Involvement of TRPM7 in cell growth as a spontaneously activated Ca2+ entry pathway in human retinoblastoma cells.
We investigated the possible involvement of the melastatin family protein TRPM7 in Ca(2+)-mediated proliferative control of human retinoblastoma (RB) cells. The growth of RB cell was facilitated by elevating the extracellular Ca(2+) concentration with a parallel increase in the magnitude of spontaneous Ca(2+) influx. Under nystatin-perforated voltage-clamp, RB cells exhibited an outward-rectifying, spontaneous cation current (I(spont)) having Ca(2+)/Mg(2+)-inhibited but -permeating properties. Various cation channel blockers inhibiting I(spont) (Gd(3+), La(3+), LOE908, 2-APB) suppressed the spontaneous Ca(2+) influx and decelerated the growth of RB cells with similar efficacies. Excision of the RB cell membrane (inside-out) into MgATP-free solution induced a 70pS single channel activity, which was effectively inhibited by millimolar concentrations of Mg(2+) or MgATP. RT-PCR and immunocytochemical experiments revealed the expression of TRPM7 mRNA and protein in RB cells, and heterologous expression of TRPM7 in HEK293 cells reproduced the key features of I(spont). In contrast, elimination of this protein from RB cells by siRNA silencing markedly reduced I(spont) density and the magnitude of spontaneous Ca(2+) influx, which was paralleled by decreased TRPM7 immunoreactivity, decelerated cell proliferation, and retarded G(1)/S cell cycle progression. These results suggest a significant regulatory role of TRPM7 for RB cell proliferation as a spontaneously activated Ca(2+) influx pathway.
Hanano
Toyohisa
T
Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Hara
Yuji
Y
Shi
Juan
J
Morita
Hiromitsu
H
Umebayashi
Chisato
C
Mori
Emiko
E
Sumimoto
Hideki
H
Ito
Yushi
Y
Mori
Yasuo
Y
Inoue
Ryuji
R
D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002413:Cations, Divalent / Q000378:metabolism; D002455:Cell Division; D002460:Cell Line; D049109:Cell Proliferation; D002675:Child, Preschool; D005260:Female; D006801:Humans; D007473:Ion Channels / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D018408:Patch-Clamp Techniques; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050053:TRPM Cation Channels; D013997:Time Factors; D014407:Tumor Cells, Cultured
D002121:Calcium Channel Blockers; D002413:Cations, Divalent; D007473:Ion Channels; D008565:Membrane Proteins; D012333:RNA, Messenger; D050053:TRPM Cation Channels; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C486583:TRPM7 protein, human; D002118:Calcium