The Canonical Transient Receptor Potential 6 Channel as a Putative Phosphatidylinositol 3,4,5-Trisphosphate-Sensitive Calcium Entry System

Abstract

We previously reported that phosphatidylinositol 3,4,5-trisphosphate (PIP₃), a lipid product of phosphoinositide 3-kinase (PI3K), induced Ca²⁺ influx via a noncapacitative pathway in platelets, Jurkat T cells, and RBL-2H3 mast cells. The identity of this Ca²⁺ influx system, however, remains unclear. Here, we investigate a potential link between PIP₃-sensitive Ca²⁺ 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 PIP₃ induces Ca²⁺ influx. First, Fura-2 fluorometric Ca²⁺ analysis shows the ability of PIP₃ to selectively stimulate [Ca²⁺]_i increase in TRPC6-expressing cells. Second, pull-down analysis indicates specific interactions between biotin-PIP₃ and TRPC6 protein. Our data indicate that PIP₃ activates store-independent Ca²⁺ entry in TRPC6 cells via a nonselective cation channel. Although the activating effect of PIP₃ on TRPC6 is reminiscent to that of 1-oleoyl-2-acetyl-sn-glycerol, this activation is not attributable to the diacylglycerol substructure of PIP₃ since other phosphoinositides failed to trigger Ca²⁺ responses. The PIP₃-activated Ca²⁺ entry is inhibited by known TRPC6 inhibitors such as Gd³⁺ and SKF96365 and is independent of IP₃ production. Furthermore, we demonstrated that TRPC6 overexpression or antisense downregulation significantly alters the amplitude of PIP₃- and anti-CD3-activated Ca²⁺ responses in Jurkat T cells. Consequently, the link between TRPC6 and PIP₃-mediated Ca²⁺ entry provides a framework to account for an intimate relationship between PI3K and PLCγ in initiating Ca²⁺ response to agonist stimulation in T lymphocytes.