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Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Lysosomes and phagosomes in murine cftr(-/-) AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, DeltaF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR DeltaF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr(-/-), as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung.

Syntaxin 1A binds to and inhibits epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels and synaptic Ca(2+) channels in addition to participating in SNARE complex assembly and membrane fusion. We exploited the isoform-specific nature of the interaction between syntaxin 1A and CFTR to identify residues in the H3 domain of this SNARE (SNARE motif) that influence CFTR binding and regulation. Mutating isoform-specific residues that map to the surface of syntaxin 1A in the SNARE complex led to the identification of two sets of hydrophilic residues that are important for binding to and regulating CFTR channels or for binding to the syntaxin regulatory protein Munc-18a. None of these mutations affected syntaxin 1A binding to other SNAREs or the assembly and stability of SNARE complexes in vitro. Conversely, the syntaxin 1A-CFTR interaction was unaffected by mutating hydrophobic residues in the H3 domain that influence SNARE complex stability and Ca(2+) channel regulation. Thus, CFTR channel regulation by syntaxin 1A involves hydrophilic interactions that are mechanistically distinct from the hydrophobic interactions that mediate SNARE complex formation and Ca(2+) channel regulation by this t-SNARE.

The interplay between activated G proteins and intracellular calcium ([Ca(2+)](i)) in the regulation of secretion was studied in the macrophage, coupling membrane capacitance with calcium-sensitive microfluorimetry. Intracellular elevation of either the nonhydrolyzable analogue of GTP, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), or [Ca(2+)](i) enhanced the amplitude and shortened the time course of stimulus-induced secretion in a dose-dependent manner. Both the ionophore- and the stimulus-induced secretory response were abolished in the presence of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). The K(d) of Ca(2+)-driven secretion was independent of GTP gamma S concentration, whereas the K(d) of the GTP gamma S-driven response decreased from 63 to 31 microM in the presence of saturating concentrations of [Ca(2+)](i). The time course of stimulus-induced secretion was dependent upon the concentration of [Ca(2+)](i). The time course of GTP gamma S-driven secretion was concentration-independent at high levels of [Ca(2+)](i), suggesting that a calcium-dependent translocation/binding step was rate-limiting. Our data strongly support a model in which [Ca(2+)](i) and activated G proteins act independently of one another in the sequential regulation of macrophage secretion. [Ca(2+)](i) appears to play a role in the recruitment and priming of vesicles from reserve intracellular pools at a step that is upstream of G protein activation. While activated, G proteins appear to play a key role in fusion of docked vesicles. Thus, secretion can result either from activating more G proteins or from elevating [Ca(2+)](i) at basal levels of G protein activation.

The multifunctional calcium/calmodulin-dependent protein kinase II, CaMKII, has been shown to regulate chloride movement and cellular function in both excitable and non-excitable cells. We show that the plasma membrane expression of a member of the ClC family of Cl(-) channels, human CLC-3 (hCLC-3), a 90-kDa protein, is regulated by CaMKII. We cloned the full-length hCLC-3 gene from the human colonic tumor cell line T84, previously shown to express a CaMKII-activated Cl(-) conductance (I(Cl,CaMKII)), and transfected this gene into the mammalian epithelial cell line tsA, which lacks endogenous expression of I(Cl,CaMKII). Biotinylation experiments demonstrated plasma membrane expression of hCLC-3 in the stably transfected cells. In whole cell patch clamp experiments, autonomously active CaMKII was introduced into tsA cells stably transfected with hCLC-3 via the patch pipette. Cells transfected with the hCLC-3 gene showed a 22-fold increase in current density over cells expressing the vector alone. Kinase-dependent current expression was abolished in the presence of the autocamtide-2-related inhibitory peptide, a specific inhibitor of CaMKII. A mutation of glycine 280 to glutamic acid in the conserved motif in the putative pore region of the channel changed anion selectivity from I(-) > Cl(-) to Cl(-) > I(-). These results indicate that hCLC-3 encodes a Cl(-) channel that is regulated by CaMKII-dependent phosphorylation.

ATP-sensitive potassium channels (KATP) are implicated in a diverse array of physiological functions. Previous work has shown that alternative usage of exons 14, 39, and 40 of the muscle-specific KATP channel regulatory subunit, sur2, occurs in tissue-specific patterns. Here, we show that exon 17 of the first nucleotide binding fold of sur2 is also alternatively spliced. RNase protection demonstrates that SUR2(Delta17) predominates in skeletal muscle and gut and is also expressed in bladder, fat, heart, lung, liver, and kidney. Polymerase chain reaction and restriction digest analysis of sur2 cDNA demonstrate the existence of at least five sur2 splice variants as follows: SUR2(39), SUR2(40), SUR2(Delta17/39), SUR2(Delta17/40), and SUR2(Delta14/39). Electrophysiological recordings of excised, inside-out patches from COS cells cotransfected with Kir6.2 and the sur2 variants demonstrated that exon 17 splicing alters KATP sensitivity to ATP block by 2-fold from approximately 40 to approximately 90 microM for exon 17 and Delta17, respectively. Single channel kinetic analysis of SUR2(39) and SUR2(Delta17/39) demonstrated that both exhibited characteristic KATP kinetics but that SUR2(Delta17/39) exhibited longer mean burst durations and shorter mean interburst dwell times. In sum, alternative splicing of sur2 enhances the observed diversity of KATP and may contribute to tissue-specific modulation of ATP sensitivity.

The mechanism by which inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5, 6)P4) regulates chloride (Cl-) secretion was evaluated in the colonic epithelial cell line T84 using whole cell voltage clamp techniques. Our studies focused on the calcium-dependent chloride conductance (gClCa) that was activated either by mobilizing intracellular calcium (Cai) stores with thapsigargin or by introduction of the autonomous, autophosphorylated calmodulin-dependent protein kinase II (CaMKII) into the cell via the patch pipette. Basal concentrations of Ins(3,4,5,6)P4 (1 microM) present in the pipette solution had no significant effect on Cl- current; however, as the concentration of the polyphosphate was increased there was a corresponding reduction in anion current, with near complete inhibition at 8-10 microM Ins(3,4,5,6)P4. Corresponding levels are found in cells after sustained receptor-dependent activation of phospholipase C. The Ins(3,4,5, 6)P4-induced inhibition of gClCa was isomer specific; neither Ins(1, 3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, nor Ins(1,3,4,5,6)P5 induced current inhibition at concentrations of up to 100 microM. Annexin IV also plays an inhibitory role in modulating gClCa in T84 cells. When 2 microM annexin IV was present in the pipette solution, a concentration that by itself has no effect on gClCa, the potency of Ins(3,4,5,6)P4 was approximately doubled. The combination of Ins(3,4,5,6)P4 and annexin IV did not alter the in vitro activity of CaMKII. These data demonstrate that Ins(3,4,5,6)P4 is an additional cellular signal that participates in the control of salt and fluid secretion, pH balance, osmoregulation, and other physiological activities that depend upon gClCa activation. Ins(3,4,5,6)P4 metabolism and action should also be taken into account when designing treatment strategies for cystic fibrosis.

Stimulation of macrophages induces the "respiratory burst" response which is associated with the generation of superoxide (O2-), a drop in cytoplasmic pH, and a pronounced depolarization of the membrane potential. The purpose of the present studies was to determine whether an increase in O2- was temporally related to changes in membrane potential and transmembrane current. Release of O2- at the single cell level was photometrically monitored during phagocytosis of immune complexes while simultaneously measuring whole-cell current. Membrane depolarization and the generation of a non-selective current followed an increase in O2- production with a variable lag time which was correlated with the state of cellular maturation in culture. In the absence of phagocytosis, the exposure of macrophages to O2- generated by a xanthine-xanthine oxidase reaction activated a non-selective current similar to that seen after phagocytosis. These results provide the first demonstration of the relationship between free radical release and the ensuing electrophysiological signaling events which are linked to particle engulfment in phagocytic cells.

Ca(2+)-activated Cl- current (ICl,Ca) in colonic T84 cells is inhibited by the specific peptide inhibitor of Ca2+/calmodulin-dependent kinase II (CaM KII). Annexin IV, a Ca(2+)-dependent phospholipid binding protein also inhibits Ca(2+)-dependent anion current activation (Kaetzel, M.A., Chan, H.-C., Dubinsky, W.P., Dedman, J.R., and Nelson, D.J. (1994) J. Biol. Chem. 269, 5297-5302). Intracellular injection of antibodies against annexin IV enhances current activation; this activation is inhibited by the peptide inhibitor of CaM KII. Intracellular application of autonomously active CaM KII in the presence of ATP induced a Cl- current similar to that activated by the Ca2+ ionophore A23187. Current activation by the exogenous kinase was completely inhibited in the presence of purified annexin IV. In vitro, annexin IV does not inhibit CaM KII activity nor does it act as a substrate for CaM KII. Thus, it appears that annexin IV inhibits phosphorylation-dependent anion conductance activation by preventing CaM KII-ion channel interaction rather than by direct interaction with the enzyme itself. These findings suggest a novel mechanism by which Ca(2+)-dependent membrane binding proteins, cytoplasmic kinases, and ion channels interact to regulate membrane conductance. The characterization of unique channel regulatory pathways in Cl- transporting epithelia may identify potential avenues of alternate therapy to compensate for the loss of functional Cl- channels in the disease of cystic fibrosis.

The cellular function of annexin IV was evaluated by correlating tissue expression, cellular localization, and whole-cell electrophysiology. Immunolocalization and biochemical data demonstrate that annexin IV is concentrated along the apical membranes of many epithelia. Introduction of purified exogenous annexin IV into colonic T84 cells through a patch pipette specifically prevented Ca(2+)-dependent Cl- current activation. Affinity-purified antibody against annexin IV applied in the same manner enhanced the activation. Reduction of the endogenous level of annexin IV with a derivatized oligodeoxynucleotide antisense to annexin IV mRNA lowered the threshold for the Ca(2+)-induced current response, mimicking the enhancement of current activation exerted by anti-annexin IV antibody. The inhibitory effect of annexin IV on Ca(2+)-dependent Cl- conductance represents a novel mechanism by which Ca(2+)-binding proteins modulate membrane channel activity.

Intestinal chloride (Cl-) secretion can be induced by the heat-stable enterotoxin (STa) from Escherichia coli via generation of cGMP. We investigated the regulatory pathway responsible for cGMP-mediated Cl- secretion in the human colonic carcinoma cell line Caco-2 using whole-cell voltage clamp techniques. Cyclic GMP or cAMP induced a 5-fold increase in Cl- conductance (gCl) in the presence of intracellular ATP and 3-isobutyl-1-methylxanthine. Current activation by cGMP persisted in the presence of the type I cGMP-dependent protein kinase (PKG) inhibitor, KT5823, but was inhibited by the specific peptide inhibitor of the cAMP-dependent protein kinase A (PKA), PKI5-24. The stimulatory effects of cGMP and cAMP on gCl were not additive. The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by intracellular ATP and by cAMP-dependent phosphorylation. In order to determine whether CFTR was involved in the cGMP-dependent increase in gCl, we tested the effect of intracellularly injected anti-CFTR505-511 antibodies previously shown to inhibit CFTR function. Antibodies introduced into individual cells via the patch pipette completely inhibited cGMP-dependent current activation. Cyclic GMP also failed to activate gCl in cystic fibrosis cells. Taken together, these studies demonstrate that activation of the CFTR via PKA-dependent phosphorylation accounts for the cGMP-mediated increase in Cl- secretion in Caco-2 cells.

The interaction of short amino acid sequences is the basis of molecular recognition and biological regulation in many cellular systems. Libraries of random peptides provide an approach to identify peptides that can be used to modulate, in a targeted fashion, the function of specific gene products. We have used a library of random peptides designed and constructed in the M13 bacteriophage to select calcium-dependent calmodulin binding-peptides. Twenty-eight independent sequences were obtained; all contained a tryptophan within the fifteen-amino acid insert. In 11 sequences, the tryptophan was located in the first possible variable position of the inserted sequence and was followed by a proline. The tryptophan-proline combination was also present in six additional isolates but at various other positions within the peptide insert. Synthetic peptides, representative of the calmodulin binding sequences, bound to calmodulin in a calcium-dependent fashion, competed with known calmodulin inhibitors and, when introduced via a patch pipette, inhibited calcium-activated chloride conductance of the colonic epithelial cell line, T84. This report demonstrates the utility of identifying modifiers of biological function and should prove to be a valuable approach in understanding the cellular role of proteins of unknown function.

The cystic fibrosis (CF) phenotype is characterized by a regulatory defect in Cl- permeability in epithelia. A gene (250,000 base pairs) that is associated with this autosomal genetic disorder has been identified. To determine the cellular function of the recently cloned gene product, the cystic fibrosis transmembrane conductance regulator (CFTR), we have produced antibody against a synthetic peptide deduced from the CFTR cDNA sequence corresponding to positions 505-511. This site includes phenylalanine 508, the deletion of which is the most commonly expressed mutation in CF. We sought to determine whether the anti-CFTR505-511 peptide antibody could modulate the activation of the volume-sensitive, Ca(2+)-dependent, as well as the cAMP-dependent Cl- conductances present in the Cl(-)-secreting human colonic T84 cell line. Affinity-purified anti-CFTR505-511 antibody was introduced into the cytoplasm of individual T84 cells and its function studied using the whole-cell patch-clamp technique. Although cAMP-dependent Cl- current activation was inhibited in cells perfused with the anti-CFTR505-511 peptide antibody, Ca(2+)-dependent anion current activation remained unaffected. Chloride current activation, which accompanies cellular swelling, was partially attenuated in anti-CFTR505-511 antibody-loaded cells as compared with control cells perfused with either saline or irrelevant antibody. These results further support a role for CFTR in anion transport in epithelial cells and suggest its possible involvement in a number of anion transport pathways in chloride secretory epithelia.

Leishmania donovani and Leishmania braziliensis grown in culture formed millimolar concentrations of allopurinol ribonucleoside 5'-monophosphate from [6-14C]allopurinol. In addition, allopurinol 1-ribonucleoside, oxipurinol riboside 5'-monophosphate, and three new metabolites of allopurinol, namely, 4-aminopyrazolo(3,4-d)pyrimidine ribonucleoside 5'-monophosphate and the corresponding di- and triphosphates (1-ribosyl 4-aminopyrazolo(3,4-d)pyrimidine 5'-diphosphate and 1-ribosyl 4-aminopyrazolo(3,4-d)pyrimidine 5'-triphosphate) were identified in the parasitic cells. They were formed via a unique amination reaction from 1-ribosyl allopurinol 5'-phosphate, analogous to the conversion of IMP to AMP. [6-14C]Allopurinol was incorporated into RNA of L. donovani in the form of 4-aminopyrazolo(3,4-d)pyrimidine. Adenine reversed the growth inhibition of allopurinol and prevented its metabolism to all of the ribonucleotide metabolites. L. donovani was 2- to 4-fold more active in its metabolism of allopurinol to ribonucleotides than L. braziliensis. 4-Aminopyrazolo(3,4-d)pyrimidine inhibited cell growth and resulted in high intracellular levels of 1-ribosyl allopurinol 5'-phosphate and smaller amounts of the 4-aminopyrazolo(3,4-d)pyrimidine ribonucleotides. The metabolism of allopurinol to 4-aminopyrazolo(3,4-d)pyrimidine ribonucleotides and its resultant cytotoxicity occurs in these parasitic protozoans, but not in mammalian cells.

The growth of Leishmania donovani promastigotes and mouse L cells is differentially inhibited by several inosine analogs with modifications in the imidazole ring. The protozoal and mammalian cells also demonstrate differential metabolism of these analogs. 7-Deazainosine, 7-thia-7,9-dideazainosine, and formycin B were converted to their respective ATP analogs by both cell types. 8-Azainosine was converted to a GTP analog by mouse L cells; L. donovani did not metabolize this nucleoside. 9-Deazainosine and allopurinol riboside were metabolized only to their respective IMP analogs by L cells. L. donovani metabolized 9-deazainosine and allopurinol riboside to their ATP analogs and also metabolized 9-deazainosine to its GTP analog. All nucleosides studied were resistant to cleavage by either organism. From metabolism studies in the presence of a specific enzyme inhibitor, it was deduced that allopurinol riboside, formycin B, and 9-deazainosine were phosphorylated by at least two different routes in the mouse L cells. The metabolism of formycin B was inhibited 65% by the adenosine kinase inhibitor, 5-iodotubercidin, whereas the metabolism of allopurinol riboside (14% inhibition) and 9-deazainosine (0% inhibition) was only slightly affected by this inhibitor. The metabolism of allopurinol riboside and 9-deazainosine by L. donovani was not affected by 5-iodotubercidin. In contrast to the results of L cells, the metabolism of formycin B by L. donovani was also not affected by 5-iodotubercidin. The abilities of mouse L cells and L. donovani to metabolize these inosine analogs to the corresponding nucleotide analogs of ATP or GTP may be considered to be an activating step and correlates well with the respective cytotoxic effects of these compounds.