The canonical transient receptor potential 6 channel as a putative phosphatidylinositol 3,4,5-trisphosphate-sensitive calcium entry system.
We previously reported that phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), a lipid product of phosphoinositide 3-kinase (PI3K), induced Ca(2+) influx via a noncapacitative pathway in platelets, Jurkat T cells, and RBL-2H3 mast cells. The identity of this Ca(2+) influx system, however, remains unclear. Here, we investigate a potential link between PIP(3)-sensitive Ca(2+) entry and the canonical transient receptor potential (TRPC) channels by developing stable human embryonic kidney (HEK) 293 cell lines expressing TRPC1, TRPC3, TRPC5, and TRPC6. Two lines of evidence support TRPC6 as a putative target by which PIP(3) induces Ca(2+) influx. First, Fura-2 fluorometric Ca(2+) analysis shows the ability of PIP(3) to selectively stimulate [Ca(2+)](i) increase in TRPC6-expressing cells. Second, pull-down analysis indicates specific interactions between biotin-PIP(3) and TRPC6 protein. Our data indicate that PIP(3) activates store-independent Ca(2+) entry in TRPC6 cells via a nonselective cation channel. Although the activating effect of PIP(3) on TRPC6 is reminiscent to that of 1-oleoyl-2-acetyl-sn-glycerol, this activation is not attributable to the diacylglycerol substructure of PIP(3) since other phosphoinositides failed to trigger Ca(2+) responses. The PIP(3)-activated Ca(2+) entry is inhibited by known TRPC6 inhibitors such as Gd(3+) and SKF96365 and is independent of IP(3) production. Furthermore, we demonstrated that TRPC6 overexpression or antisense downregulation significantly alters the amplitude of PIP(3)- and anti-CD3-activated Ca(2+) responses in Jurkat T cells. Consequently, the link between TRPC6 and PIP(3)-mediated Ca(2+) entry provides a framework to account for an intimate relationship between PI3K and PLCgamma in initiating Ca(2+) response to agonist stimulation in T lymphocytes.
Tseng
Ping-Hui
PH
Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, USA.
Lin
Ho-Pi
HP
Hu
Hongzhen
H
Wang
Chunbo
C
Zhu
Michael Xi
MX
Chen
Ching-Shih
CS
D001692:Biological Transport / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002217:Carbachol / Q000378:metabolism; D002460:Cell Line; D002614:Chelating Agents / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D050052:TRPC Cation Channels; D010738:Type C Phospholipases / Q000378:metabolism
D015220:Calcium Channels; D002614:Chelating Agents; D005456:Fluorescent Dyes; D018129:Phosphatidylinositol Phosphates; D020033:Protein Isoforms; D050052:TRPC Cation Channels; C060974:phosphatidylinositol 3,4,5-triphosphate; D002217:Carbachol; D010738:Type C Phospholipases; D002118:Calcium; D016257:Fura-2