Selected Publications Compartmentalized cAMP at the plasma membrane clusters PDE3A and CFTR into microdomains. Penmasta, H., Zhang, W., Yarlagadda, S., Li, C., Conoley, V.G., Yue, J., Bahouth, S.W., Buddington, R.K., Zhang, G., Nelson, D.J., Sonecha, M.D., Manganiello, V., Wine, J.J., Naren, A.P. 2010. In Press Molecular Biology of the Cell.
Disease causing mutations in the cystic fibrosis transmembrane conductance regulator determine the functional responses of alveolar macrophages. Deriy, L.V., Gomez, E.A., Zhang, G., Beacham, D., Hopson, J.A., Gallan, A.J., Shevchenko, P., Bindokas, V.P., and Nelson, D.J. 2009. J. Biol. Chem. The granular chloride channel ClC-3 is permissive for insulin secretion. Deriy, L.V., Gomez, E.A., Jacobson, D.A., Wang, X.., Hopson, J.A., Liu, X.Y., Zhang, G., Bindokas, V.P., Philipson, L.H., and Nelson,, D.J. 2009. Cell Metabolism. The granular chloride channel CLC-3 is permissive for insulin secrection. Deriy LV, Gomez EA, Jacobson DA, Wang X, Hopson JA, Liu XY, Zhang G, Bindokas VP, Philipson LH, and Nelson DJ 2009. In Press. Cell Metabolism. An expanded biological repertoire for Ins(3,4,5,6)P4 through its modulation of ClC-3 function. Mitchell J, Wang X, Zhang G, Gentzsch M, Nelson DJ, Shears SB. Curr Biol. 2008 Oct 28;18(20):1600-5. Summary Ins(3,4,5,6)P4 inhibits plasma membrane Cl− flux in secretory epithelia [1]. However, in most other mammalian cells, receptor-dependent elevation of Ins(3,4,5,6)P4 levels is an “orphan” response that lacks biological significance [2]. We set out to identify Cl− channel(s) and/or transporter(s) that are regulated by Ins(3,4,5,6)P4 in vivo. Several candidates [3,4,5] were excluded through biophysical criteria, electrophysiological analysis, and confocal immunofluorescence microscopy. Then, we heterologously expressed ClC-3 in the plasma membrane of HEK293-tsA201 cells; whole-cell patch-clamp analysis showed Ins(3,4,5,6)P4 to inhibit Cl− conductance through ClC-3. Next, we heterologously expressed ClC-3 in the early endosomal compartment of BHK cells; by fluorescence ratio imaging of endocytosed FITC-transferrin, we recorded intra-endosomal pH, an in situ biosensor for Cl− flux across endosomal membranes [6]. A cell-permeant, bioactivatable Ins(3,4,5,6)P4 analog elevated endosomal pH from 6.1 to 6.6, reflecting inhibition of ClC-3. Finally, Ins(3,4,5,6)P4 inhibited endogenous ClC-3 conductance in postsynaptic membranes of neonatal hippocampal neurones. Among other ClC-3 functions that could be regulated by Ins(3,4,5,6)P4 are tumor cell migration [7], apoptosis [8], and inflammatory responses [9]. Ins(3,4,5,6)P4 is a ubiquitous cellular signal with diverse biological actions. Platel-activating factor-induced chloride channel activation is associated with intracellular acidosis and apoptosis of intestinal epithelial cells. Claud EC, Lu J, Wang XQ, Abe M, Petrof EO, Sun J, Nelson DJ, Marks J, Jilling T. Am J Physiol Gastrointest Liver Physiol. 2008 May;294(5):G1191-200. Epub 2008 Mar 13. Abstract Platelet-activating factor (PAF) is a phospholipid inter- and intracellular mediator implicated in intestinal injury primarily via induction of an inflammatory cascade. We find that PAF also has direct pathological effects on intestinal epithelial cells (IEC). PAF induces Cl(-) channel activation, which is associated with intracellular acidosis and apoptosis. Using the rat small IEC line IEC-6, electrophysiological experiments demonstrated that PAF induces Cl(-) channel activation. This PAF-activated Cl(-) current was inhibited by Ca(2+) chelation and a calcium calmodulin kinase II inhibitor, suggesting PAF activation of a Ca(2+)-activated Cl(-) channel. To determine the pathological consequences of Cl(-) channel activation, microfluorimetry experiments were performed, which revealed PAF-induced intracellular acidosis, which is also inhibited by the Cl(-) channel inhibitor 4,4'diisothiocyanostilbene-2,2'disulfonic acid and Ca(2+) chelation. PAF-induced intracellular acidosis is associated with caspase 3 activation and DNA fragmentation. PAF-induced caspase activation was abolished in cells transfected with a pH compensatory Na/H exchanger construct to enhance H(+) extruding ability and prevent intracellular acidosis. As ClC-3 is a known intestinal Cl(-) channel dependent on both Ca(2+) and calcium calmodulin kinase II phosphorylation, we generated ClC-3 knockdown cells using short hairpin RNA. PAF induced Cl(-) current; acidosis and apoptosis were all significantly decreased in ClC-3 knockdown cells. Our data suggest a novel mechanism of PAF-induced injury by which PAF induces intracellular acidosis via activation of the Ca(2+)-dependent Cl(-) channel ClC-3, resulting in apoptosis of IEC.Spatiotemporal coupling of cAMP transporter to CTFR chloride channel function in the gut epithelia. Claud EC, Lu J, Wang XQ, Abe M, Petrof EO, Sun J, Nelson DJ, Marks J, Jilling T. Am J Physiol Gastrointest Liver Physiol. 2008 May;294(5):G1191-200. Epub 2008 Mar 13. Summary 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. CFTR surface expression and chloride currents are decreased by inhibitors of N-WASP and actin polymerization. Ganeshan R, Nowotarski K, Di A, Nelson DJ, Kirk KL. Biochim Biophys Acta. 2007 Feb;1773(2):192-200. Epub 2006 Oct 3. Summary The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid turnover at the plasma membrane in various cell types. The ubiquitously expressed N-WASP promotes actin polymerization and regulates endocytic trafficking of other proteins in response to signaling molecules such as Rho-GTPases. In the present study we investigated the effects of wiskostatin, an N-WASP inhibitor, on the surface expression and activity of CFTR. We demonstrate, using surface biotinylation methods, that the steady-state surface CFTR pool in stably transfected BHK cells was dramatically decreased following wiskostatin treatment with a corresponding increase in the amount of intracellular CFTR. Similar effects were observed for latrunculin B, a specific actin-disrupting reagent. Both reagents strongly inhibited macroscopic CFTR-mediated Cl− currents in two cell types including HT29-Cl19A colonic epithelial cells. As previously reported, CFTR internalization from the cell surface was strongly inhibited by a cyclic-AMP cocktail. This effect of cyclic-AMP was only partially blunted in the presence of wiskostatin, which raises the possibility that these two factors modulate different steps in CFTR traffic. In kinetic studies wiskostatin appeared to accelerate the initial rate of CFTR endocytosis as well as inhibit its recycling back to the cell surface over longer time periods. Our studies implicate a role for N-WASP-mediated actin polymerization in regulating CFTR surface expression and channel activity.
 CLC-3 channels modulate excitatory synaptic transmission in hippocampal neurons. Wang XQ, Deriy LV, Foss S, Huang P, Lamb FS, Kaetzel MA, Bindokas V, Marks JD, Nelson DJ. Neuron. 2006 Oct 19;52(2):321-33. AbstractIt is well established that ligand-gated chloride flux across the plasma membrane modulates neuronal excitability. We find that a voltage-dependent Cl(-) conductance increases neuronal excitability in immature rodents as well, enhancing the time course of NMDA receptor-mediated miniature excitatory postsynaptic potentials (mEPSPs). This Cl(-) conductance is activated by CaMKII, is electrophysiologically identical to the CaMKII-activated CLC-3 conductance in nonneuronal cells, and is absent in clc-3(-/-) mice. Systematically decreasing [Cl(-)](i) to mimic postnatal [Cl(-)](i) regulation progressively decreases the amplitude and decay time constant of spontaneous mEPSPs. This Cl(-)-dependent change in synaptic strength is absent in clc-3(-/-) mice. Using surface biotinylation, immunohistochemistry, electron microscopy, and coimmunoprecipitation studies, we find that CLC-3 channels are localized on the plasma membrane, at postsynaptic sites, and in association with NMDA receptors. This is the first demonstration that a voltage-dependent chloride conductance modulates neuronal excitability. By increasing postsynaptic potentials in a Cl(-) dependent fashion, CLC-3 channels regulate neuronal excitability postsynaptically in immature neurons.  CFTR regulates phagosome acidification in macrophages and alerts bacterial activity.
Di A, Brown ME, Deriy LV, Li C, Szeto FL, Chen Y, Huang P, Tong J, Naren AP, Bindokas V, Palfrey HC, Nelson DJ. Nat Cell Biol. 2006 Sep;8(9):933-44. Epub 2006 Aug 20. Abstract: 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.
Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions. Li C, Dandridge
KS, Di A, Marrs KL, Harris EL, Roy K, Jackson JS, Makarova NV, Fujiwara
Y, Farrar PL, Nelson DJ, Tigyi GJ, Naren AP. J Exp Med. 2005 October 3; 202(7): 975–986. Abstract 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.Infectious diarrhea disease is second only to cardiovascular disease as cause of death (1) and is one of the major causes of morbidity and mortality among children. It accounts for 21% of deaths of children who are younger than 5 yr of age in the developing world, and causes 2.5 million deaths per year (2). Although incidence rates increase with a country's decreasing socioeconomic status, infectious diarrhea also is a common illness in Western industrialized societies, particularly among vulnerable groups of young children, elderly adults, and people who have underlying diseases (3). Two major organisms that cause infectious diarrhea in humans are Escherichia coli and Vibrio cholerae; their secreted toxins (heat stable or heat labile toxin, and cholera toxin [CTX], respectively) influence gastrointestinal epithelial cell function and induce diarrhea by numerous mechanisms (4). CTX is likely the most recognizable enterotoxin that causes secretory diarrhea by stimulating transepithelial Cl– secretion, thereby increasing the osmotic impetus for fluid secretion (5). Diarrhea also can result from an increased immunoinflammatory response that is triggered by cytokine or mediator (e.g., adenosine; ADO) secretion from intestinal mucosal inflammatory cells in response to luminal factors (e.g., dietary or bacterial antigens). This also is a common symptom that is associated with the major gastrointestinal disorders, such as inflammatory bowel disease (IBD) afflicting >1 million Americans (6), and irritable bowel syndrome, which affects 15 million Americans (7). Mucosal inflammation plays an essential role in the pathogenesis of irritable bowel syndrome and IBD (8, 9). ADO, an inflammatory mediator, stimulated chloride secretion in several types of secretory epithelia, including mammalian ileum and colon (10, 11). Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel that is localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands (12); it is well-established that CFTR plays a pivotal role in CTX-mediated diarrhea (13). Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods (14, 15), acts on the LPA receptors (LPA1, LPA2, and LPA3), which are G-protein coupled receptors that belong to the endothelial cell differentiation gene family (16–18). LPA was reported to play an essential role in a variety of conditions involving gastrointestinal wound repair, apoptosis, IBD, and diarrhea (19–23). In the present study, we investigated how LPA-elicited LPA2-mediated signaling might regulate CFTR function in the gut, and evaluated the usefulness of LPA in preventing CTX-induced secretory diarrhea in mice. Full text [PDF] 5-amino-imidazole carboxamide riboside acutely potentiates glucose-stimulated insulin secretion from mouse pancreatic islets by KATP channel-dependent and -independent pathways. Wang CZ, Wang Y, Di A, Magnuson MA, Ye H, Roe MW, Nelson DJ, Bell GI, Philipson LH. Biochem Biophys Res Commun. 2005 May 20;330(4):1073-9. Abstract AMP-activated protein kinase (AMPK) is an important signaling effector that couples cellular metabolism and function. The effects of AMPK activation on pancreatic β-cell function remain unresolved. We used 5-amino-imidazole carboxamide riboside (AICAR), an activator of AMPK, to define the signaling mechanisms linking the activation of AMPK with insulin secretion. Application of 300 μM AICAR to mouse islets incubated in 5–14 mM glucose significantly increased AMPK activity and potentiated insulin secretion. AICAR inhibited ATP-sensitive K+ (KATP) channels and increased the frequency of glucose-induced calcium oscillations in islets incubated in 8–14 mM glucose. At lower glucose concentration (5 mM) AICAR did not affect KATP activity or intracellular ([Ca2+]i). AICAR also did not inhibit 86Rb+ efflux from islets isolated from Sur1−/− mice that lack KATP channels yet significantly potentiated glucose stimulated insulin secretion. Our data suggest that AICAR stimulates insulin secretion by both KATP channel-dependent and -independent pathways.
Mutation of critical GIRK subunit residues disrupts N- and C-termini association and channel function. Sarac R, Hou P, Hurley KM, Hriciste D, Cohen NA, Nelson DJ. J Neurosci. 2005 Feb 16;25(7):1836-46. Abstract The subfamily of G-protein-linked inwardly rectifying potassium channels (GIRKs) is coupled to G-protein receptors throughout the CNS and in the heart. We used mutational analysis to address the role of a specific hydrophobic region of the GIRK1 subunit. Deletion of the GIRK1 C-terminal residues 330-384, as well as the point mutation I331R, resulted in a decrease in channel function when coexpressed with GIRK4 in oocytes and in COS-7 cells. Surface protein expression of GIRK1 I331R coexpressed with GIRK4 was comparable with wild type, indicating that subunits assemble and are correctly localized to the membrane. Subsequent mutation of homologous residues in both the GIRK4 subunit and Kir2.1 (G  -independent inward rectifier) also resulted in a decrease in channel function. Intracellular domain associations resulted in the coimmunoprecipitation of the GIRK1 N and C termini and GIRK4 N and C termini. The point mutation I331R in the GIRK1 C terminus or L337R in the GIRK4 C terminus decreased the association between the N and C termini. Mutation of a GIRK1 N-terminal hydrophobic residue, predicted structurally to interact with the C-terminal domain, also resulted in a decrease in channel function and termini association. We hypothesize that the hydrophobic nature of this GIRK1 subunit region is critical for interaction between adjacent termini and is permissive for channel gating. In addition, the homologous mutation in cytoplasmic domains of Kir2.1 (L330R) did not disrupt association, suggesting that the overall structural integrity of this region is critical for inward rectifier function.
A process for controlling intracellular bacterial infections induced by membrane injury. Roy D, Liston DR, Idone VJ, Di A, Nelson DJ, Pujol C, Bliska JB, Chakrabarti S, Andrews NW. Science. 2004 Jun 4;304(5676):1515-8. Abstract Strategies for inhibiting phagolysosome fusion are essential for the intracellular survival and replication of many pathogens. We found that the lysosomal synaptotagmin Syt VII is required for a mechanism that promotes phagolysosomal fusion and limits the intracellular growth of pathogenic bacteria. Syt VII was required for a form of Ca2+-dependent phagolysosome fusion that is analogous to Ca2+-regulated exocytosis of lysosomes, which can be triggered by membrane injury. Bacterial type III secretion systems, which permeabilize membranes and cause Ca2+ influx in mammalian cells, promote lysosomal exocytosis and inhibit intracellular survival in Syt VII +/+ but not –/– cells. Thus, the lysosomal repair response can also protect cells against pathogens that trigger membrane permeabilization.
Identification of an N-terminal amino acid of the CLC-3 chloride channel critical in phosphorylation-dependent activation of a CaMKII-activated chloride current. Robinson NC, Huang P, Kaetzel MA, Lamb FS, Nelson DJ. J Physiol. 2004 Apr 15;556(Pt 2):353-68. Epub 2004 Jan 30. Abstract CLC-3, a member of the CLC family of chloride channels, mediates function in many cell types in the body. The multifunctional calcium–calmodulin-dependent protein kinase II (CaMKII) has been shown to activate recombinant CLC-3 stably expressed in tsA cells, a human embryonic kidney cell line derivative, and natively expressed channel protein in a human colonic tumour cell line T84. We examined the CaMKII-dependent regulation of CLC-3 in a smooth muscle cell model as well as in the human colonic tumour cell line, HT29, using whole-cell voltage clamp. In CLC-3-expressing cells, we observed the activation of a Cl− conductance following intracellular introduction of the isolated autonomous CaMKII into the voltage-clamped cell via the patch pipette. The CaMKII-dependent Cl− conductance was not observed following exposure of the cells to 1 μm autocamtide inhibitory peptide (AIP), a selective inhibitor of CaMKII. Arterial smooth muscle cells express a robust CaMKII-activated Cl− conductance; however, CLC-3−/− cells did not. The N-terminus of CLC-3, which contains a CaMKII consensus sequence, was phosphorylated by CaMKII in vitro, and mutation of the serine at position 109 (S109A) abolished the CaMKII-dependent Cl− conductance, indicating that this residue is important in the gating of CLC-3 at the plasma membrane. Chloride channels are found ubiquitously in the body and control a myriad of cellular functions, including excitability, secretion, volume regulation, and salt balance. Historically, chloride channels have been classified based on their single-channel conductance, anion selectivity, cellular localization, or mechanism of regulation; yet none of these alone can completely and accurately categorize the enormous diversity of anion channels identified. However, one chloride channel gene family that has been well defined is the CLC family (Jentsch et al. 1999, 2002). The CLC family of voltage-gated chloride channels consists of nine cloned mammalian members, including CLC-3, which shares approximately 80% homology with the intracellular chloride channels CLC-4 and CLC-5 (Jentsch, 1996). CLC-3 has a ubiquitous expression pattern, but is found highly concentrated in brain, most notably in the olfactory bulb, hippocampus, and cerebellum (Kawasaki et al. 1994). The CLC-3−/− knockout mouse phenotype displays blindness and severe neurodegeneration, specifically of the hippocampus (Stobrawa et al. 2001). In addition to its neuronal prevalence, CLC-3 is also notably expressed in secretory epithelia in the kidney and colon. While tissue expression of CLC-3 is well characterized, cellular localization has been more elusive. Although the highly homologous CLC-4 and CLC-5 have been localized internally (Gunther et al. 1998), CLC-3 has been shown to be both an intracellular (Stobrawa et al. 2001) and plasma membrane-resident protein (Huang et al. 2001; Weylandt et al. 2001). Recent studies report the existence of alternate splice variant products of CLC-3, CLC-3A and CLC-3B, each with different subcellular localizations (Gentzsch et al. 2002; Ogura et al. 2002). While broad expression and physiological importance of CLC-3 are established, the mechanism of channel activation remains controversial. CLC-3 has been proposed to be the ubiquitous volume-regulated channel (Duan et al. 1997; Yamazaki et al. 1998; von Weikersthal et al. 1999). However, experiments in cells lacking CLC-3 have concluded that CLC-3 is not the swelling-activated chloride channel (Li et al. 2000; Huang et al. 2001; Stobrawa et al. 2001; Weylandt et al. 2001). Additionally, it has been reported that CLC-3 is Ca2+ regulated, either via a direct pathway (Kawasaki et al. 1995) or through the multifunctional Ca2+–calmodulin-dependent protein kinase II (CaMKII) (Huang et al. 2001), while a knockout study reports no loss of an ionophore-induced Ca2+-activated Cl− conductance (Arreola et al. 2002). It should be noted that CLC-3 has multiple splice variants and the differential expression of each of these in various cell types may lead to diverse regulation of the channel. Regulated Ca2+-dependent Cl− conductances have been described in a diversity of cell types, including neurones, smooth muscle and secretory epithelia. The multifunctional CaMKII is a major mediator of Ca2+ signalling and is abundant in the brain, accounting for approximately 2% of total protein in the hippocampus (Erondu & Kennedy, 1985). Phosphorylation-dependent gating of Cl− channels by CaMKII is well established in many cell types (Wagner et al. 1991; Worrell & Frizzell, 1991; Chan et al. 1994; Holevinsky et al. 1994; Kaetzel et al. 1994; Xie et al. 1996, 1998). Previous work in our laboratory has demonstrated that CLC-3 exhibits CaMKII-dependent channel gating in a stable cell line expressing recombinant human CLC-3 and in T84 cells expressing CLC-3 endogenously. In both cell types, the currents activated by autonomously active CaMKII are identical in their biophysical properties. While CaMKII activates the long form of human CLC-3 (hCLC-3) when expressed at the plasma membrane, the mechanism of channel activation when expressed in the cytoplasmic compartment remains uninvestigated. High levels of CLC-3 expression in the brain, kidney and colon emphasize the cellular and physiological importance of this channel in secretory processes. In the present study, we set out to establish the molecular identity of the endogenous CaMKII-activated Cl− current (ICl,CaMKII). Using a combination of both molecular biological as well as electrophysiological studies, we determined that ICl,CaMKII native in smooth muscle and transfected tsA cells is due to the activation of CLC-3. The channel localizes to both cytoplasmic as well as a plasma membrane site in HT29 cells, where its activation by the kinase is not dependent upon a translocation event between the two compartments. Furthermore, the serine at position 109 in hCLC-3 is necessary for phosphorylation and kinase activation of CLC-3, and mutation of this serine to an alanine results in a loss of CaMKII-activated Cl− conductance. Taken together, these data indicate that CLC-3 expressed at the plasma membrane is a Cl− conductance activated by CaMKII-dependent phosphorylation at S109.
Dynamin regulates focal exocytosis in phagocytosing macrophages. Di A, Nelson DJ, Bindokas V, Brown ME, Libunao F, Palfrey HC. Mol Biol Cell. 2003 May;14(5):2016-28. Epub 2003 Feb 21. Abstract Phagocytosis in macrophages is thought to involve insertion of cytoplasmic vesicles at sites of membrane expansion before particle ingestion ("focal" exocytosis). Capacitance (Cm) measurements of cell surface area were biphasic, with an initial rise indicative of exocytosis followed by a fall upon phagocytosis. Unlike other types of regulated exocytosis, the Cm rise was insensitive to intracellular Ca2+, but was inhibited by guanosine 5'-O-(2-thio)diphosphate. Particle uptake, but not Cm rise, was affected by phosphatidylinositol 3-kinase inhibitors. Inhibition of actin polymerization eliminated the Cm rise, suggesting possible coordination between actin polymerization and focal exocytosis. Introduction of anti-pan-dynamin IgG blocked Cm changes, suggesting that dynamin controls focal exocytosis and thereby phagocytosis. Similarly, recombinant glutathione S-transferase amphiphysin-SH3 domain, but not a mutated form
that cannot bind to dynamin, inhibited both focal exocytosis and phagocytosis.
Immunochemical analysis of endogenous dynamin distribution in macrophages
revealed a substantial particulate pool, some of which localized to a
presumptive endosomal compartment. Expression of enhanced green fluorescent
proteindynamin-2 showed a motile dynamin pool, a fraction of which
migrated toward and within the phagosomal cup. These results suggest that
dynamin is involved in the production and/or movement of vesicles from an
intracellular organelle to the cell surface to support membrane expansion
around the engulfed particle.
The interaction between syntaxin 1A and cystic fibrosis transmembrane conductance regulator Cl- channels is mechanistically distinct from syntaxin 1A-SNARE interactions. Ganeshan R, Di A, Nelson DJ, Quick MW, Kirk KL. J Biol Chem. 2003 Jan 31;278(5):2876-85. Epub 2002 Nov 22. Abstract Syntaxin 1A binds to and inhibits epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels and synaptic Ca2+ 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 Ca2+ 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 Ca2+ channel regulation by this t-SNARE. The cystic fibrosis transmembrane conductance regulator (CFTR)1 is a cyclic AMP-activated anion channel that mediates salt and fluid transport across epithelial cells (1). The CFTR protein consists of a symmetric arrangement of two membrane-spanning regions, two nucleotide binding domains (nucleotide binding domains 1 and 2), and a central regulatory domain with multiple phosphorylation sites (2). The amino-terminal cytoplasmic tail binds to syntaxin 1A, a component of the membrane traffic machinery, and this interaction is blocked by Munc-18, a high affinity, syntaxin-binding protein (3). Syntaxin 1A inhibits CFTR-mediated chloride currents in a variety of cell types and expression systems. This effect of syntaxin 1A on CFTR channel activity may be due to the fact that the amino-terminal cytoplasmic tail of CFTR, to which syntaxin 1A binds, regulates channel gating apparently by interacting with the regulatory domain and/or nucleotide binding domain 1 (4). Syntaxin 1A is a t-SNARE that is highly expressed in neurons and, to a lesser extent, in a variety of epithelial cells (5). Together with SNAP-25 (another t-SNARE) and VAMP-2/synaptobrevin (a v-SNARE), syntaxin 1A assembles into core SNARE complexes that regulate membrane fusion at the presynaptic membrane in neurons. The ternary SNARE complex consists of a parallel four-helix bundle containing one coiled-coil domain each from syntaxin and VAMP-2/synaptobrevin and two from SNAP-25 (6). The four associating α-helices contain hydrophobic residues that are grouped into “layers” that zipper together the four-helix bundle, resulting directly or indirectly in bilayer mixing. This paradigm applies to nonneuronal as well as to neuronal fusion events; in the former case, other SNARE isoforms are involved. The interactions among v-SNAREs and t-SNAREs are typically nonselective, at least in vitro (7), presumably because hydrophobic residues that are common to multiple isoforms mediate these interactions. The helical region of syntaxin 1A that participates in SNARE complex assembly, termed the SNARE motif or the H3 domain (8), is proximal to the COOH-terminal membrane anchor region and lies downstream of another helical domain referred to as Habc. The SNARE motif, which forms an amphipathic helix, is composed of residues that are arranged in a heptad repeat manner designated a–g. The residues that map to heptad positions a and d are typically hydrophobic, well conserved across various syntaxin isoforms, and usually buried in the hydrophobic core of the SNARE complex. Conversely, residues at the b, c, andf heptad positions are more variable and are exposed on the surface of the ternary complex. The cytoplasmic domain of syntaxin 1A, encompassing the Habc and H3 domains, also interacts with Munc-18a with high affinity, and this interaction prevents syntaxin 1A from participating in SNARE complex assembly in vitro (9). In addition to its role in the assembly of SNARE complexes, syntaxin 1A also has been reported to modulate multiple types of ion channels and transporters (3, 10-12). For example, syntaxin 1A inhibits the activities of several types of voltage-gated Ca2+ channels in a variety of expression systems and cell types. It has been proposed that the simultaneous association of syntaxin 1A with synaptic vesicle proteins and voltage-gated Ca2+ channels may help couple SNARE complex formation/dissociation to the influx of Ca2+at neurotransmitter release sites (13). With respect to CFTR-syntaxin interactions, syntaxin 1A, but not any other isoform that has been tested (i.e. syntaxins 2–5), binds to the CFTR amino-terminal tail (N-tail) and inhibits channel activity (5).2 In addition, CFTR binds to the H3 domain of syntaxin 1A, and soluble syntaxin 1A peptides that lack the transmembrane region but include the H3 region can rescue CFTR-mediated currents from inhibition by membrane-anchored syntaxin 1A (,3, 5). As argued for the syntaxin 1A-Ca2+ channel interaction, the interaction between this t-SNARE and CFTR channels may help synchronize the activity of this channel with protein traffic in epithelial cells. Thus, the ability of syntaxin 1A to influence the function of ion channels and to couple their activity to membrane traffic may be a general phenomenon. However, it is not clear if the structural basis of the interactions between syntaxin 1A and different ion channels is similar. In this regard, Bezprozvanny et al.(14) have reported that the regulation of voltage-gated Ca2+ channels by syntaxin 1A is disrupted by point mutations in specific hydrophobic residues in the H3 domain that are also implicated in SNARE complex stability. In the present study, we exploited the isoform specificity of the syntaxin 1A interaction with CFTR to identify unique residues in the H3 domain of syntaxin 1A that participate in CFTR binding and channel regulation. These residues are hydrophilic and are located in the outer shell of the SNARE complex structure (i.e. distinct from those in the hydrophobic layers that stabilize the SNARE complex). Mutating these residues diminished both the physical and functional interactions of syntaxin 1A with CFTR but had no effect on the biochemical association of syntaxin 1A with other SNARE proteins or with Munc-18. Conversely, the CFTR-syntaxin 1A interaction was not affected by mutations in specific hydrophobic residues of the H3 domain that disrupt Ca2+ channel regulation and compromise SNARE stability. Our results indicate that syntaxin 1A regulates CFTR channels by a protein-protein interaction that is mechanistically distinct from how syntaxin 1A participates in SNARE complex assembly or regulates voltage-gated calcium channels.
CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex. Cormet-Boyaka E, Di A, Chang SY, Naren AP, Tousson A, Nelson DJ, Kirk KL. Proc Natl Acad Sci U S A. 2002 Sep 17;99(19):12477-82. Epub 2002 Sep 3. Abstract Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion reactions in eukaryotic cells by assembling into complexes that link vesicle-associated SNAREs with SNAREs on target membranes (t-SNAREs). Many SNARE complexes contain two t-SNAREs that form a heterodimer, a putative intermediate in SNARE assembly. Individual t-SNAREs (e.g., syntaxin 1A) also regulate synaptic calcium channels and cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial chloride channel that is defective in cystic fibrosis. Whether the regulation of ion channels by individual t-SNAREs is related to SNARE complex assembly and membrane fusion is unknown. Here we show that CFTR channels are coordinately regulated by two cognate t-SNAREs, SNAP-23 (synaptosome-associated protein of 23 kDa) and syntaxin 1A. SNAP-23 physically associates with CFTR by binding to its amino-terminal tail, a region that modulates channel gating. CFTR-mediated chloride currents are inhibited by introducing excess SNAP-23 into HT29-Cl.19A epithelial cells. Conversely, CFTR activity is stimulated by a SNAP-23 antibody that blocks the binding of this t-SNARE to the CFTR amino-terminal tail. The physical and functional interactions between SNAP-23 and CFTR depend on syntaxin 1A, which binds to both proteins. We conclude that CFTR channels are regulated by a t-SNARE complex that may tune CFTR activity to rates of membrane traffic in epithelial cells. The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel that localizes to the apical membranes of epithelial cells lining the airways, intestine, and exocrine glands (1). A major role of CFTR is to mediate salt and water secretion in submucosal glands and intestinal crypts, which lubricates the mucosal surface and helps deliver simultaneously secreted proteins out of the gland or crypt. Excessive activation of the CFTR chloride channel causes secretory diarrhea whereas defective synthesis or function leads to cystic fibrosis (2, 3). The CFTR polypeptide contains two cytoplasmic tails, two membrane-spanning domains, two nucleotide binding domains (NBDs), and a large regulatory domain (R domain) that is phosphorylated by cAMP-dependent protein kinase (4). The NBDs and R domain are the major domains that control channel gating, although the amino-terminal tail modulates channel activity as well (5, 6). The amino-terminal tail also binds syntaxin 1A, a t-SNARE (t for target and SNARE for soluble N-ethylmaleimide-sensitive factor attachment protein receptor) that mediates membrane fusion reactions in cells (7, 8). The role of syntaxin 1A in membrane fusion has been best characterized for neurons, where this t-SNARE localizes to the presynaptic plasma membrane. Syntaxin 1A forms a ternary complex with another t-SNARE at the plasma membrane, SNAP-25 (synaptosome-associated protein of 25 kDa), and with vesicle-associated membrane protein-2/synaptobrevin, a vesicle-associated SNARE on synaptic vesicles (9, 10). The ternary SNARE complex is an important mediator of synaptic vesicle fusion, a paradigm that applies to membrane fusion reactions in all types of eukaryotic cells. The t-SNAREs themselves are capable of forming heterodimers that have been argued to be intermediates in SNARE complex assembly and membrane fusion (11, 12). In addition to regulating membrane fusion, syntaxin 1A binds to and modulates several ion channels including CFTR, voltage-gated calcium channels, and epithelial sodium channels (13–19). Syntaxin 1A inhibits CFTR-mediated chloride currents in heterologous expression systems (7, 8). Reagents that block the physical interaction between syntaxin 1A and the amino-terminal tail of CFTR potentiate CFTR-mediated currents in epithelial cells. The kinetic properties of voltage-gated calcium channels are modified by coexpression with syntaxin 1A or SNAP-25 in Xenopus oocytes or HEK293 cells (14, 15, 18, 20). For example, Zhong et al. (18) reported that SNAP-25 inhibited the activity of P/Q-type Ca2+ channels by shifting the voltage dependence of inactivation to more negative potentials. Coexpressing syntaxin 1A with SNAP-25 partially restored the normal voltage dependence of inactivation. Zhong et al. (18) interpreted these results to indicate that individual t-SNAREs bind to and inhibit voltage-gated calcium channels possibly as a means to couple calcium channels to the vesicle fusion machinery at sites of neurotransmitter release. The fact that syntaxin 1A reversed the effects of SNAP-25 on the gating of P/Q-type calcium channels implies that individual t-SNAREs rather than the t-SNARE heterodimer are more potent regulators of this ion channel. Here we determined whether CFTR channels are regulated by a t-SNARE complex in epithelial cells, or alternatively, if this ion channel is reciprocally regulated by individual t-SNAREs as argued for P/Q-type calcium channels. We focused on SNAP-23, a SNAP-25 homologue, as a potential regulator of CFTR because: (i) this t-SNARE is expressed in many tissues including lung, intestine, and pancreas, tissues that also express CFTR (21, 22) and (ii) SNAP-23 binds to syntaxin 1A in vitro (21) and can be cross-linked to syntaxin 1A in epithelial cells in vivo (unpublished results). Our results indicate that epithelial CFTR channels are regulated by a t-SNARE complex composed of both SNAP-23 and syntaxin 1A. The interaction of CFTR with a putative intermediate in SNARE complex assembly may link its activity to protein secretion in epithelial tissues.
Quantal release of free radicals during exocytosis of phagosomes. Di A, Krupa B, Bindokas VP, Chen Y, Brown ME, Palfrey HC, Naren AP, Kirk KL, Nelson DJ. Nat Cell Biol. 2002 Apr;4(4):279-85. Abstract Secretion of lysosomes and related organelles is important for immune system function. High-resolution membrane capacitance techniques were used to track changes in membrane area in single phagocytes during opsonized polystyrene bead uptake and release. Secretagogue stimulation of cells preloaded with beads resulted in immediate vesicle discharge, visualized as step increases in capacitance. The size of the increases were consistent with phagosome size. This hypothesis was confirmed by direct observation of dye release from bead-containing phagosomes after secretagogue stimulation. Capacitance recordings of exocytosis were correlated with quantal free radical release, as determined by amperometry. Thus, phagosomes undergo regulated secretion in macrophages, one function of which may be to deliver sequestered free radicals to the extracellular space. Full text [PDF]Mechanisms of CFTR regulation by syntaxin 1A and PKA. Chang SY, Di A, Naren AP, Palfrey HC, Kirk KL, Nelson DJ. J Cell Sci. 2002 Feb 15;115(Pt 4):783-91. Summary Activation of the chloride selective anion channel CFTR is stimulated by cAMP-dependent phosphorylation and is regulated by the target membrane t-SNARE syntaxin 1A. The mechanism by which SNARE proteins modulate CFTR in secretory epithelia is controversial. In addition, controversy exists as to whether PKA activates CFTR-mediated Cl- currents (ICFTR) by increasing the number of channels in the plasma membrane and/or by stimulating membrane-resident channels. SNARE proteins play a well known role in exocytosis and have recently been implicated in the regulation of ion channels; therefore this investigation sought to resolve two related issues: (a) is PKA activation or SNARE protein modulation of CFTR linked to changes in membrane turnover and (b) does syntaxin 1A modulate CFTR via direct effects on the gating of channels residing in the plasma membrane versus alterations in membrane traffic. Our data demonstrate that syntaxin 1A inhibits CFTR as a result of direct protein-protein interactions that decrease channel open probability (Po) and serves as a model for other SNARE protein-ion channel interactions. We also show that PKA activation can enhance membrane trafficking in some epithelial cell types, and this is independent from CFTR activation or syntaxin 1A association. Priming of insulin granules for exocytosis by granular Cl(-) uptake and acidification. Barg S, Huang P, Eliasson L, Nelson DJ, Obermüller S, Rorsman P, Thévenod F, Renström E. J Cell Sci. 2001 Jun;114(Pt 11):2145-54. Summary ATP-dependent priming of the secretory granules precedes Ca2+-regulated neuroendocrine secretion, but the exact nature of this reaction is not fully established in all secretory cell types. We have further investigated this reaction in the insulin-secreting pancreatic B-cell and demonstrate that granular acidification driven by a V-type H+-ATPase in the granular membrane is a decisive step in priming. This requires simultaneous Cl- uptake through granular ClC-3 Cl- channels. Accordingly, granule acidification and priming are inhibited by agents that prevent transgranular Cl- fluxes, such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and an antibody against the ClC-3 channels, but accelerated by increases in the intracellular ATP:ADP ratio or addition of hypoglycemic sulfonylureas. We suggest that this might represent an important mechanism for metabolic regulation of Ca2+-dependent exocytosis that is also likely to be operational in other secretory cell types.
Calcium-G protein interactions in the regulation of macrophage secretion. Di A, Krupa B, Nelson DJ. J Biol Chem. 2001 Oct 5;276(40):37124-32. Epub 2001 Jul 30. Abstract The interplay between activated G proteins and intracellular calcium ([Ca2+]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) (GTPγS), or [Ca2+]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) (GDPβS). TheK d of Ca2+-driven secretion was independent of GTPγS concentration, whereas theK d of the GTPγS-driven response decreased from 63 to 31 μM in the presence of saturating concentrations of [Ca2+]i. The time course of stimulus-induced secretion was dependent upon the concentration of [Ca2+]i. The time course of GTPγS-driven secretion was concentration-independent at high levels of [Ca2+]i, suggesting that a calcium-dependent translocation/binding step was rate-limiting. Our data strongly support a model in which [Ca2+]i and activated G proteins act independently of one another in the sequential regulation of macrophage secretion. [Ca2+]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 [Ca2+]i at basal levels of G protein activation. The class of phagocytic inflammatory cells encompasses the mononuclear phagocytes, including circulating monocytes, tissue macrophages, neutrophils, and eosinophils. The macrophage is the primary differentiated cell of this system and plays a major role in host defense against microbial infections. Macrophages possess a diversity of plasma membrane receptors that recognize and bind both particulate and soluble stimuli found in body fluids. In addition, hematopoietic cells in general and macrophages in particular contain several distinct secretory organelles that appear to be selectively mobilized for secretion by different stimuli. In macrophages this may include different types of dense core granules as well as phagosomes and phagolysosomes. Although the initial steps in the intracellular signal transduction cascades from the receptors that eventuate secretion and/or phagocytosis are known, the Ca2+ and G protein dependence of the pathway(s) that links receptor binding and cross-linking to vesicle fusion is still unclear. In the studies described in this investigation, heat-aggregated immunoglobulin G (HAIGG),1which binds to and aggregates Fc receptors, was used to mimic immune complex stimulation. When Fcγ receptors are engaged at the cell surface by the opsonized surface of interacting particles, the resultant receptor clustering initiates tyrosine phosphorylation of cytoplasmic residues in the immunoreceptor tyrosine activation motifs by Src-family kinases. The resultant tyrosine-phosphorylated residues form anchoring sites for SH2 domain-containing proteins, the most important of which seems to the non-receptor tyrosine kinase Syk, which can phosphorylate several downstream substrates involved in the phagocytic response. One such substrate is the Rho family guanine nucleotide exchange factor Vav, which activates the small G proteins Rac and Cdc42. HAIGG, thus, was used in our studies to maximize G protein turnover contributing to actin polymerization and, as our data demonstrate, the exocytotic fusion of vesicles. The studies were carried out to elucidate the relative roles of intracellular calcium ([Ca2+]i) and activated G proteins in mediating the secretory response in the activated macrophage. Membrane capacitance is proportional to cell surface area, and therefore, the measurement of membrane capacitance has become an important technique for studying exocytosis and endocytosis in a wide variety of secretory cells (1-11). Macrophage stimulation is accompanied by a complex series of capacitance changes reflective of exocytosis and phagocytosis (12). In this study we have taken advantage of the differential sensitivity of phagocytosis and exocytosis to temperature and [Ca2+]i to study the regulation of the stimulus-induced secretory response in isolation. Classically, in cells of neurosecretory origin, regulated secretory responses are triggered by an increase in the [[Ca2+]i] through the opening of voltage-activated Ca2+ channels. GTP-binding proteins clearly play a role, but they appear to be subservient to the primary Ca2+ signal (very little secretion occurs with GTPγS at resting [Ca2+]i levels). By contrast, the regulated secretory response of hematopoietic cells is substantially dependent on G proteins, whereas Ca2+ seems to play a more modulatory role (6, 13, 14). Studies in permeabilized mast cells, neutrophils, and eosinophils show that secretion can be induced by GTPγS in the effective absence of [Ca2+]i(<10−9 m) (15). In contrast to mast cells, very little is known about the regulation of secretion in phagocytes, and our studies represent a first approach to this problem. Previous studies suggested that the regulated secretory response of the phagocyte, as with other cells in the immune system, are triggered by guanine nucleotides and shaped in time course by local gradients in [Ca2+]i (6, 13, 14). However, the interplay between the two signaling elements has made it difficult to study their individual effects, since in both mast cells and neutrophils, GTPγS induces a transient increase in [Ca2+]i in cells that are weakly Ca2+-buffered (6, 13, 16) by a mechanism that is still unknown. Therefore, to elucidate the role of [Ca2+]i and GTP-binding proteins in the signal transduction cascade, we studied their effects on secretion in isolation. Cells were stimulated through three pathways; they are 1) surface Fc receptor ligation by multivalent ligands that enhanced the turnover of G proteins in the presence of elevated [Ca2+]i, 2) exposure to the Ca2+ionophore A23187 in Ca2+-containing external solutions in the absence of enhanced G protein activation, and 3) intracellular application of GTPγS in highly Ca2+-buffered internal solutions with and without receptor stimulation. Previous data obtained from the mast cell indicated that [Ca2+]iinfluenced the latency and rise time but not the amplitude of the degranulation response (6). In our studies, both the time course and amplitude of secretion in the macrophage is a sensitive function of [Ca2+]i, suggesting that Ca2+-mediated regulatory control of secretion is present at least in some cells of hematopoietic origin. Our data suggest that, like the mast cell, Ca2+ may be permissive and GTP may be essential to the secretory process. Thus, GTPγS can elicit a capacitance increase in the nominal absence of Ca2+, and Ca2+-induced secretion is ablated by GDPβS (a GDP analogue that prevents G proteins from binding GTP). Moreover, the presence of GTPγS does not alter the Ca2+ sensitivity of secretion, whereas the K d for GTPγS is reduced in the presence of optimal [[Ca2+]i]. It has become clear that the final stages of exocytosis in cells of hematopoietic origin are determined by the activation of GTP-binding proteins. The nature of the specific G proteins involved in hematopoietic cell secretion is, however, still not entirely clear. Early investigators proposed the existence of an elusive GEprotein, implying that it was hetrotrimeric in nature (17, 18). However, there is a growing body of evidence that one or more of the small GTP-binding proteins, including members of the Rho family (Rho, Rac, Cdc42) as well as Ras, Rab, and ARF family members may be involved in vesicle mobilization, docking, and/or membrane fusion in cells of hematopoietic origin (15, 19-22). For example, antagonism of Rho family function using specific proteins appears to block mast cell degranulation in a permeabilized cell model (20, 23). Rab GTPases appear to recruit tethering and docking proteins from the cytosol to the membrane after an interconversion between inactive, GDP-bound forms and active, GTP-bound forms, a process that does not appear to require GTP hydrolysis (22). Rab not only appears to regulate vesicle docking but also has been found to enable SNARE complex formation preceding and, finally, permitting vesicle fusion (for review, see Ref. 22). Consistent with these findings, our data suggest a role for a GTPase-dependent step in the final stages of vesicle fusion. The rationale for the present studies was the unresolved role that activated G proteins and Ca2+ play in the regulation of exocytosis in the macrophage. The controversy resides in Ca2+ versus the GTP sensitivity of the response. The early studies of Neher (6) on the mast cell suggest that elevations in [Ca2+]i do not lead to secretion in the absence of GTPγS. Instead it was proposed that GTPγS was involved in priming the Ca2+-regulated exocytotic response. Although Ca2+ did not initiate secretion, the response was interpreted as ultimately Ca2+-regulated in that increases in [Ca2+]i accelerated the time course of degranulation. However, the dose dependence of secretion on the levels of activated G proteins was not investigated. Our studies were carried out under conditions in which we examined the sensitivity of the G protein regulatory arm of the response in isolation from the Ca2+-dependent arm. We demonstrate that the pathway linking receptor binding and exocytosis induced by human HAIGG involves both G protein activation and calcium signaling as independent yet synergistic secretory stimuli. Although activated G proteins are both necessary and sufficient stimuli for secretion, the response in the macrophage is highly sensitive to changes in [Ca2+]i both in amplitude and time course. The fact that GTPγS increases the amplitude but not the sensitivity of Ca2+-driven exocytosis suggests that the Ca2+regulatory step is independent and upstream of the G protein-sensitive step and that the two regulatory pathways are additive. The observation that Ca2+ shifts the sensitivity of GTPγS-induced secretion indicates that the activated G protein-sensitive step is downstream of the Ca2+ regulatory step, and its sensitivity can be enhanced by Ca2+ priming. Therefore, unlike secretion in the mast cell, where GTP increases the Ca2+sensitivity of exocytosis (6) and G proteins are thought to prime a Ca2+ regulated response, in our studies of stimulus-induced secretion in the macrophage, Ca2+ enhances the sensitivity of a GTP-dependent process.
Oxidants potentiate Ca++ and c-AMP stimulated Cl- secretion in intestinal epithelial T84 cells. Sugi K, Musch MW, Nelson DJ, and Chang EB. Gastroenterology. 2001. 120: 89-98Summary Diarrhea is one of the major complications of inflammatory bowel disease. The role of oxidants in promoting net intestinal secretion is important, but the cellular mechanisms underlying their effects are unclear. We examined the effects and defined the cellular actions of the oxidant monochloramine (NH(2)Cl) on anion secretion in human colonic T84 cells. METHODS: Effects of NH(2)Cl on basal and agonist-stimulated short-circuit current (Isc) of T84 monolayers were determined. Apical Cl(-) and basolateral K(+) conductances were measured by efflux of (125)I(-) and (86)Rb(+), respectively. RESULTS: NH(2)Cl alone had little effect on Isc and (125)I(-) efflux. However, pretreatment with NH(2)Cl led to a concentration-dependent potentiation of the Ca(2+)-mediated Isc and of submaximal cAMP-mediated responses. These effects were associated with increased basolateral K(+) channel conductance and were blocked by increasing cellular Ca(2+) buffering capacity with Quin-2. Whole-cell voltage clamp experiments showed that NH(2)Cl potentiated Ca(2+) activation of basolateral K(+) channel conductance. CONCLUSIONS: Oxidants potentiate both Ca(2+)- and cAMP-stimulated Cl(-) secretion by a direct effect on calcium-activated basolateral K(+) channel conductance, lowering its Ca(2+) activation threshold. This effect may play an important role in amplifying and prolonging the secretory response of inflamed intestinal mucosa and enhancing the severity of diarrhea. Full text [PDF] Regulation of human CLC-3 channels by multifunctional Ca2+/calmodulin-dependent protein kinase. Huang P, Liu J, Di A, Robinson NC, Musch MW, Kaetzel MA, Nelson DJ. J Biol Chem. 2001 Jun 8;276(23):20093-100. Epub 2001 Mar 26. Abstract 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 cells1, 2, 3, 4. 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 neurosecretion5, 6, 7 and calcium-channel gating8, 9, 10, 11 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. Impermeability of the GIRK2 weaver channel to divalent cations. Hou P, Di A, Huang P, Hansen CB, Nelson DJ. Am J Physiol Cell Physiol. 2000 May;278(5):C1038-46. Abstract A single amino acid mutation (G156S) in the putative pore-forming region of the G protein-sensitive, inwardly rectifying K+ channel subunit, GIRK2, renders the conductance constitutively active and nonselective for monovalent cations. The mutant channel subunit (GIRK2wv) causes the pleiotropic weaver disease in mice, which is characterized by the selective vulnerability of cerebellar granule cells and Purkinje cells, as well as dopaminergic neurons in the mesencephalon, to cell death. It has been proposed that divalent cation permeability through constitutively active GIRK2wv channels contributes to a rise in internal calcium in the GIRK2wv-expressing neurons, eventually leading to cell death. We carried out comparative studies of recombinant GIRK2wv channels expressed in Xenopus oocytes and COS-7 cells to determine the magnitude and relative permeability of the GIRK2wv conductance to Ca2+. Data from these studies demonstrate that the properties of the expressed current differ in the two systems and that when recombinant GIRK2wv is expressed in mammalian cells it is impermeable to Ca2+. Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl(-) currents. Naren AP, Di A, Cormet-Boyaka E, Boyaka PN, McGhee JR, Zhou W, Akagawa K, Fujiwara T, Thome U, Engelhardt JF, Nelson DJ, Kirk KL. J Clin Invest. 2000 Feb;105(3):377-86. Abstract The CFTR Cl– channel controls salt and water transport across epithelial tissues. Previously, we showed that CFTR-mediated Cl– currents in the Xenopus oocyte expression system are inhibited by syntaxin 1A, a component of the membrane trafficking machinery. This negative modulation of CFTR function can be reversed by soluble syntaxin 1A peptides and by the syntaxin 1A binding protein, Munc-18. In the present study, we determined whether syntaxin 1A is expressed in native epithelial tissues that normally express CFTR and whether it modulates CFTR currents in these tissues. Using immunoblotting and immunofluorescence, we observed syntaxin 1A in native gut and airway epithelial tissues and showed that epithelial cells from these tissues express syntaxin 1A at >10-fold molar excess over CFTR. Syntaxin 1A is seen near the apical cell surfaces of human bronchial airway epithelium. Reagents that disrupt the CFTR-syntaxin 1A interaction, including soluble syntaxin 1A cytosolic domain and recombinant Munc-18, augmented cAMP-dependent CFTR Cl– currents by more than 2- to 4-fold in mouse tracheal epithelial cells and cells derived from human nasal polyps, but these reagents did not affect CaMK II–activated Cl– currents in these cells. The inwardly rectifying K(+) channel subunit GIRK1 rescues the GIRK2 weaver phenotype. Hou P, Yan S, Tang W, Nelson DJ. J Neurosci. 1999 Oct 1;19(19):8327-36. Abstract The weaver (wv) gene has been identified as a glycine to serine substitution at residue 156 in the H5 region of inwardly rectifying K+ channel, GIRK2. The mutation is permissive for the expression of homotetrameric channels that are nonselective for cations and G-protein-independent. Coexpression of GIRK2wv with GIRK1, GIRK2, or GIRK3 in Xenopus oocytes along with expression of subunit combinations linked as dimers and tetramers was used to investigate the effects of the pore mutation on channel selectivity and gating as a function of relative subunit position and number within a heterotetrameric complex. GIRK1 formed functional, K+ selective channels with GIRK2 and GIRK3. Coexpression of GIRK2wv with GIRK1 gave rise to a component of K+-selective, G-protein-dependent current. Currents resulting from coexpression of GIRK2wv with GIRK2 or GIRK3 were weaver-like. Current from dimers of GIRK1-GIRK2wv, GIRK2-GIRK2wv, and GIRK3-GIRK2wv was phenotypically similar to that obtained from coexpression of monomers. Linked tetramers containing GIRK1 and GIRK2wv in an alternating array gave rise to wild-type, K+-selective currents. When two mutant subunits were arranged adjacently in a tetramer, currents were weaver-like. These results support the hypothesis that in specific channel stoichiometries, GIRK1 rescues the weaver phenotype and suggests a basis for the selective neuronal vulnerability that is observed in the weaver mouse. Membrane capacitance changes associated with particle uptake during phagocytosis in macrophages. Holevinsky KO, Nelson DJ. Biophys J. 1998 Nov;75(5):2577-86. Abstract 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. Regulation of Ca2+-dependent Cl- conductance in a human colonic epithelial cell line (T84): cross-talk between Ins(3,4,5,6)P4 and protein phosphatases. Xie W, Solomons KR, Freeman S, Kaetzel MA, Bruzik KS, Nelson DJ, Shears SB. J Physiol. 1998 Aug 1;510 ( Pt 3):661-73. Abstract 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. Gating of the chloride channel, CLC-3, which is expressed in brain and chloride (Cl−) secretory epithelial tissues has remained controversial since its initial identification and characterization (1). The 760-amino acid protein encoded by theClc-3 gene was originally cloned from rat kidney and showed abundant expression in rat brain, most notably in the olfactory bulb, hippocampus, and cerebellum (1). When expressed in a stably transfected cell line, the rat kidney isoform of the channel showed basal activation, inhibition by phorbol esters, and Ca2+sensitivity (2). Duan and colleagues (3, 4) characterized the functional expression of a cardiac clone of guinea pigclc-3, which when expressed in NIH 3T3 cells resulted in a large basally active Cl− conductance that was activated by an increase in cell volume, inhibited by phorbol esters and exhibited biophysical properties at the single channel level identical to the swelling activated current in native cardiac myocytes. Shimada and colleagues (5) examined rat hepatocytes for Clc-3 expression and found that mRNA for two different isoforms was present; a short form corresponding to the guinea pig clone, and a long form containing a putative 58-amino acid addition at the N terminus of the protein. Both isoforms gave rise to current expression with identical selectivity when transiently expressed in CHO-K1 cells; however, they differed in the degree of outward rectification and voltage-dependent inactivation (5). Adding a further dimension to the controversy, Friedrich and colleagues (6) report in a mutational analysis of Clc-4 and Clc-5 that they were unable to detect currents upon Clc-3 expression in Xenopus oocytes or in transfected HEK293 cells. Recent evidence from the studies of Stobrawa et al. (7) in transgenic mice with disrupted Clc-3 demonstrates that the chloride channel is broadly expressed and present in endosomal compartments and neuronal synaptic vesicles. Although theClc-3 knockout mice remained smaller than wild type littermates, they nonetheless remained viable for approximately 1 year. Among the most dramatic effects which Stobrawa and colleagues (7) demonstrated in the Clcn-3−/− knockout mice were the nearly complete developmental loss of the hippocampus after birth as well as the complete loss of photoreceptors (7). The loss of both the hippocampus and photoreceptors was attributed to inadequate acidification of synaptic vesicles. Defects observed in theClc-3-deficient mice lead Stobrawa and colleagues (7) to hypothesize that Clc-3 acts as an anion shunt pathway which maintains charge balance for the proton pump which acidifies the vesicle interior prior to membrane fusion (7). The finding that Clc-3 is an intracellular chloride channel expressed in both synaptic as well as endosomal vesicles suggests that it may be in dynamic equilibrium with the plasma membrane at a level which is dependent upon vesicular cycling in the various tissues in which it is expressed. Hence, expression of Clc-3 at surface sites will depend upon a balance between vesicle fusion with the plasma membrane and membrane retrieval. Regulated Ca2+-dependent Cl−conductances have been described in cells types as diverse as neurons, lymphocytes, and secretory epithelia. They regulate cellular volume, excitability, and salt balance. The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII)1 is a major mediator of Ca2+ signaling, is abundantly expressed in the brain, and is found in almost every cell type. Regulation of Cl−channels by CaMKII has been shown in cells from the human colonic tumor cell line, T84 (8-13), airway epithelia (14), T lymphocytes (15), human macrophages (16), biliary epithelial cells (17), and cystic fibrosis-derived pancreatic epithelial cells (18). The molecular identity of the channel or channels mediating the CaMKII-dependent conductance has remained unknown. Given the current controversy in the literature as to the activation pathway for heterologously expressed Clc-3, the ubiquitous expression of the CaMKII-activated Cl− conductance in native cells, and the presence of three possible CaMKII consensus sequences in the predicted amino acid sequence, our aim in the present study was to test the hypothesis that hCLC-3 is a Cl−channel regulated by CaMKII. For this study, we cloned a full-length hCLC-3 gene from T84 cells. This gene was 92% identical to the rat long form ofClc-3 reported by Shimada and colleagues (5). Surface biotinylation experiments demonstrated that at least a portion of hCLC-3 is expressed at the surface of the stably transfected cells. Immunohistochemistry data showed that an increase in intracellular Ca2+ shifted the distribution of CLC-3 from a cytoplasmic, vesicular compartment to apparent surface sites. This translocation step was not required for current expression in the stably transfected cells as demonstrated in high resolution membrane capacitance measurements. We used the whole cell voltage-clamp technique to characterize the gating and selectivity of recombinant hCLC-3 channels stably expressed in a large-T antigen stabilized human embryonic kidney cell line HEK293 (tsA) cells. Our data show that the functional expression of the recombinant hCLC-3 induces Cl− conductance which is regulated by CaMKII with phenotypic properties of endogenous ICl,CaMKII in secretory epithelia. A mutation in the putative pore region, G280E, produced a characteristic change in anion selectivity from the I− > Br− > Cl− to Cl− > Br− > I− further demonstrating that the transfected channel was responsible for the current in the stably transfected cell line. Cytoskeletal actin gates a Cl- channel in neocortical astrocytes. Lascola CD, Nelson DJ, Kraig RP. J Neurosci. 1998 Mar 1;18(5):1679-92.] Abstract Increases in astroglial Cl The frequency of observing both spontaneous and inducible
Cl Syntaxin 1A inhibits CFTR chloride channels by means of domain-specific protein-protein interactions. Naren PP, Quick MW, Collawn JF, Nelson DJ, and Kirk KL. Physiology.1998 September; pp. 10972-10977 Abstarct Previously we showed that the functional activity of the epithelial chloride channel that is encoded by the cystic fibrosis gene (CFTR) is reciprocally modulated by two components of the vesicle fusion machinery, syntaxin 1A and Munc-18. Here we report that syntaxin 1A inhibits CFTR chloride channels by means of direct and domain-specific protein-protein interactions. Syntaxin 1A stoichiometrically binds to the N-terminal cytoplasmic tail of CFTR, and this binding is blocked by Munc-18. The modulation of CFTR currents by syntaxin 1A is eliminated either by deletion of this tail or by injecting this tail as a blocking peptide into coexpressing Xenopus oocytes. The CFTR binding site on syntaxin 1A maps to the third predicted helical domain (H3) of this membrane protein. Moreover, CFTR Cl- currents are effectively inhibited by a minimal syntaxin 1A construct (i.e., the membrane-anchored H3 domain) that cannot fully substitute for wild-type syntaxin 1A in membrane fusion reactions. We also show that syntaxin 1A binds to and inhibits the activities of disease-associated mutants of CFTR, and that the chloride current activity of recombinant DeltaF508 CFTR (i.e., the most common cystic fibrosis mutant) can be potentiated by disrupting its interaction with syntaxin 1A in cultured epithelial cells. Our results provide evidence for a direct physical interaction between CFTR and syntaxin 1A that limits the functional activities of normal and disease-associated forms of this chloride channel. Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms. Naren AP, Nelson DJ, Xie W, Jovov B, Pevsner J, Bennett MK, Benos DJ, Quick MW, Kirk KL. Nature. 1997 Nov 20;390(6657):302-5. Abstract
N-type Inactivation in the Mamalian shaker K+ channel Kv1.4 Lee TE, Philipson LH, Nelson DJ J. Membrane Biol. 1996 Febr; 151, 225-235 Abstract Mammalian voltage-gated K+ channels are oligomeric proteins, some of which may be composed in vivo of subunits derived from several similar genes. We have studied N-type inactivation in the rapidly inactivating Kv1.4 channel and, in specific, heteromultimers of this gene product with Kv1.5 noninactivating subunits. Heteromultimeric channels were analyzed for the stoichiometry of Kv1.4:Kv1.5 subunits by observing shifts in the midpoints of steady-state availability from that of homomultimeric channels. This analysis was employed to examine inactivation of heteromultimeric channels expressed in Xenopus oocytes using two model systems: by expression of a Kv1.4–Kv1.5 tandem fusion construct and by coexpression of native Kv1.4 and Kv1.5 channels across a wide relative concentration range of microinjected mRNA. Additionally, inactivation was examined in coexpression experiments of N-terminal deletion mutants of Kv1.4. We found that (i) a single inactivating subunit conferred inactivation in all hetero-multimers studied; (ii) the rate of inactivation could not be distinguished in channels containing two inactivating subunits from those containing one inactivating subunit; and (iii) large deletions in the linker region between the N-terminal inactivation region and the first membrane-spanning domain had no effect on the rate of inactivation. These data confirm the importance of the proximal N-terminal region in the inactivation of mammalian Kv1.4 channels, and suggest that the inactivation particle remains in close proximity to the permeation pathway even when the channel is in the open state.
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