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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Phosphatidylinositol (4,5)Bisphosphate Inhibits K⁺-Efflux Channel Activity in NT1 Tobacco Cultured Cells^1,[W],[OA]

Robert H. Smith Institute for Plant Sciences and Genetics in Agriculture, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot 76100, Israel (X.M., O.S., S.D., L.Y., N.M.); and Department of Plant Biology, North Carolina State University, Raleigh, North Carolina 27695–7649 (Y.J.I., I.P., A.L., W.F.B.)

In the animal world, the regulation of ion channels by phosphoinositides (PIs) has been investigated extensively, demonstrating a wide range of channels controlled by phosphatidylinositol (4,5)bisphosphate (PtdInsP₂). To understand PI regulation of plant ion channels, we examined the in planta effect of PtdInsP₂ on the K⁺-efflux channel of tobacco (Nicotiana tabacum), NtORK (outward-rectifying K channel). We applied a patch clamp in the whole-cell configuration (with fixed "cytosolic" Ca²⁺ concentration and pH) to protoplasts isolated from cultured tobacco cells with genetically manipulated plasma membrane levels of PtdInsP₂ and cellular inositol (1,4,5)trisphosphate: "Low PIs" had depressed levels of these PIs, and "High PIs" had elevated levels relative to controls. In all of these cells, K channel activity, reflected in the net, steady-state outward K⁺ currents (I_K), was inversely related to the plasma membrane PtdInsP₂ level. Consistent with this, short-term manipulations decreasing PtdInsP₂ levels in the High PIs, such as pretreatment with the phytohormone abscisic acid (25 µM) or neutralizing the bath solution from pH 5.6 to pH 7, increased I_K (i.e. NtORK activity). Moreover, increasing PtdInsP₂ levels in controls or in abscisic acid-treated high-PI cells, using the specific PI-phospholipase C inhibitor U73122 (2.5–4 µM), decreased NtORK activity. In all cases, I_K decreases stemmed largely from decreased maximum attainable NtORK channel conductance and partly from shifted voltage dependence of channel gating to more positive potentials, making it more difficult to activate the channels. These results are consistent with NtORK inhibition by the negatively charged PtdInsP₂ in the internal plasma membrane leaflet. Such effects are likely to underlie PI signaling in intact plant cells.

² Present address: Department of Psychiatry and Psychotherapy, Institute of Neurophysiology, Charité Medical University, Berlin 10117, Germany.

³ Present address: Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322.

⁴ Present address: Department of Genetics, University of Wisconsin, Madison, WI 53706–1580.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Wendy F. Boss (wendy_boss{at}ncsu.edu).

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.129007

^* Corresponding author; e-mail nava.moran{at}huji.ac.il.

Received September 1, 2008; accepted November 24, 2008; published December 3, 2008.

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