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J W Hanrahan

Publications and source records attributed to J W Hanrahan.

At least 55 records · Page 3Linked to original sources
PubMed1996

Flickery block of single CFTR chloride channels by intracellular anions and osmolytes.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and nucleotide-dependent chloride channel. Single CFTR currents recorded on cell show slight outward rectification, which has previously been suggested to be due to an asymmetrical chloride ion gradient or to a specific interaction between permeant intracellular anions and the channel. Using a single-channel recording from Chinese hamster ovary cells stably expressing CFTR, we have found that both the sparingly permeant anion glutamate and the impermeant anion gluconate cause a rapid, voltage-dependent block of CFTR channels when applied to the intracellular, but not the extracellular, face of excised patches. Both the affinity and the voltage dependence of block were affected by the extracellular chloride concentration in a manner consistent with chloride ions being able to repel these blocking ions from the pore. These results are discussed in terms of previous models of CFTR current outward rectification, and it is suggested that this rectification may result from a combination of asymmetrical chloride concentrations and voltage-dependent block of the channel by large cytoplasmic anions. In addition, we find that CFTR conductance is decreased by high concentrations of intracellular sucrose, sorbitol, and urea in a manner consistent with a rapid block of the channel by these molecules.

Animals↗
PubMed1995

Plasma membrane Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways.

Na+/H+ exchanger (NHE) activity is regulated by several types of receptors directly coupled to distinct classes (i.e. Gs, Gi, Gq, and G12) of heterotrimeric (alpha beta gamma) GTP-binding proteins (G proteins), which, upon activation, modulate production of various second messengers (e.g. cAMP, cGMP, diacylglycerol, inositol trisphosphate, and Ca2+). Recently, four isoforms of the rat Na+/H+ exchanger were identified by molecular cloning. To examine their intrinsic responsiveness to G protein and second messenger stimulation, three of these isoforms, NHE-1, -2, and -3, were stably expressed in mutant Chinese hamster ovary cells devoid of endogenous NHE activity (AP-1 cells). Incubation of cells with either AIF4-, a general agonist of G proteins, or cholera toxin, a selective activator of G alpha s that stimulates adenylate cyclase, accelerated the rates of amiloride-inhibitable 22Na+ influx mediated by NHE-1 and -2, whereas they inhibited that by NHE-3. Similarly, short term treatment with phorbol 12-myristate 13-acetate, which mimics diacylglycerol activation of protein kinase C (PKC), or with agents (i.e. forskolin, 8-(4-chlorophenylthio)-cAMP, and isobutylmethylxanthine) that lead to activation of cAMP-dependent protein kinase (PKA) also stimulated transport by NHE-1 and NHE-2 but depressed that by NHE-3. The effects of phorbol 12-myristate 13-acetate were blocked by depleting cells of PKC or by inhibiting PKC using chelerythrine chloride, confirming a role for PKC in modulating NHE isoform activities. Likewise, the PKA antagonist, H-89, attenuated the effects of elevated cAMPi on NHE-1, -2, and -3, further demonstrating the regulation by PKA. Unlike cAMPi, elevation of cGMPi by treatment with dibutyryl-cGMP or 8-bromo-cGMP had no influence on NHE isoform activities, thereby excluding the possibility of a role for cGMP-dependent protein kinase in these cells. These data support the concept that the NHE isoforms are differentially responsive to agonists of the PKA and PKC pathways.

Animals↗
PubMed1995

cAMP-dependent protein kinase-mediated phosphorylation of cystic fibrosis transmembrane conductance regulator residue Ser-753 and its role in channel activation.

Hormonal regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is largely mediated via cAMP-dependent protein kinase (PKA). CFTR contains 10 dibasic consensus sites for potential PKA phosphorylation ((R/K) (R/K)X(S*/T*)). Previous studies (Chang, X.-B., Tabcharani, J. A., Hou, Y.-X., Jensen, T. J., Kartner, N., Alon, N., Hanrahan, J. W., and Riordan, J.R (1993) J. Biol. Chem. 268, 11304-11311) showed that approximately 25% of the CFTR wild-type response to PKA activation remained upon inhibition of most detectable phosphorylation by in vitro mutagenesis of all 10 dibasic consensus sites (10SA CFTR). To identify potential additional sites responsible for the residual activity, large amounts of this mutant CFTR were phosphorylated with PKA using high specific activity [gamma-32P]ATP. Cyanogen bromide cleavage indicated that a large portion of the observed PKA phosphorylation occurred within a 5.8-kDa fragment of the R domain between residues 722-773. Removal of serines at potential PKA sites in this fragment showed that Ser-753 accounted for all of the gamma-32P labeling of the 5.8-kDa peptide. Replacement of Ser-753 with alanine reduced the level of residual CFTR activity by a further 40%, indicating that phosphorylation at this previously unidentified site contributes to the activation of 10SA CFTR.

Amino Acid Sequence↗
PubMed1994

Phosphatase inhibitors activate normal and defective CFTR chloride channels.

The cystic fibrosis transmembrane conductance 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 mechanisms responsible for the dephosphorylation and spontaneous deactivation ("rundown") of CFTR chloride channels excised from transfected Chinese hamster ovary (CHO) and human airway epithelial cells. We report that the alkaline phosphatase inhibitors bromotetramisole, 3-isobutyl-1-methylxanthine, theophylline, 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 channels 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 delta F508 after cooling). These results suggest that CFTR dephosphorylation is dynamic and that membrane-associated phosphatase activity may be a potential therapeutic target for the treatment of cystic fibrosis.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Basolateral K channel activated by carbachol in the epithelial cell line T84.

Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (Isc), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T84 cells. Carbachol had little effect on Isc when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent Isc response to calcium ionophores. Carbachol (100 microM) transiently elevated intracellular free calcium ([Ca2+]i) by approximately 3-fold in confluent cells cultured on glass coverslips with a time course resembling the Isc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (-54 mV) with pipette solution containing 150 mM KCl (37 degrees C). This rectification persisted when patches were bathed in symmetrical 150 mM KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na >> Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mM barium. It is referred to as the KBIC channel based on its most distinctive properties (Ba-insensitive, inwardly rectifying, Ca-activated). Like single KBIC channels, the carbachol-stimulated Isc was relatively insensitive to several blockers on the basolateral side and was unaffected by barium. These comparisons between the properties of the macroscopic current and single channels suggest that the KBIC channel mediates basolateral membrane K conductance in T84 cell monolayers during stimulation by cholinergic secretagogues.

4-Aminopyridine↗
PubMed1994

Regulation of an inwardly rectifying K channel in the T84 epithelial cell line by calcium, nucleotides and kinases.

Agonists that elevate calcium in T84 cells stimulate chloride secretion by activating KBIC, an inwardly rectifying K channel in the basolateral membrane. We have studied the regulation of this channel by calcium, nucleotides and phosphorylation using patch clamp and short-circuit current (ISC) techniques. Open probability (Po) was independent of voltage but declined spontaneously with time after excision. Rundown was slower if patches were excised into a bath solution containing ATP (10 microM-5 mM), ATP (0.1 mM)+protein kinase A (PKA; 180 nM), or isobutylmethylxanthine (IBMX; 1 mM). Analysis of event durations suggested that the channel has at least two open and two closed states, and that rundown under control conditions is mainly due to prolongation of the long closed time. Channel activity was restimulated after rundown by exposure to ATP, the poorly hydrolyzable ATP analogue AMP-PNP, or ADP. Activity was further enhanced when PKA was added in the presence of MgATP, but only if free calcium concentration was elevated (400 nM). Nucleotide stimulation and inward rectification were both observed in nominally Mg-free solutions. cAMP modulation of basolateral potassium conductance in situ was confirmed by measuring currents generated by a transepithelial K gradient after permeabilization of the apical membrane using alpha-toxin. Finally, protein kinase C (PKC) inhibited single KBIC channels when it was added directly to excised patches. These results suggest that nonhydrolytic binding of nucleotides and phosphorylation by PKA and PKC modulate the responsiveness of the inwardly rectifying K channel to Ca-mediated secretagogues.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Effects of EGTA on calcium signaling in airway epithelial cells.

We have studied the effect of the extracellular calcium buffers ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) as well as varying extracellular calcium concentration on the intracellular free calcium response to histamine in a cystic fibrosis airway epithelial cell line (CF/T43). Histamine stimulates a rapid transient increase in cell calcium followed by a slower second peak lasting several minutes. Bathing cells in nominally calcium-free medium (no added calcium) did not abolish the second peak in the biphasic response to histamine. We examined the effect of including either 1 mM EGTA or BAPTA in the perfusate to investigate whether influx might have been supported by trace amounts of calcium in the nominally calcium-free medium. The second histamine-stimulated peak had a shorter duration but similar amplitude in the presence of BAPTA. In contrast, the second peak was completely abolished by EGTA. To examine if the different histamine responses were due to EGTA's lower dissociation constant or some pharmacological effect of unbound EGTA, extracellular calcium was removed by pretreating the saline with BAPTA covalently bound to polystyrene beads, and effluent from the imaging chamber was collected and analyzed for calcium contamination. Histamine stimulation still produced a biphasic calcium response when extracellular free calcium was approximately 7 nM, a concentration that should reverse the electrochemical gradient for calcium at the plasma membrane. These data suggest that the unbound form of EGTA can interfere with calcium signaling in cells through inhibition of its release from intracellular stores.

Calcium↗
PubMed1994

The molecular basis of chloride transport in shark rectal gland.

Transepithelial Cl- secretion in vertebrates is accomplished by a secondary active transport process brought about by the coordinated activity of apical and basolateral transport proteins. The principal basolateral components are the Na+/K(+)-ATPase pump, the Na+/K+/2Cl- cotransporter (symporter) and a K+ channel. The rate-limiting apical component is a cyclic-AMP-stimulated Cl- channel. As postulated nearly two decades ago, the net Cl- movement from the blood to the lumen involves entry into the epithelial cells with Na+ and K+, followed by active Na+ extrusion via the pump and passive K+ exit via a channel. Intracellular [Cl-] is raised above electrochemical equilibrium and exits into the lumen when the apical Cl- channel opens. Cl- secretion is accompanied by a passive paracellular flow of Na+. The tubules of the rectal glands of elasmobranchs are highly specialized for secreting concentrated NaCl by this mechanism and hence have served as an excellent experimental model in which to characterize the individual steps by electrophysiological and ion flux measurements. The recent molecular cloning and heterologous expression of the apical Cl- channel and basolateral cotransporter have enabled more detailed analyses of the mechanisms and their regulation. Not surprisingly, since hormones acting through kinases control secretion, both the Cl- channel, which is the shark counterpart of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), and the cotransporter are regulated by phosphorylation and dephosphorylation. The primary stimulation of secretion by hormones employing cyclic AMP as second messenger activates CFTR via the direct action of protein kinase A (PKA), which phosphorylates multiple sites on the R domain. In contrast, phosphorylation of the cotransporter by as yet unidentified kinases is apparently secondary to the decrease in intracellular chloride concentration caused by anion exit through CFTR.

Animals↗
PubMed1993

Multi-ion pore behaviour in the CFTR chloride channel.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a non-rectifying, low-conductance channel regulated by ATP and phosphorylation, which mediates apical chloride conductance in secretory epithelia and malfunctions in cystic fibrosis (CF). Mutations at Lys 335 and Arg 347 in the sixth predicted transmembrane helix of CFTR alter its halide selectivity in whole-cell studies and its single channel conductance, but the physical basis of these alterations is unknown and permeation in CFTR is poorly understood. Here we present evidence that wild-type CFTR can contain more than one anion simultaneously. The conductance of CFTR passes through a minimum when channels are bathed in mixtures of two permeant anions. This anomalous mole fraction effect can be abolished by replacing Arg 347 with an aspartate and can be toggled on or off by varying the pH after the same residue is replaced with a histidine. Thus the CFTR channel should provide a convenient model in which to study multi-ion pore behaviour and conduction. The loss of multiple occupancy may explain how naturally occurring CF mutations at this site cause disease.

Amino Acid Sequence↗
PubMed1993

Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites.

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in transepithelial ion transport by acting as a tightly regulated apical chloride channel. Regulation is achieved by the concerted action of ATP at conserved nucleotide binding folds and serine phosphorylation at multiple sites by protein kinases A (PKA) and C (PKC). A previous investigation concluded that activation by PKA is critically dependent on phosphorylation at four of the nine predicted PKA sites in the R domain (S660A, S737A, S795A, S813A), because a "Quad" mutant lacking these sites could not be activated. We show in the present work that not only can this mutant be phosphorylated and activated, but a mutant in which all 10 predicted PKA sites have been altered still retains significant PKA-activated function. Potentiation of the PKA response by PKC is also preserved in this mutant. Thus CFTR may be regulated by cryptic PKA sites which also mediate interactions between different kinases. Such hierarchical phosphorylation of CFTR by obvious and cryptic PKA sites could provide a metered response to secretagogues.

Amino Acid Sequence↗
PubMed1993

Histamine stimulates a biphasic calcium response in the human tracheal epithelial cell line CF/T43.

We have studied the cytosolic free Ca2+ concentration ([Ca2+]i) response to histamine and other inflammatory mediators in a cystic fibrosis tracheal epithelial cell line (CF/T43) using digital fluorescence imaging. Brief pulses of histamine increased [Ca2+]i in a concentration-dependent manner with threshold, half-maximal, and maximal responses at approximately 5 microM, 120 microM, and 10 mM, respectively. The calcium response to sustained histamine exposure was markedly biphasic, consisting of an early peak (to approximately 2.4 microM [Ca2+]i) followed by a smaller second peak that lasted 45-60 s. Neither peak was directly dependent on Ca2+ influx. In contrast, stimulation with bradykinin gave a single peak followed by a smooth decay back to baseline levels. Sustained perfusion with histamine did not affect the bradykinin response, which is known to be mediated by inositol 1,4,5-trisphosphate (IP3). The H1-type histamine receptor blockers mepyramine, diphenhydramine, and (+)-chlorpheniramine were potent antagonists of the histamine response. Diphenhydramine was also a weak agonist at high concentrations (> or = 1 mM) and gave a biphasic response similar to that with histamine. The H2-type receptor blocker cimetidine and the H3-type receptor blocker thioperamide had no effect. Indomethacin failed to inhibit the second phase of the histamine-stimulated [Ca2+]i response, suggesting that the second [Ca2+]i peak is not due to secondary production of prostaglandins. Neomycin, which inhibits IP3 production, completely abolished the [Ca2+]i response to bradykinin stimulation but did not affect the second phase of the histamine response. The biphasic nature of the histamine response, the insensitivity of the second [Ca2+]i peak to neomycin, and the independence of bradykinin and histamine responses suggest that histamine may modulate [Ca2+]i through multiple IP3 and non-IP3 pathways.

Adenosine Triphosphate↗
PubMed1991

Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene.

The expression of the cystic fibrosis (CF) gene on its introduction into nonepithelial somatic cells has recently been shown to result in the appearance of distinctive low conductance chloride channels stimulated by cyclic AMP (Kartner, N., Hanrahan, J.W., Jensen, T.J., Naismith, A.L., Sun, S., Ackerley, C.A., Reyes, E.F., Tsui, L.-C., Rommens, J.M., Bear, C.E., and Riordan, J.R. (1991) Cell 64, 681-691; Anderson, M. P., Rich, D.P., Gregory, R.J., Smith, A.E., and Welsh, M.J. (1991) Science 251, 679-682). Since Xenopus oocytes provide a powerful system for ion channel characterization, we have examined whole cell and single channel currents in them after injection of cRNA to program the synthesis of the cystic fibrosis transmembrane conductance regulator (CFTR). This has enabled the direct demonstration that the cyclic AMP activation is mediated by protein kinase A and that CFTR is without effect on the endogenous calcium-activated chloride channels of the oocyte, which have been well characterized previously and widely used as reporters of the expression of G-protein-coupled receptors. These findings strengthen the argument that the CF gene codes for a novel regulated chloride channel rather than a regulatory protein which can modulate separate chloride channel molecules.

Animals↗
PubMed1991

Phosphorylation-regulated Cl- channel in CHO cells stably expressing the cystic fibrosis gene.

A cyclic AMP-stimulated chloride conductance appears when the cystic fibrosis gene is expressed in non-epithelial cells by infection with recombinant viruses. Cyclic AMP-stimulated conductance in this system is mediated by the same ohmic, low-conductance Cl- channel as in human secretory epithelia, but control of this channel by phosphorylation has not been directly demonstrated. Here we report the appearance of the low-conductance Cl- channel in Chinese hamster ovary cells after stable transfection with the cystic fibrosis gene. The channel is regulated on-cell by membrane-permeant analogues of cAMP and off-cell by protein kinases A and C and by alkaline phosphatase. These results are further evidence that the cystic fibrosis transmembrane regulator is a Cl- channel which can be activated by specific phosphorylation events and inactivated by dephosphorylation; they reveal an unsuspected synergism between converging kinase regulatory pathways.

Animals↗
PubMed1991

Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductance.

The nature of involvement of the cystic fibrosis gene product (CFTR) in epithelial anion transport is not yet understood. We have expressed CFTR in Sf9 insect cells using the baculovirus expression vector system. Reactivity with antibodies against 12 different epitopes spanning the entire sequence suggested that the complete polypeptide chain was synthesized. Immunogold labeling showed localization to both cell-surface and intracellular membranes. Concomitant with CFTR expression, these cells exhibited a new cAMP-stimulated anion permeability. This conductance, monitored both by radioiodide efflux and patch clamping, strongly resembled that present in several CFTR-expressing human epithelial cells. These findings demonstrate that CFTR can function in heterologous nonepithelial cells and lend support to the possibility that CFTR may itself be a regulated anion channel.

Animals↗
PubMed1991

On the activation of outwardly rectifying anion channels in excised patches.

Previous studies have shown that outwardly rectifying anion channels can be activated in excised patches by exposure to protein kinases or large depolarizing voltage pulses and by raising the bath temperature to 37 degrees C. These maneuvers presumably induce some conformational change in the channel or a regulatory molecule. However, the mechanisms underlying stimulation have not been defined. We have tested several procedures known to influence the structure and solubility of proteins for their effects on activation of anion channels in excised patches. Spontaneous and voltage-induced activation of outward rectifiers was enhanced by increasing the ionic strength or the pH of the bath solution. These maneuvers had no effect on the activity of calcium-activated cation nonselective channels. Activation of outward rectifiers depended on the anion used and followed the (inverse) Hofmeister series consistent with a salting-in process. Divalent cations also enhanced activation with relative potencies Ba greater than Ca greater than Mg but at a much lower concentration (4 meq/l). Exposing patches from T84 cells to purified catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent kinase had no effect on the outward rectifier but did activate the low-conductance Cl channel. The results indicate that the outward rectifier is labile and raise the possibility that nonspecific physical mechanisms may contribute to its activation in excised patches by kinases and other stimuli.

Adenosine Triphosphate↗
PubMed1991

Anion channels in the apical membrane of mammalian corneal epithelium primary cultures.

Chloride ion (Cl-) secretion by the rabbit corneal epithelium involves a catecholamine-stimulated Cl- conductance at the apical membrane, but the characteristics of the ion channel(s) responsible are unknown. A primary cell culture system was developed that induces stratified epithelial differentiation of rat and rabbit corneal epithelial cells, as detected by differential interference contrast light and transmission electron microscopy. In patch clamp studies, gigaOhm seal, were obtained easily during the first 1-4 days in vitro, and patches contained a voltage-dependent outwardly rectifying channel. Single-channel conductance at 24 degrees C was about 10, 29, and 72 pS in symmetric 150 mM NaCl at membrane potentials of -60, 0, and +60 mV, respectively. The current-voltage relationship measured with 75 mM NaCl and sucrose in the bath indicated anion selectivity. Increasing the temperature to 37 degrees C and replacing HEPES buffer with tricine, increased channel conductance and decreased rectification. Characteristics of this channel and a low conductance Cl- channel are compared with those of anion channels of other vertebrate epithelia.

Animals↗