Preferential phosphorylation of R-domain Serine 768 dampens activation of CFTR channels by PKA

CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (P(o)) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted P(o)-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals.     

Nucleoside triphosphates are required to open the CFTR chloride channel.

The CFTR Cl- channel contains two predicted nucleotide-binding domains (NBD1 and NBD2); therefore, we examined the effect of ATP on channel activity. Once phosphorylated by cAMP-dependent protein kinase (PKA), channels required cytosolic ATP to open. Activation occurred by a PKA-independent mechanism. ATP gamma S substituted for ATP in PKA phosphorylation, but it did not open the channel. Several hydrolyzable nucleotides (ATP greater than GTP greater than ITP approximately UTP greater than CTP) reversibly activated phosphorylated channels, but nonhydrolyzable analogs and Mg(2+)-free ATP did not. Studies of CFTR mutants indicated that ATP controls channel activity independent of the R domain and suggested that hydrolysis of ATP by NBD1 may be sufficient for channel opening. The finding that nucleoside triphosphates regulate CFTR begins to explain why CF-associated mutations in the NBDs block Cl- channel function.     

Phosphorylation of the cystic fibrosis transmembrane conductance regulator.

Regulation of epithelial chloride flux, which is defective in patients with cystic fibrosis, may be mediated by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Part of the R-domain of CFTR (termed CF-2) was expressed in and purified from Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC, cyclic GMP-dependent protein kinase (PKG), and calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino acid sequencing and peptide mapping of CF-2 revealed that serines 660, 700, 737, and 813 as well as serine 768, serine 795, or both were phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same sites that were phosphorylated in CF-2. Kinetic analysis of phosphorylation of CF-2 and of synthetic peptides confirmed that these sites were excellent substrates for PKA, PKC, or PKG. CFTR was immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation was stimulated in response to agents that activated either PKA or PKC. Peptide mapping confirmed that CFTR was phosphorylated at several sites identified in vitro. Thus, regulation of CFTR is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by different second messenger pathways.     

PKC phosphorylation modulates PKA-dependent binding of the R domain to other domains of CFTR

Activity of the CFTR channel is regulated by phosphorylation of its regulatory domain (RD). In a previous study, we developed a bicistronic construct called DeltaR-Split CFTR, which encodes the front and back halves of CFTR as separate polypeptides without the RD. These fragments assemble to form a constitutively active CFTR channel. Coexpression of the third fragment corresponding to the missing RD restores regulation by PKA, and this is associated with dramatically enhanced binding of the phosphorylated RD. In the present study, we examined the effect of PKC phosphorylation on this PKA-induced interaction. We report here that PKC alone enhanced association of the RD with DeltaR-Split CFTR and that binding was further enhanced when the RD was phosphorylated by both kinases. Mutation of all seven PKC consensus sequences on the RD (7CA-RD) did not affect its association under basal (unphosphorylated) conditions but abolished phosphorylation-induced binding by both kinases. Iodide efflux responses provided further support for the essential role of RD binding in channel regulation. The basal activity of DeltaR-Split/7CA-RD channels was similar to that of DeltaR-Split/wild type (WT)-RD channels, whereas cAMP-stimulated iodide efflux was greatly diminished by removal of the PKC sites, indicating that 7CA-RD binding maintains channels in an inactive state that is unresponsive to PKA. These results suggest a novel mechanism for CFTR regulation in which PKC modulates PKA-induced domain-domain interactions.     

Phosphorylation of Na,K-ATPase alpha-subunits in microsomes and in homogenates of Xenopus oocytes resulting from the stimulation of protein kinase A and protein kinase C.

The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was characterized in purified enzyme preparations of Bufo marinus kidney and duck salt gland and in microsomes of Xenopus oocytes. In addition, we have examined cAMP and phorbol esters, which are stimulators of PKA and PKC, respectively, for their ability to provoke the phosphorylation of alpha-subunits of Na,K-ATPase in homogenates of Xenopus oocytes. In the enzyme from the duct salt gland, phosphorylation by PKA and PKC occurs on serine and threonine residues, whereas in the enzyme from B. marinus kidney and Xenopus oocytes, phosphorylation by PKA occurs only on serine residues. Phosphopeptide analysis indicates that a site phosphorylated by PKA resides in a 12-kDa fragment comprising the C terminus of the polypeptide. Studies of phosphorylation performed on homogenates of Xenopus oocytes show that not only endogenous oocyte Na,K-ATPase but also exogenous Xenopus Na,K-ATPase expressed in the oocyte by microinjection of cRNA can be phosphorylated in response to stimulation of oocyte PKA and PKC. In conclusion, these data are consistent with the possibility that the alpha-subunit of Na,K-ATPase can serve as a substrate for PKA and PKC in vivo.     

Kv4.2 is a locus for PKC and ERK/MAPK cross-talk

Transient outward K+ currents are particularly important for the regulation of membrane excitability of neurons and repolarization of action potentials in cardiac myocytes. These currents are modulated by PKC (protein kinase C) activation, and the K+- channel subunit Kv4.2 is a major contributor to these currents. Furthermore, the current recorded from Kv4.2 channels expressed in oocytes is reduced by PKC activation. The mechanism underlying PKC regulation of Kv4.2 currents is unknown. In the present study, we determined that PKC directly phosphorylates the Kv4.2 channel protein. In vitro phosphorylation of the intracellular N- and C-termini of Kv4.2 GST (glutathione transferase) tagged fusion protein revealed that the C-terminal of Kv4.2 was phosphorylated by PKC, whereas the N-terminal was not. Amino acid mapping and site-directed mutagenesis revealed that the phosphorylated residues on the Kv4.2 C-terminal were Ser447 and Ser537. A phospho-site-specific antibody showed that phosphorylation at the Ser537 site was increased in the hippocampus in response to PKC activation. Surface biotinylation experiments revealed that mutation to alanine of both Ser447 and Ser537 in order to block phosphorylation at both of the PKC sites increased surface expression compared with wild-type Kv4.2. Electrophysiological recordings of the wild-type and both the alanine and aspartate mutant Kv4.2 channels expressed with KChIP3 (Kv4 channel-interacting protein 3) revealed no significant difference in the half-activation or half-inactivation voltage of the channel. Interestingly, Ser537 lies within a possible ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) recognition (docking) domain in the Kv4.2 C-terminal sequence. We found that phosphorylation of Kv4.2 by PKC enhanced ERK phosphorylation of the channel in vitro. These findings suggest the possibility that Kv4.2 is a locus for PKC and ERK cross-talk.     

Modulation of Xenopus oocyte-expressed phospholemman-induced ion currents by co-expression of protein kinases.

Phospholemman (PLM), the major sarcolemmal substrate for phosphorylation by cAMP-dependent kinase (PKA) protein kinase C (PKC) and NIMA kinase in muscle, induces hyperpolarization-activated anion currents in Xenopus oocytes, most probably by enhancing endogenous oocyte currents. PLM peptides from the cytoplasmic tail are phosphorylated by PKA at S68, by NIMA kinase at S63, and by PKC at both S63 and S68. We have confirmed the phosphorylation sites in the intact protein, and we have investigated the role of phosphorylation in the regulatory activity of PLM using oocyte expression experiments. We found: (1) the cytoplasmic domain is not essential for inducing currents in oocytes; (2) co-expression of PKA increased the amplitude of oocyte currents and the amount of PLM in the oocyte membrane largely, but not exclusively, through phosphorylation of S68; (3) co-expression of PKA had no effect on a PLM mutant in which all putative phosphorylation sites had been inactivated by serine to alanine mutation (SSST 62, 63, 68, 69 AAAA); (4) co-expression of PKC had no effect in this system; (5) co-expression of NIMA kinase increased current amplitude and membrane protein level, but did not require PLM phosphorylation. These findings point to a role for phosphorylation in the function of PLM.     

Mechanism of activation of Xenopus CFTR by stimulation of PKC

PKA-mediated phosphorylation of the regulatory (R) domain plays a major role in the activation of the human cystic fibrosis transmembrane conductance regulator (hCFTR). In contrast, the effect of PKC-mediated phosphorylation is controversial, smaller than that of PKA, and dependent on the cell type. In the present study, we expressed Xenopus CFTR (XCFTR) and hCFTR in Xenopus oocytes and examined their responses (i.e., macroscopic membrane conductance) to maximal stimulation by PKC and PKA agonists. With XCFTR, the average response to PKC was approximately sixfold that of PKA stimulation. In contrast, with hCFTR, the response to PKC was 90% of the response to PKA stimulation. The reason for these differences was the small response of XCFTR to PKA stimulation. Using the substituted cysteine accessibility method, we found no evidence for insertion of functional CFTR channels in the plasma membrane in response to PKC stimulation. The increase in macroscopic conductance in response to PKC stimulation of XCFTR was due to an approximately fivefold increase in single-channel open probability, with a minor (30%) increase in single-channel conductance. The responses of XCFTR to PKC stimulation and of hCFTR to PKA stimulation were mediated by similar increases in P_o. In both instances, there were no changes in the number of channels in the membrane. We speculate that in animals other than humans, PKC stimulation may be the dominant mechanism for activation of CFTR. chloride channel; channel regulation; cystic fibrosis transmembrane conductance regulator gating; cystic fibrosis; phosphorylation; protein kinase A     

Dual effects of ADP and adenylylimidodiphosphate on CFTR channel kinetics show binding to two different nucleotide binding sites.

The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl)ethyl]ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay, whereas ATP-P3-[1-(2-nitrophenyl)ethyl]ester only decelerated the conductance increase upon ATP addition. ADP decelerated the conductance increase upon ATP addition and accelerated the conductance decay upon ATP withdrawal. The results present the first direct evidence that AMP-PNP binds to two sites on the CFTR. The effects of ADP also suggest two different binding sites because of the two different modes of inhibition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either reflect binding to NBD-A (i.e., product inhibition in the hydrolysis cycle) or allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NBD-A.     

Inhibition of heart calcium and chloride currents by sodium iodide. Specific attenuation in cAMP-dependent protein kinase-mediated regulation.

The enzymes cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) regulate the activity of cardiac ion channel proteins. In this study the whole-cell arrangement of the patch clamp technique was used to examine the effect of NaI on PKA-stimulated Cl- and Ca2+ channels in isolated guinea pig ventricular myocytes. Cl- currents (ICl) activated either by the beta-adrenergic agonist isoproterenol or the membrane-soluble cAMP analogue, 8-chlorphenylthio (8-CPT) cAMP, were greatly reduced in amplitude after substitution of an external solution containing 140 mM NaCl with a solution containing 140 mM NaI. This reduction was accompanied by a shift of -7 mV in the reversal potential (Erev) for ICl and could be reversed upon return to the NaCl external solution. Inhibition of ICl by NaI occurred in a concentration-dependent manner and was more pronounced for inward ICl (IC50 = 19 mM at -60 mV) than for outward ICl (IC50 = 60 mM at +60 mV). In contrast to ICl activated by PKA, ICl activated by PKC was slightly augmented in the presence of NaI and the Erev was found to shift by -15 mV. Based on these data, the relative permeability of I- to Cl- (PI/PCl) for this channel was calculated to be 1.79. NaI produced no change in the amplitude of inward calcium currents (ICa) recorded under basal conditions, but strongly inhibited ICa augmented by isoproterenol and 8-CPT cAMP, and during dialysis of cells with the catalytic subunit of PKA (CS). The in vitro incorporation of [gamma-32P]ATP into histone IIA and Kemptide, measured in the presence of PKA and cAMP, was not significantly different in assay mixtures containing salts of Cl- and I-. However, the ability of isoproterenol to augment basal ICa in whole-cell experiments was attenuated when experiments were carried out entirely in NaI external solution. Thus, the reduction in ICl and ICa observed in this study may result from a direct effect of I- on the phosphorylation/dephosphorylation of cardiac ion channel proteins or associated regulatory proteins.     

Stable knockdown of CFTR establishes a role for the channel in P2Y receptor-stimulated anion secretion

P2Y receptor regulation of anion secretion was investigated in porcine endometrial gland (PEG) epithelial cells. P2Y2, P2Y4, and P2Y6 receptors were detected in monolayers of PEG cells and immunocytochemistry indicated that P2Y4 receptors were located in the apical membrane. Apical membrane current measurements showed that Ca2+-dependent and PKC-dependent Cl- channels were activated following treatment with uridine triphosphate (UTP) (5 microM). Current-voltage relationships comparing calcium-dependent and PKC-dependent UTP responses under biionic conditions showed significant differences in selectivity between Cl-)and I- for the PKC-dependent conductance (P(I)/P(Cl) = 0.76), but not for Ca2+-dependent conductance (PI/P(Cl) = 1.02). The I-/Cl- permeability ratio for the PKC-dependent conductance was identical to that measured for 8-cpt cAMP. Furthermore, PKC stimulation using phorbol 12-myristate 13-acetate (PMA) activated an apical membrane Cl- conductance that was blocked by the CFTR selective inhibitor, CFTRinh-172. CFTR silencing, accomplished by stable expression of small hairpin RNAs (shRNA), blocked the PKC-activated conductance associated with UTP stimulation and provided definitive evidence of a role for CFTR in anion secretion. CFTR activation increased the initial magnitude of Cl- secretion, and provided a more sustained secretory response compared to conditions where only Ca2+-activated Cl- channels were activated by UTP. Measurements of [cAMP]i following UTP and PMA stimulation were not significantly different than untreated controls. Thus, these results demonstrate that UTP and PMA activation of CFTR occurs independently of increases in intracellular cAMP and extend the findings of earlier studies of CFTR regulation by PKC in Xenopus oocytes to a mammalian anion secreting epithelium.     

Protein kinase C inhibits ROMK1 channel activity via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism

The activity of apical K(+) channels in cortical collecting duct (CCD) is stimulated and inhibited by protein kinase A (PKA) and C (PKC), respectively. Direct interaction between phosphatidylinositol 4,5-bisphosphate (PIP(2)) and the cloned CCD K(+) channel, ROMK1, is critical for channel opening. We have found previously that phosphorylation of ROMK1 by PKA increases affinity of the channel for PIP(2) and mutation of PKA sites reduces the affinity of ROMK1 for PIP(2). In this study we investigate the molecular mechanism for PKC regulation of ROMK and report that mutants of ROMK1 with reduced PIP(2) affinity exhibit an increased sensitivity to inhibition by phorbol myristate acetate (PMA). The effect of PMA can be prevented by pretreatment with calphostin-C. Activation of PKC by carbachol in Xenopus oocytes co-expressing M1 muscarinic receptors also causes inhibition of the channels. Calphostin-C prevents carbachol-induced inhibition, suggesting that activation of PKC is necessary for inhibition of the channels. PMA reduces open probability of the channel in cell-attached patch clamp recordings. After inhibition by PMA in cell-attached recordings, application of PIP(2) to the cytoplasmic face of excised inside-out membranes restores channel activity. PMA reduces PIP(2) content in oocyte membrane and calphostin-C prevents the reduction. These results suggest that reduction of membrane PIP(2) content contributes to the inhibition of ROMK1 channels by PKC. This mechanism may underscore the inhibition of K(+) secretion in CCD by hormones that activate PKC.     

Modulation of cardiac Ca2+ channels in Xenopus oocytes by protein kinase C.

L-Type calcium channel was expressed in Xenopus laevis oocytes injected with RNAs coding for different cardiac Ca2+ channel subunits, or with total heart RNA. The effects of activation of protein kinase C (PKC) by the phorbol ester PMA (4 beta-phorbol 12-myristate 13-acetate) were studied. Currents through channels composed of the main (alpha 1) subunit alone were initially increased and then decreased by PMA. A similar biphasic modulation was observed when the alpha 1 subunit was expressed in combination with alpha 2/delta, beta and/or gamma subunits, and when the channels were expressed following injection of total rat heart RNA. No effects on the voltage dependence of activation were observed. The effects of PMA were blocked by staurosporine, a protein kinase inhibitor. beta subunit moderate the enhancement caused by PMA. We conclude that both enhancement and inhibition of cardiac L-type Ca2+ currents by PKC are mediated via an effect on the alpha 1 subunit, while the beta subunit may play a mild modulatory role.     

Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating

CFTR, the protein defective in cystic fibrosis, functions as a Cl- channel regulated by cAMP-dependent protein kinase (PKA). CFTR is also an ATPase, comprising two nucleotide-binding domains (NBDs) thought to bind and hydrolyze ATP. In hydrolyzable nucleoside triphosphates, PKA-phosphorylated CFTR channels open into bursts, lasting on the order of a second, from closed (interburst) intervals of a second or more. To investigate nucleotide interactions underlying channel gating, we examined photolabeling by [alpha32P]8-N3ATP or [gamma32P]8-N3ATP of intact CFTR channels expressed in HEK293T cells or Xenopus oocytes. We also exploited split CFTR channels to distinguish photolabeling at NBD1 from that at NBD2. To examine simple binding of nucleotide in the absence of hydrolysis and gating reactions, we photolabeled after incubation at 0 degrees C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30 degrees C, with extensive washing, also at 30 degrees C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30 degrees C nucleotide remained tightly bound at NBD1 for many minutes, in the form of nonhydrolyzed nucleoside triphosphate. As nucleotide-dependent gating of CFTR channels occurred on the time scale of seconds under comparable conditions, this suggests that the nucleotide interactions, including hydrolysis, that time CFTR channel opening and closing occur predominantly at NBD2. Vanadate also appeared to act at NBD2, presumably interrupting its hydrolytic cycle, and markedly delayed termination of channel open bursts. Vanadate somewhat increased the magnitude, but did not alter the rate, of the slow loss of nucleotide tightly bound at NBD1. Kinetic analysis of channel gating in Mg8-N3ATP or MgATP reveals that the rate-limiting step for CFTR channel opening at saturating [nucleotide] follows nucleotide binding to both NBDs. We propose that ATP remains tightly bound or occluded at CFTR's NBD1 for long periods, that binding of ATP at NBD2 leads to channel opening wherupon its hydrolysis prompts channel closing, and that phosphorylation acts like an automobile clutch that engages the NBD events to drive gating of the transmembrane ion pore.     

Palmitoylation and Membrane Association of the Stress Axis Regulated Insert (STREX) Controls BK Channel Regulation by Protein Kinase C

Large-conductance, calcium- and voltage-gated potassium (BK) channels play an important role in cellular excitability by controlling membrane potential and calcium influx. The stress axis regulated exon (STREX) at splice site 2 inverts BK channel regulation by protein kinase A (PKA) from stimulatory to inhibitory. Here we show that palmitoylation of STREX controls BK channel regulation also by protein kinase C (PKC). In contrast to the 50% decrease of maximal channel activity by PKC in the insertless (ZERO) splice variant, STREX channels were completely resistant to PKC. STREX channel mutants in which Ser(700), located between the two regulatory domains of K(+) conductance (RCK) immediately downstream of the STREX insert, was replaced by the phosphomimetic amino acid glutamate (S700E) showed a ～50% decrease in maximal channel activity, whereas the S700A mutant retained its normal activity. BK channel inhibition by PKC, however, was effectively established when the palmitoylation-mediated membrane-anchor of the STREX insert was removed by either pharmacological inhibition of palmitoyl transferases or site-directed mutagenesis. These findings suggest that STREX confers a conformation on BK channels where PKC fails to phosphorylate and to inhibit channel activity. Importantly, PKA which inhibits channel activity by disassembling the STREX insert from the plasma membrane, allows PKC to further suppress the channel gating independent from voltage and calcium. Our results present an important example for the cross-talk between ion channel palmitoylation and phosphorylation in regulation of cellular excitability.     

SPI-0211 activates T84 cell chloride transport and recombinant human ClC-2 chloride currents

The purpose of this study was to determine the mechanism of action of SPI-0211 (lubiprostone), a novel bicyclic fatty acid in development for the treatment of bowel dysfunction. Adult rabbit intestine was shown to contain mRNA for ClC-2 using RT-PCR, Northern blot analysis, and in situ hybridization. T84 cells grown to confluence on permeable supports were shown to express ClC-2 channel protein in the apical membrane. SPI-0211 increased electrogenic Cl- transport across the apical membrane of T84 cells, with an EC50 of approximately 18 nM measured by short-circuit current (Isc) after permeabilization of the basolateral membrane with nystatin. SPI-0211 effects on Cl- currents were also measured by whole cell patch clamp using the human embryonic kidney (HEK)-293 cell line stably transfected with either recombinant human ClC-2 or recombinant human cystic fibrosis transmembrane regulator (CFTR). In these studies, SPI-0211 activated ClC-2 Cl- currents in a concentration-dependent manner, with an EC50 of approximately 17 nM, and had no effect in nontransfected HEK-293 cells. In contrast, SPI-0211 had no effect on CFTR Cl- channel currents measured in CFTR-transfected HEK-293 cells. Activation of ClC-2 by SPI-0211 was independent of PKA. Together, these studies demonstrate that SPI-0211 is a potent activator of ClC-2 Cl- channels and suggest a physiologically relevant role for ClC-2 Cl- channels in intestinal Cl- transport after SPI-0211 administration.     

Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by cAMP-dependent phosphorylation and by intracellular ATP. Intracellular ATP also regulates a class of K+ channels that have a distinct pharmacology: they are inhibited by sulfonylureas and activated by a novel class of drugs called K+ channel openers. In search of modulators of CFTR Cl- channels, we examined the effect of sulfonylureas and K+ channel openers on CFTR Cl- currents in cells expressing recombinant CFTR. The sulfonylureas, tolbutamide and glibenclamide, inhibited whole-cell CFTR Cl- currents at half-maximal concentrations of approximately 150 and 20 microM, respectively. Inhibition by both agents showed little voltage dependence and developed slowly; > 90% inhibition occurred 3 min after adding 1 mM tolbutamide or 100 microM glibenclamide. The effect of tolbutamide was reversible, while that of glibenclamide was not. In contrast to their activating effect on K+ channels, the K+ channel openers, diazoxide, BRL 38227, and minoxidil sulfate inhibited CFTR Cl- currents. Half-maximal inhibition was observed at approximately 250 microM diazoxide, 50 microM BRL 38227, and 40 microM minoxidil sulfate. The rank order of potency for inhibition of CFTR Cl- currents was: glibenclamide < BRL 38227 approximately equal to minoxidil sulfate > tolbutamide > diazoxide. Site-directed mutations of CFTR in the first membrane-spanning domain and second nucleotide-binding domain did not affect glibenclamide inhibition of CFTR Cl- currents. However, when part of the R domain was deleted, glibenclamide inhibition showed significant voltage dependence. These agents, especially glibenclamide, which was the most potent, may be of value in identifying CFTR Cl- channels. They or related analogues might also prove to be of value in treating diseases such as diarrhea, which may involve increased activity of the CFTR Cl- channel.     

Severed channels probe regulation of gating of cystic fibrosis transmembrane conductance regulator by its cytoplasmic domains

Opening and closing of a CFTR Cl(-) channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634-836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1-633 and 837-1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them.     

Identification of a Protein Kinase C-dependent phosphorylation site involved in sensitization of TRPV4 channel

Transient Receptor Potential (TRP) proteins are non-selective cation channels performing diverse cellular functions. TRPV1 and TRPV4, two calcium-permeable channels of the vanilloid subfamily of TRP proteins, are activated by various physical and chemical stimuli, including noxious heat and mechanical stress, respectively. These channels are also required for exaggerated sensation of painful stimuli, condition referred to as hyperalgesia, which is frequently associated with inflammation. Phosphorylation of TRPV1, involving Protein Kinase C (PKC) and Protein Kinase A (PKA), appears to be the predominant mechanism for channel sensitization and development of heat hyperalgesia. PKC and PKA pathways have also been implicated in the sensitization of TRPV4, but the respective phosphorylation sites remain unknown. Using mass spectrometry, we report now that TRPV4 is phosphorylated on serine 824 by the PKC-activating phorbol 12-myristate 13-acetate. This phosphorylation is prevented by a PKC inhibitor, confirming the involvement of PKC. Ser824, located in the carboxy-terminal cytosolic tail of TRPV4, is also phosphorylated after activation of the PKA pathway by forskolin, albeit less potently. Substitution of Ser824 with aspartic acid, mimicking phosphorylation at this site, increased TRPV4-mediated calcium influx in resting and in stimulated cells, underlining the importance of this residue in TRPV4 regulation. Thus PKC, and possibly PKA, phosphorylate TRPV4 at Ser824 leading to the enhancement of TRPV4 channel function. Our findings suggest an important role of this phosphorylation in TRPV4 sensitization and the development of hyperalgesia.     

Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels

Background potassium channels determine membrane potential and input resistance and serve as prominent effectors for modulatory regulation of cellular excitability. TREK-1 is a two-pore domain background K+ channel (KCNK2, K2P2.1) that is sensitive to a variety of physicochemical and humoral factors. In this work, we used a recombinant expression system to show that activation of G alpha(q)-coupled receptors leads to inhibition of TREK-1 channels via protein kinase C (PKC), and we identified a critical phosphorylation site in a key regulatory domain that mediates inhibition of the channel. In HEK 293 cells co-expressing TREK-1 and either the thyrotropin-releasing hormone receptor (TRHR1) or the Orexin receptor (Orx1R), agonist stimulation induced robust channel inhibition that was suppressed by a bisindolylmaleimide PKC inhibitor but not by a protein kinase A blocker ((R(p))-cAMP-S). Channel inhibition by agonists or by direct activators of PKC (phorbol dibutyrate) and PKA (forskolin) was disrupted not only by alanine or aspartate mutations at an identified PKA site (Ser-333) in the C terminus, but also at a more proximal regulatory site in the cytoplasmic C terminus (Ser-300); S333A and S300A mutations enhanced basal TREK-1 current, whereas S333D and S300D substitutions mimicked phosphorylation and strongly diminished currents. When studied in combination, TREK-1 current density was enhanced in S300A/S333D but reduced in S300D/S333A mutant channels. Channel mutants were expressed and appropriately targeted to cell membranes. Together, these data support a sequential phosphorylation model in which receptor-induced kinase activation drives modification at Ser-333 that enables subsequent phosphorylation at Ser-300 to inhibit TREK-1 channel activity.