Odor-stimulated phosphatidylinositol 3-kinase in lobster olfactory receptor cells. J Neurophysiol 85: [2537][2538][2539][2540][2541][2542][2543][2544] 2001. Two antagonists of phosphoinositide 3-OH kinases (PI3Ks), LY294002 and Wortmannin, reduced the magnitude of the receptor potential in lobster olfactory receptor neurons (ORNs) recorded by patch clamping the cells in vivo. An antibody directed against the c-terminus of human PI3K-P110␤ detected a molecule of predicted size in the outer

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... tes of the ORNs. Two 3-phosphoinositides, PI(3,4)P 2 (1-4 M) and PI(3,4,5)P 3 (1-4 M) applied to the cytoplasmic side of inside-out patches taken from cultured lobster ORNs, reversibly activated a Na ϩ -gated channel previously implicated in the transduction cascade in these cells. 3-Phosphoinositides were the most effective phosphoinositide (1 M) in enhancing the open probability of the channel. Collectively, these results implicate 3-phosphoinositides in lobster olfactory transduction and raise the need to consider the 3-phosphoinositide pathway in olfactory transduction. I N T R O D U C T I O N Phosphoinositides are integral membrane constituents in eukaryotic cells that also function in transmembrane signaling (review: Zhang and Majerus 1998). The discovery of D-3 phosphorylated inositol lipids and their synthesis by a family of phosphoinositide 3-OH kinases (PI3Ks) has fostered important new insight into cell signal transduction (Fruman et al. 1998; Toker and Cantley 1997) . With different specificity, these enzymes phosphorylate phosphatidylinositol (PI) itself, phosphatidylinositol 4-phosphate [PI(4)P], and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] in the D-3 position of the inositol ring to generate phosphatidylinositol 3-phosphate [PI(3)P], phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ], and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ], respectively. In higher animals, at least, there is a relatively large, constitutive pool of PI(3)P present in resting cells, in contrast to endogenously low levels of PI(3,4)P 2 and PI(3,4,5)P 3 that are transiently and rapidly elevated in response to a wide range of external stimuli and are thought to have signaling function (review: Kapeller and Cantley 1994). The latter 3-phosphoinositides are generated by a family of heterodimeric PI3Ks, isoforms of p110 PI3Ks, consisting of a 110-kDa catalytic subunit and an associated 50-to 100-kDa noncatalytic, regulatory subunit. While they are capable of phosphorylating PI and PI(4)P in vitro, they exhibit a preference for PI(4,5)P 2 as a substrate within cells (Hawkins et al. 1992 ). These enzymes can be activated by a wide array of ligands such as hormones, neurotransmitters, growth factors, and cytokines acting through both tyrosine kinases and G-protein-coupled receptors, depending on the particular isoform (Tang and Downes 1997). The G-protein-activated PI3K (p110gamma) appears to be a distinct form of the enzyme. It associates with a noncatalytic p101 subunit that is responsible for the substrate selectivity of the enzyme by sensitizing the catalytic subunit toward G ␤␥ in the presence of PI(4,5)P 2 (Maier et al. 1999) . In general, the downstream components of PI3K-dependent signaling pathways are still being identified. 3-Phosphoinositides have yet to be implicated in sensory transduction, although other phosphoinositides can modulate ion channels implicated in olfactory transduction in lobsters (Zhainazarov and Ache 1999) and in vertebrate phototransduction (Womack et al. 2000). In olfaction, the role of phosphatidylinositol (PI) signaling in general is unclear (reviews: Schild and Restrepo 1998; Zhainazarov and Ache 1995), and even controversial (Brunet et al. 1996; Gold 1999) . Interest in PI signaling in olfaction, however, has focused on the canonical phosphoinositide turnover pathway in which PI(4,5)P 2 is cleaved into inositol(1,4,5)trisphosphate (IP 3 ) and diacylglycerol by odor-stimulated phospholipase C. It remains to be determined whether PI signaling in olfactory transduction could be mediated at least in part through activation of one or more PI3Ks. The involvement of PI signaling in olfaction is perhaps best established in lobster olfactory receptor neurons (ORNs), where it has been possible to both functionally and molecularly implicate a plasma membrane-associated IP 3 receptor in activation of the cells (Fadool and Ache 1992; Munger et al. 2000) . We therefore attempted to implicate the 3-phosphoinositide pathway in these cells. We report that two antagonists of PI3Ks, LY294002 and Wortmannin, reduce the magnitude of the receptor potential in lobster ORNs. We show that an antibody directed against the c-terminus of human PI3K-P110␤ detected a molecule of appropriate molecular weight in the outer dendrite (transduction zone) of the lobster ORNs. We also show that two 3-phosphoinositides, PI(3,4)P 2 (1-4 M) and PI(3,4,5)P 3 (1-4 M), applied to the cytoplasmic side of inside-out patches taken from cultured lobster ORNs reversibly Address for reprint requests: B. W. Ache,