Chemotactic peptide fMetLeuPhe induces translocation of the TRPV2 channel in macrophages.
The present study was conducted to characterize the regulation and function of TRPV2 in macrophages. Among six members of the TRPV family channels, only the expression of TRPV2 was detected in macrophages. We then determined localization of TRPV2 using TtT/M87 macrophages transfected with TRPV2-EGFP. In serum-free condition, most of the TRPV2 signal was located in the cytoplasm and colocalized with the endoplasmic reticulum marker. Treatment with serum induced translocation of some of the TRPV2-EGFP to the plasma membrane. Serum-induced translocation was blocked by transfection of short-form TRPV2 (s-TRPV2) lacking a pore-forming region and the sixth transmembrane domain. Addition of a chemotactic peptide formyl Met-Leu-Phe (fMLP) also induced translocation of TRPV2-EGFP to the plasma membrane. The fMLP-induced translocation was blocked by an inhibitor of PI 3-kinase, LY294002, and pertussis toxin. Whole-cell patch clamp analysis showed a Cs+ current in the TtT/M87 cell, which was blocked by an addition of ruthenium red and transfection of either s-TRPV2 or siRNA for TRPV2. fMLP increased the Cs+ current. fMLP induced a rapid and sustained elevation of cytoplasmic Ca2+ ([Ca2+]C), the sustained phase of which was abolished by removal of extracellular calcium. The sustained elevation of [Ca2+]C was also blocked by ruthenium red, and transfection of either s-TRPV2 or siRNA. Finally, fMLP-induced migration of macrophage was blocked by ruthenium red or transfection of s-TRPV2. These results suggest that fMLP induces translocation of TRPV2 from intracellular compartment to the plasma membrane, and this translocation is critical for fMLP-induced calcium entry.
Nagasawa
Masahiro
M
Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan.
Nakagawa
Yuko
Y
Tanaka
Shigeyasu
S
Kojima
Itaru
I
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002630:Chemotactic Factors / Q000494:pharmacology*; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D037342:Pertussis Toxin / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D050916:TRPV Cation Channels / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology
D015220:Calcium Channels; D002630:Chemotactic Factors; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; D050916:TRPV Cation Channels; C485717:Trpv2 protein, mouse; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019284:Thapsigargin; D037342:Pertussis Toxin; D002118:Calcium