PKD2, or polycystin 2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease, belongs to the transient receptor potential channel superfamily and has been shown to function as a nonselective cation channel in the plasma membrane. However, the mechanism of PKD2 activation remains elusive. We show that PKD2 overexpression increases epidermal growth factor (EGF)-induced inward currents in LLC-PK1 kidney epithelial cells, while the knockdown of endogenous PKD2 by RNA interference or the expression of a pathogenic missense variant, PKD2-D511V, blunts the EGF-induced response. Pharmacological experiments indicate that the EGF-induced activation of PKD2 occurs independently of store depletion but requires the activity of phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K). Pipette infusion of purified phosphatidylinositol-4,5-bisphosphate (PIP2) suppresses the PKD2-mediated effect on EGF-induced conductance, while pipette infusion of phosphatidylinositol-3,4,5-trisphosphate (PIP3) does not have any effect on this conductance. Overexpression of type Iα phosphatidylinositol-4-phosphate 5-kinase [PIP(5)Kα], which catalyzes the formation of PIP2, suppresses EGF-induced currents. Biochemical experiments show that PKD2 physically interacts with PLC-γ2 and EGF receptor (EGFR) in transfected HEK293T cells and colocalizes with EGFR and PIP2 in the primary cilium of LLC-PK1 cells. We propose that plasma membrane PKD2 is under negative regulation by PIP2. EGF may reduce the threshold of PKD2 activation by mechanical and other stimuli by releasing it from PIP2-mediated inhibition.

Mutations in pkd2 result in the type 2 form of autosomal dominant polycystic kidney disease, which accounts for ϳ15% of all cases of the disease. PKD2, the protein product of pkd2, belongs to the transient receptor potential superfamily of cation channels, and it can function as a mechanosensitive channel in the primary cilium of kidney cells, an intracellular Ca 2؉ release channel in the endoplasmic reticulum, and/or a nonselective cation channel in the plasma membrane. We have identified mDia1/Drf1 (mammalian Diaphanous or Diaphanous-related formin 1 protein) as a PKD2-interacting protein by yeast two-hybrid screen. mDia1 is a member of the RhoA GTPase-binding formin homology protein family that participates in cytoskeletal organization, cytokinesis, and signal transduction. We show that mDia1 and PKD2 interact in native and in transfected cells, and binding is mediated by the cytoplasmic C terminus of PKD2 binding to the mDia1 N terminus. The interaction is more prevalent in dividing cells in which endogenous PKD2 and mDia1 co-localize to the mitotic spindles. RNA interference experiments reveal that endogenous mDia1 knockdown in HeLa cells results in the loss of PKD2 from mitotic spindles and alters intracellular Ca 2؉ release. Our results suggest that mDia1 facilitates the movement of PKD2 to a centralized position during cell division and has a positive effect on intracellular Ca 2؉ release during mitosis. This may be important to ensure equal segregation of PKD2 to the daughter cell to maintain a necessary level of channel activity. Alternatively, PKD2 channel activity may be important in the cell division process or in cell fate decisions after division.

Depletion of intracellular Ca2؉ stores leads to the activation of Ca 2؉ inflow through store-operated Ca 2؉ channels. Although the identity of these channels is unknown, there is considerable evidence that the transient receptor potential channel 1 (TRPC1) participates in the formation of these channels. We show that TRPC1 physically interacts with the a-isoform of the inhibitor of the myogenic family (I-mfa), a known inhibitor of basic helix-loop-helix transcription factors, in vitro and in vivo. The interaction is mediated by the C-terminal cytoplasmic tail of TRPC1 and the C-terminal cysteine-rich domain of I-mfa. Using the whole cell configuration of the patch clamp technique, we show that ectopic expression of I-mfa in CHO-K1 cells reduces native store-activated Ca 2؉ currents, whereas knock-down of endogenous Imfa in A431 cells by RNA interference enhances these currents. Pipette perfusion of purified recombinant Imfa rescues the effect of I-mfa knock-down on storeoperated conductance. Finally, cell dialysis with a monoclonal antibody specific to TRPC1 results in the suppression of store-activated conductance in cells lacking I-mfa, but not in I-mfa expressing cells. We propose that I-mfa functions as a molecular switch to suppress the store dependence of TRPC1.G protein-coupled receptors and receptor tyrosine kinases comprise a very large group of cell surface receptors that elicit their physiological responses through the production of inositol (1,4,5)-trisphosphate (IP 3 ) 1 (1). Upon receptor activation, newly synthesized IP 3 acts on IP 3 receptors (IP 3 Rs) to trigger a rapid increase in the intracellular Ca 2ϩ concentration by releasing free Ca 2ϩ from intracellular stores (2). Intracellular Ca 2ϩ concentration returns to normal levels by extrusion of cytoplasmic Ca 2ϩ to the extracellular space by plasma membrane Ca 2ϩ -ATPase and Na ϩ -Ca 2ϩ exchangers, re-admission of Ca 2ϩ into the endoplasmic reticulum by the SERCA pump, and Ca 2ϩ entry via the store-operated Ca 2ϩ channels (3, 4). Whereas all of these pathways contribute to the maintenance of normal Ca 2ϩ homeostasis, store-operated Ca 2ϩ entry is of particular interest because it is responsible for the regulation of diverse cellular functions (5) ranging from cell proliferation and gene expression in T lymphocytes (6) to endothelial cell function (7) and regulation of the acrosome reaction in germ cells (8).Despite intensive investigation, the identity of the storeoperated channels and the cellular mechanisms underlying the coupling of store depletion and Ca 2ϩ entry remain elusive (9, 10). Mammalian TRPCs have been proposed to function as store-operated channels (11,12). However, it is not clear whether they are store-, receptor-operated (13, 14), and/or channels serving other functions.TRPC1 was the first mammalian TRP channel cloned (15, 16) and there has been considerable evidence to support its role in store-operated Ca 2ϩ entry. First, genetic elimination of TRPC1 by homologous recombination significantly reduces Ca 2ϩ release-activated...

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We have investigated the isozymes of a phosphoinositide‐specific phospholipase C (PLC) in bovine retina using several monoclonal antisera to PLCβ1, γ1, and δ1. Immunoblot analysis showed that all three isozymes were present in the retina. Immunocytochemical localization in frozen bovine retina sections showed that PLCγ1 was present in the photoreceptor cell layer, outer plexiform cell layer, inner plexiform cell layer, and ganglion cell layer. Immunoreaction within the photoreceptor cell layer was dependent on dark/light adaptation state of retinas. Immunoblot analysis of rod outer segments (ROS) with monoclonal or polyclonal antibodies to PLCγ1 showed the presence of an immunoreactive band of 140 kDa. ROS prepared from retinas light‐adapted in vitro had more PLCγ1 on immunoblots than ROS from dark‐adapted retinas. PLC enzyme activity in ROS from light‐adapted retinas was 69 and 46% higher than ROS from dark‐adapted retinas, when assayed in the presence and absence of ATP, respectively. This increase in enzyme activity was observed at [Ca2+]free between 0.32 and 100 µM. These results demonstrate the presence of PLCγ1 in bovine ROS and show that ROS prepared from light‐adapted retinas are enriched in this isozyme, suggesting that light may promote the binding of this isozyme to bleached ROS membranes.