Deborah J. Nelson

CFTR regulates phagosome acidification in macrophages and alters bactericidal activity

Acidification of phagosomes has been proposed to have a key role in the microbicidal function of phagocytes. Here, we show that in alveolar macrophages the cystic fibrosis transmembrane conductance regulator Cl− channel (CFTR) participates in phagosomal pH control and has bacterial killing capacity. Alveolar macrophages from Cftr−/− mice retained the ability to phagocytose and generate an oxidative burst, but exhibited defective killing of internalized bacteria. Lysosomes from CFTR-null macrophages failed to acidify, although they retained normal fusogenic capacity with nascent phagosomes. We hypothesize that CFTR contributes to lysosomal acidification and that in its absence phagolysosomes acidify poorly, thus providing an environment conducive to bacterial replication.

Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms.

The cystic fibrosis gene encodes a cyclic AMP-gated chloride channel (CFTR) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. The molecular mechanisms that modulate CFTR activity in epithelial tissues are poorly understood. Here we show that CFTR is regulated by an epithelially expressed syntaxin (syntaxin 1A), a membrane protein that also modulates neurosecretion and calcium-channel gating in brain. Syntaxin 1A physically interacts with CFTR chloride channels and regulates CFTR-mediated currents both in Xenopus oocytes and in epithelial cells that normally express these proteins. The physical and functional interactions between syntaxin 1A and CFTR are blocked by a syntaxin-binding protein of the Munc18 protein family (also called n-Sec1; refs 12,13,14). Our results indicate that CFTR function in epithelial cells is regulated by an interplay between syntaxin and Munc18 isoforms.

Spatiotemporal Coupling of cAMP Transporter to CFTR Chloride Channel Function in the Gut Epithelia

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized at apical cell membranes and exists in macromolecular complexes with a variety of signaling and transporter molecules. Here, we report that the multidrug resistance protein 4 (MRP4), a cAMP transporter, functionally and physically associates with CFTR. Adenosine-stimulated CFTR-mediated chloride currents are potentiated by MRP4 inhibition, and this potentiation is directly coupled to attenuated cAMP efflux through the apical cAMP transporter. CFTR single-channel recordings and FRET-based intracellular cAMP dynamics suggest that a compartmentalized coupling of cAMP transporter and CFTR occurs via the PDZ scaffolding protein, PDZK1, forming a macromolecular complex at apical surfaces of gut epithelia. Disrupting this complex abrogates the functional coupling of cAMP transporter activity to CFTR function. Mrp4 knockout mice are more prone to CFTR-mediated secretory diarrhea. Our findings have important implications for disorders such as inflammatory bowel disease and secretory diarrhea.

Regulation of Human CLC-3 Channels by Multifunctional Ca2+/Calmodulin-dependent Protein Kinase

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 (ICl,CaMKII), and transfected this gene into the mammalian epithelial cell line tsA, which lacks endogenous expression of ICl,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.

Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands; it is well established that CFTR plays a pivotal role in cholera toxin (CTX)-induced secretory diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving gastrointestinal wound repair, apoptosis, inflammatory bowel disease, and diarrhea. Here we show, for the first time, that type 2 LPA receptors (LPA2) are expressed at the apical surface of intestinal epithelial cells, where they form a macromolecular complex with Na+/H+ exchanger regulatory factor–2 and CFTR through a PSD95/Dlg/ZO-1–based interaction. LPA inhibited CFTR-dependent iodide efflux through LPA2-mediated Gi pathway, and LPA inhibited CFTR-mediated short-circuit currents in a compartmentalized fashion. CFTR-dependent intestinal fluid secretion induced by CTX in mice was reduced substantially by LPA administration; disruption of this complex using a cell-permeant LPA2-specific peptide reversed LPA2-mediated inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea.

The distribution, activity, and function of the cilia in the frog brain

1. The distribution, activity, and function of the cilia in the brain was studied using in vitro preparations of the frog choroid plexuses and ependyma.

Alternative splicing of sur2 exon 17 regulates nucleotide sensitivity of the ATP-sensitive potassium channel

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(Δ17) 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(Δ17/39), SUR2(Δ17/40), and SUR2(Δ14/39). 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 ≈40 to ≈90 μm for exon 17 and Δ17, respectively. Single channel kinetic analysis of SUR2(39) and SUR2(Δ17/39) demonstrated that both exhibited characteristic KATP kinetics but that SUR2(Δ17/39) 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.

Membrane Capacitance Changes Associated with Particle Uptake during Phagocytosis in Macrophages

We report the use of capacitance measurements to monitor particle uptake after cellular exposure to phagocytic stimuli. In these studies, human monocyte-derived macrophages (HMDMs) and cells from the murine macrophage-like cell line J774.1 were exposed to immune complexes or sized latex particles (0.8 or 3.2 micron in diameter). An average decrease in cell capacitance of 8 pF was seen after exposure of the cells to immune complexes. Cells in which particle uptake was inhibited by cytochalasin B treatment before exposure to immune complexes showed an average increase of 0.5 pF. The decrease in membrane capacitance after exposure of cells to particulate stimuli was absent with the soluble stimulus, platelet-activating factor, further confirming that decreases in membrane capacitance were due to particle uptake. Exposure of cells to sized latex particles resulted in a graded, stepwise decrease in membrane capacitance. The average step size for 0.8-micron particles was 250 fF, and the average step change for the larger 3.2-micron particles was 480 fF, as calculated from Gaussian fits to the step size amplitude histograms. The predicted step size for the individual particles based upon the minimum amount of membrane required to enclose a particle and a specific capacitance of 10 fF/micron2 was 20 and 320 fF, respectively. The step size for the smaller particles deviates significantly from the predicted size distribution, indicating either a possible lower limit to the size of the phagocytic vacuole or multiple particles taken up within a single phagosome. Dynamic interaction between phagocytosis and exocytosis was observed in a number of cells as a biphasic response consisting of an initial rapid increase in capacitance, consistent with cellular exocytosis, followed by stepwise decreases in capacitance.

Structural determinant for assembly of mammalian K+ channels

K+ channel function is regulated through the assembly of channel subunit isoforms into either homo- or heterotetrameric structures each characterized by distinct pharmacologic and kinetic properties. In studying the molecular basis of subunit association in mammalian Shaker-like K+ channels, we constructed deletion mutants of the inactivating K+ channel hKv1.4 alone and in tandem with hKv1.5 and examined the functional properties electrophysiologically in Xenopus oocytes. Deletion of 255 amino acids in the amino-terminal domain of hKv1.4 prevented the formation of hybrid channels within the subfamily but had no effect on homomultimerization or voltage-dependent gating. The amino-terminal deletion mutant of Kv2.1, a noninactivating K+ channel from a distantly related subfamily also forms functional homomultimeric channels. Although members of different K+ channel subfamilies do not coassemble, coexpression of the amino-terminal deletion mutants of hKv1.4 and Kv2.1 resulted in the formation of functional hybrid channels. These results demonstrate that the amino-terminal region of mammalian K+ channels subserves two functions. It provides a recognition site necessary for hetero- but not homomultimeric channel assembly within a subfamily and prevents coassembly between subfamilies.

Disease causing mutations in the cystic fibrosis transmembrane conductance regulator determine the functional responses of alveolar macrophages

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, ΔF508, 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 ΔF508 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.