title: TRPC4 expression determines sensitivity of the platelet-type capacitative Ca2+ entry channel to intracellular alkalosis.
abstract: The present study was designed to analyze the molecular basis of the intracellular pH-dependent capacitative Ca2+ entry (CCE) of human platelets and megakaryocytic cells, specifically to test the hypothesis that members of the classical transient receptor potential (TRPC) protein family are involved in the CCE pathway that is promoted by intracellular alkalosis. Human platelets as well as the tested megakaryocytic cell lines (CMK cells, MEG-01 cells) and HEK293 cells displayed thapsigargin-induced CCE and responded to monensin with comparable elevation in intracellular pH. Promotion of CCE by monensin-induced intracellular alkalosis, however, was profound in mature platelets, moderate in CMK cells and lacking in MEG-01 cells as well as in HEK293 cells. Analysis of the TRPC expression pattern by immunoblotting revealed that mature platelets and CMK cells express TRPC4 along with TRPC1 and TRPC3, while TRPC4 is lacking in MEG-01 cells. HEK293 cells displayed CCE characteristics as well as lack of TRPC4 expression similar to MEG-01 cells. Over-expression of TRPC4 in HEK293 cells was found to result in a gain of pH-sensitivity of CCE with clearly detectable promotion of CCE in response to monensin. These results suggest that platelet CCE channel complexes contain TRPC4 as a molecular component that determines sensitivity of CCE to intracellular alkalosis.
authors.affiliation: Department of Hygiene and Preventive Medicine, Yamagata University School of Medicine, Yamagata 990-9585, Japan. wakabaya@med.id.yamagata-u.ac.jp
authors.forename: Ichiro
authors.initials: I
authors.lastname: Wakabayashi
authors.forename: Mikio
authors.initials: M
authors.lastname: Marumo
authors.forename: Annarita
authors.initials: A
authors.lastname: Graziani
authors.forename: Michael
authors.initials: M
authors.lastname: Poteser
authors.forename: Klaus
authors.initials: K
authors.lastname: Groschner
mesh_terms: D000471:Alkalosis; D001773:Blood Cells; D001792:Blood Platelets / Q000737:chemistry*; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000032:analysis*; D002460:Cell Line; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008533:Megakaryocytes / Q000737:chemistry; D008985:Monensin / Q000494:pharmacology; D050052:TRPC Cation Channels / Q000032:analysis*; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; D019284:Thapsigargin; D008985:Monensin; D002118:Calcium