasmic reticulum (ER), we also examined whether it can be phosphorylated in cells and what influence if any this might have on its susceptibility to degradation. Teleologically, activation by phosphorylation of CFTR Cl(-) channels in the ER might be undesirable to the cell. Using various phosphorylation site mutants and kinase and phosphatase inhibitors in pulse-chase experiments, we have found that although nascent CFTR can be phosphorylated at the ER, this is without effect on its ability to mature and avoid proteolysis. Furthermore, we found that microsomes from cells expressing CFTR processing mutants such as DeltaF508 do not generate Cl(-) active channels when fused with planar bilayers unless maturation is promoted, e.g. by growth of cells at reduced temperature or other means. We conclude that the ER-retained mutant nascent chains which are incapable of maturation may be phosphorylated but do not form active channels. Stimulation by PKA of the insertion of CFTR containing vesicles into the plasma membrane as part of the mechanism of stimulation of chloride secretion has been reported, as has an influence of CFTR on the balance between endocytosis and exocytosis but these findings have not been universally confirmed.

Amino Acid Sequence↗
PubMed1999

Association of cystic fibrosis transmembrane conductance regulator and protein phosphatase 2C.

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are rapidly deactivated by a membrane-bound phosphatase activity. The efficiency of this regulation suggests CFTR and protein phosphatases may be associated within a regulatory complex. In this paper we test that possibility using co-immunoprecipitation and cross-linking experiments. A monoclonal anti-CFTR antibody co-precipitated type 2C protein phosphatase (PP2C) from baby hamster kidney cells stably expressing CFTR but did not co-precipitate PP1, PP2A, or PP2B. Conversely, a polyclonal anti-PP2C antibody co-precipitated CFTR from baby hamster kidney membrane extracts. Exposing baby hamster kidney cell lysates to dithiobis (sulfosuccinimidyl propionate) caused the cross-linking of histidine-tagged CFTR (CFTR(His10)) and PP2C into high molecular weight complexes that were isolated by chromatography on Ni(2+)-nitrilotriacetic acid-agarose. Chemical cross-linking was specific for PP2C, because PP1, PP2A, and PP2B did not co-purify with CFTR(His10) after dithiobis (sulfosuccinimidyl propionate) exposure. These results suggest CFTR and PP2C exist in a stable complex that facilitates regulation of the channel.

Animals↗
PubMed1999

Characterization of the inhibitory effect of boiled rice on intestinal chloride secretion in guinea pig crypt cells.

BACKGROUND & AIMS: When rice is incorporated into oral rehydration therapy for patients with secretory diarrhea, clinical outcomes improve. We have shown that a factor purified from boiled rice (RF) blocks the secretory response of intestinal crypt cells to adenosine 3',5'-cyclic monophosphate (cAMP). Now we report that the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the cellular target for this rice inhibitor. METHODS: We used RF, the same previously described extract prepared from boiled rice, to assess chloride channel activation in vitro, measuring (1) cell volume regulation of guinea pig intestinal crypt epithelial cell suspensions using standard Coulter counter technology, (2) transepithelial chloride current in monolayers of T84 cells mounted in Ussing chambers, and (3) whole-cell and single-channel currents using the patch-clamp technique in cells transfected to express CFTR. RESULTS: RF inhibited activation by cAMP of CFTR chloride channels in all experimental preparations; RF did not block volume-stimulated Cl- secretion, suggesting that its effect might be specific for CFTR chloride channels. RF inhibited transepithelial cAMP-stimulated Cl- current in T84 cells and inhibited forskolin (i.e., cAMP)-induced current in cells transfected with CFTR. Excised patch and single-channel patch-clamp recordings supported the view that the response was a direct effect on CFTR rather than on cAMP signal transduction. CONCLUSIONS: RF exerts a specific inhibitory effect on CFTR chloride channels, blocking activation from the luminal surface of the cell and reversing established activation. Many major diarrheal states are based on cAMP-induced CFTR activation, leading to excessive gut secretion; our findings could have clinical relevance.

Animals↗
PubMed1999

Substrates of multidrug resistance-associated proteins block the cystic fibrosis transmembrane conductance regulator chloride channel.

1. The effects of physiological substrates of multidrug resistance-associated proteins (MRPs) on cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel currents were examined using patch clamp recording from CFTR-transfected mammalian cell lines. 2. Two MRP substrates, taurolithocholate-3-sulphate (TLCS) and beta-estradiol 17-(beta-D-glucuronide) (E217betaG) caused a voltage-dependent block of macroscopic CFTR Cl- currents when applied to the intracellular face of excised membrane patches, with mean apparent dissociation constants (KDs) of 96+/-10 and 563+/-103 microM (at 0 mV) respectively. The unconjugated bile salts taurocholate and cholate were also effective CFTR channel blockers under these conditions, with KDs of 453+/-44 and 3760+/-710 microM (at 0 mV) respectively. 3. Reducing the extracellular Cl- concentration from 154 to 20 mM decreased the KD for block intracellular TLCS to 54+/-1 microM, and also significantly reduced the voltage dependence of block, by suggesting that TLCS blocks Cl- permeation through CFTR by binding within the channel pore. 4. Intracellular TLCS reduced the apparent amplitude of CFTR single channel currents, suggesting that the duration of block is very fast compared to the gating of the channel. 5. The apparent affinity of block by TLCs is comparable to that of other well-known CFTR channel blockers, suggesting that MRP substrates may comprise a novel class of probes of the CFTR channel pore. 6. These results also suggest that the related proteins CFTR and MRP may share a structurally similar anion binding site at the cytoplasmic face of the membrane.

ATP-Binding Cassette Transporters↗
PubMed1998

Non-pore lining amino acid side chains influence anion selectivity of the human CFTR Cl- channel expressed in mammalian cell lines.

1. The effects of individually mutating two adjacent threonine residues in the sixth membrane-spanning region (TM6) of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel on permeation properties were examined using patch clamp recording from mammalian cell lines stably expressing human CFTR. 2. A number of mutations of T338 significantly affected the permeation properties of the channel. Increases and decreases in single channel conductance were observed for different mutants. Anion selectivity was strongly affected, with no two channel variants sharing the same selectivity sequence. Several mutations led to strong inward rectification of the macroscopic current-voltage relationship. The effects of these mutations on permeation properties were correlated with the size of the amino acid side chain substituted, rather than its chemical nature. 3. Most mutations of T339 resulted in a lack of functional channel expression and apparent misprocessing of the protein. One mutant, T339V, was characterized in detail; its permeation properties were significantly altered, although these effects were not as strong as for T338 mutations. 4. These results suggest an important role for T338 in controlling the permeation properties of the CFTR Cl- channel. It is suggested that mutation of this residue alters the interaction between permeating anions and the channel pore via an indirect effect on the orientation of the TM6 helix.

Amino Acid Substitution↗
PubMed1998

The CFTR chloride channel: nucleotide interactions and temperature-dependent gating.

The gating cycle of CFTR (Cystic Fibrosis Transmembrane conductance Regulator) chloride channels requires ATP hydrolysis and can be interrupted by exposure to the nonhydrolyzable nucleotide AMP-PNP. To further characterize nucleotide interactions and channel gating, we have studied the effects of AMP-PNP, protein kinase C (PKC) phosphorylation, and temperature on gating kinetics. The rate of channel locking increased from 1.05 x 10(-3) sec-1 to 58.7 x 10(-3) sec-1 when AMP-PNP concentration was raised from 0.5 to 5 mM in the presence of 1 mM MgATP and 180 nM protein kinase A catalytic subunit (PKA). Although rapid locking precluded estimation of Po or opening rate immediately after the addition of AMP-PNP to wild-type channels, analysis of locking rates in the presence of high AMP-PNP concentrations revealed two components. The appearance of a distinct, slow component at high [AMP-PNP] is evidence for AMP-PNP interactions at a second site, where competition with ATP would reduce Po and thereby delay locking. All channels exhibited locking when they were strongly phosphorylated by PKA, but not when exposed to PKC alone. AMP-PNP increased Po at temperatures above 30 degrees C but did not cause locking, evidence that the stabilizing interactions between domains, which have been proposed to maintain CFTR in the open burst state, are relatively weak. The temperature dependence of normal CFTR gating by ATP was strongly asymmetric, with the opening rate being much more temperature sensitive (Q10 = 9.6) than the closing rate (Q10 = 3.6). These results are consistent with a cyclic model for gating of phosphorylated CFTR.

Adenosine Triphosphate↗
PubMed1998

Dibasic protein kinase A sites regulate bursting rate and nucleotide sensitivity of the cystic fibrosis transmembrane conductance regulator chloride channel.

1. The relationship between phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and its gating by nucleotides was examined using the patch clamp technique by comparing strongly phosphorylated wild-type (WT) channels with weakly phosphorylated mutant channels lacking four (4SA) or all ten (10SA) dibasic consensus sequences for phosphorylation by protein kinase A (PKA). 2. The open probability (Po) of strongly phosphorylated WT channels in excised patches was about twice that of 4SA and 10SA channels, after correcting for the number of functional channels per patch by addition of adenylylimidodiphosphate (AMP-PNP). The mean burst durations of WT and mutant channels were similar, and therefore the elevated Po of WT was due to its higher bursting rate. 3. The ATP dependence of the 10SA mutant was shifted to higher nucleotide concentrations compared with WT channels. The relationship between Po and [ATP] was noticeably sigmoid for 10SA channels (Hill coefficient, 1.8), consistent with positive co-operativity between two sites. Increasing ATP concentration to 10 mM caused the Po of both WT and 10SA channels to decline. 4. Wild-type and mutant CFTR channels became locked in open bursts when exposed to mixtures of ATP and the non-hydrolysable analogue AMP-PNP. The rate at which the low phosphorylation mutants became locked open was about half that of WT channels, consistent with Po being the principal determinant of locking rate in WT and mutant channels. 5. We conclude that phosphorylation at 'weak' PKA sites is sufficient to sustain the interactions between the ATP binding domains that mediate locking by AMP-PNP. Phosphorylation of the strong dibasic PKA sites controls the bursting rate and Po of WT channels by increasing the apparent affinity of CFTR for ATP.

Adenosine Triphosphate↗
PubMed1998

Structural basis for specificity and potency of xanthine derivatives as activators of the CFTR chloride channel.

1. On the basis of their structure, we compared the ability of 35 xanthine derivatives to activate the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel stably expressed in chinese hamster ovary (CHO) cells using the cell-attached patch clamp and iodide efflux techniques. 2. Activation of CFTR channels was obtained with 3-mono, 1,3-di or 1,3,7-tri-substituted alkyl xanthine derivatives (enprofylline, theophylline, aminophylline, IBMX, DPMX and pentoxifylline). By contrast, xanthine derivatives substituted at the C8- or N9-position failed to open CFTR channels. 3. The CFTR chloride channel activity was blocked by glibenclamide (100 microM) but not by DIDS (100 microM). 4. Activation of CFTR by xanthines was not mimicked by the calcium ionophore A23187, adenosine, UTP, ATP or the specific phosphodiesterase inhibitors rolipram, Ro 20-1724 and milrinone. In addition, we found no correlation between the effect of xanthines on CFTR and on the cellular cyclic AMP or ATP levels. 5. We then synthesized a series of 3,7-dimethyl-1-alkyl xanthine derivatives; among them, 3,7-dimethyl-1-propyl xanthine and 3,7-dimethyl-1-isobutyl xanthine both activated CFTR channels without increasing the intracellular cyclic AMP level, while the structurally related 3,7-dimethyl-1-(2-propenyl) xanthine and 3,7-dimethyl-1-(oxiranyl methyl) xanthine were inactive. 6. Our findings delineate a novel function for xanthine compounds and identify the molecular features that enable xanthine activation of CFTR. These results may be useful in the development of new molecules for studying the pharmacology of chloride channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗