title: Cytoskeletal reorganization internalizes multiple transient receptor potential channels and blocks calcium entry into human neutrophils.
abstract: Store-operated calcium entry (SOCE) is required for polymorphonuclear neutrophil (PMN) activation in response to G protein-coupled agonists. Some immunocytes express proteins homologous to the Drosophila transient receptor potential gene (trp) calcium channel. TRP proteins assemble into heterotetrameric ion channels and are known to support SOCE in overexpression systems, but the evidence that TRP proteins support SOCE and are functionally important in wild-type cells remains indirect. We therefore studied the expression and function of TRP proteins in primary human PMN. TRPC1, TRPC3, TRPC4, and TRPC6 were all expressed as mRNA as well as membrane proteins. Immunofluorescence microscopy demonstrated localization of TRPC1, TRPC3, and TRPC4 to the PMN cell membrane and their internalization after cytoskeletal reorganization by calyculin A (CalyA). Either TRPC internalization by CalyA or treatment with the inositol triphosphate receptor inhibitor 2-aminoethoxydiphenyl borane resulted in the loss of PMN SOCE. Cytochalasin D (CytoD) disrupts actin filaments, thus preventing cytoskeletal reorganization, and pretreatment with CytoD rescued PMN SOCE from inhibition by CalyA. Comparative studies of CytoD and 2-aminoethoxydiphenyl borane inhibition of PMN cationic entry after thapsigargin or platelet-activating factor suggested that SOCE occurs through both calcium-specific and nonspecific pathways. Taken together, these studies suggest that the multiple TRPC proteins expressed by human PMN participate in the formation of at least two store-operated calcium channels that have differing ionic permeabilities and regulatory characteristics.
authors.affiliation: Department of Surgery, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Itagaki
authors.forename: Kolenkode B
authors.initials: KB
authors.lastname: Kannan
authors.forename: Brij B
authors.initials: BB
authors.lastname: Singh
authors.forename: Carl J
authors.initials: CJ
authors.lastname: Hauser
mesh_terms: D002118:Calcium / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000276:immunology; D027682:Cation Transport Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects / Q000276:immunology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007473:Ion Channels / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008387:Marine Toxins; D018375:Neutrophil Activation / Q000276:immunology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists; D050052:TRPC Cation Channels; D050053:TRPM Cation Channels
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D027682:Cation Transport Proteins; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007473:Ion Channels; D008387:Marine Toxins; D010080:Oxazoles; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D043562:Receptors, G-Protein-Coupled; D050052:TRPC Cation Channels; D050053:TRPM Cation Channels; C475667:TRPM3 protein, human; C437888:TRPM4 protein, human; C459082:TRPM6 protein, human; C096351:transient receptor potential cation channel, subfamily C, member 1; C059041:calyculin A; D002118:Calcium