Abstract
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 (IK), 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 microM) or neutralizing the bath solution from pH 5.6 to pH 7, increased IK (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 microM), decreased NtORK activity. In all cases, IK 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.
Highlights
Many environmental and internal signals induce increased metabolism of phosphoinositides (PIs) in both animals and plants cells
Our results strongly suggest the inhibition of Nicotiana tabacum Outward-Rectifying K channel (NtORK) by PtdInsP2
In an attempt to resolve the possible effects of PtdInsP2 on NtORK into effects on properties of the open channel vs. effects on channel gating, we examined the voltage dependence of its chord conductance, G'K, extracted from I'K (Eq 1, Materials and Methods)
Summary
Many environmental and internal signals induce increased metabolism of phosphoinositides (PIs) in both animals and plants cells. PIs are involved in numerous cellular processes important for cell development and growth, including secretion of metabolites, vesicular transport, organization of the cytoskeleton and regulation of ion channels and transporters (reviewed by Hilgemann, 2003; Meijer and Munnik, 2003; Suh and Hille, 2005; Huang, 2007; Im et al, 2007). While DAG operates within the plane of the membrane, production of InsP3 is practically synonymous with further signaling through mobilization of Ca2+ from internal stores (Berridge and Irvine, 1984; Berridge et al, 1998). Detailed descriptions have accumulated about PtdInsP2 interactions with membrane ion channels and other ion transporters, highlighting its important roles in conveying information about physical and chemical stimuli and in maintaining cellular ionic homeostasis (reviewed by Suh and Hille, 2005; Huang, 2007)
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