Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) is a key component in signal transduction, being a precursor to other signalling molecules and itself associated with roles in signal transduction and cell biology. Tubby is a membrane bound transcription factor whose dysfunction results in obesity in mice. It contains a domain that selectively binds PtdIns(4,5)P 2 . We have investigated the use of a fluorescently tagged version of this domain to monitor changes in PtdIns(4,5)P 2 concentration in living cells and compared it to the pleckstrin homology domain of PLCδ1. Our results show that selected mutants of this domain report receptor-mediated changes in cellular PtdIns(4,5)P 2 . In contrast to the pleckstrin homology domain of PLCδ1 it does not have a significant affinity for inositol 1,4,5-trisphosphate (IP 3 ). Using a selected mutant, we examine the regulation of ATP-sensitive K + channels via a G q/11 -coupled receptor. These experiments reveal a correlation between reporter translocation and the onset of current inhibition whilst the recovery of current after agonist removal is delayed when compared to the reporter. Furthermore our studies reveal the importance of Ca 2+ in determining the overall activity of phospholipase C in living cells. This probe may be valuable in examining changes in PtdIns(4,5)P 2 distinct from those of IP 3 in intact cells in a variety of physiological settings.

Abstract-The activation of ATP-sensitive K ϩ channels by protein kinase A in vascular smooth muscle is an important component of the action of vasodilators. In this study, we examine the molecular mechanisms of regulation of the cloned equivalent of this channel comprising the sulfonylurea receptor 2B and the inward rectifier 6.1 subunit (SUR2B/Kir6.1). Specifically, we focus on whether the channel is directly phosphorylated and the sites at which this occurs in the protein complex. We identify one site in Kir6.1 (S385) and two sites in SUR2B (T633 and S1465) using a combination of biochemical and functional assays. Our work supports a model in which multiple sites in the channel complex have to be phosphorylated before activation occurs. A TP-sensitive potassium (K ATP ) channels are found in a variety of tissues, and one of their roles is to allow the metabolic state of the cell to be reported at the plasma membrane. [1][2][3][4] The channels exist in the cell membrane as an octomer of 4 pore-forming inward rectifier K ϩ (Kir6.X) subunits with 4 sulfonylurea receptor (SUR) subunits. 5,6 The channels are closed by raised intracellular ATP, stimulated by MgADP, and are sensitive to K ϩ channel openers such as pinacidil and diazoxide. In addition, the channels are blocked by antidiabetic sulfonylureas. 1-4 The differences in physiological and pharmacological properties in specific tissues are accounted for by differential subunit expression. [7][8][9] The phosphorylation of ion channels by protein kinases is an important mechanism by which membrane excitability is regulated by cell signaling pathways. 10 This is of particular physiological importance for K ATP channels in vascular smooth muscle, where they control smooth muscle tone in response to hormones and neurotransmitters. [11][12][13] In particular, endogenous vasodilators such as calcitonin gene-related peptide and adenosine have been shown to act via protein kinase A (PKA) to stimulate glibenclamide-sensitive currents in vascular smooth muscle. 14 -16 What is the molecular counterpart of the channel in smooth muscle? The pharmacological properties of the native channel are compatible with the known properties of SUR2B-containing channel complexes. 9 Furthermore, channel activity in many native smooth muscles is absolutely dependent on nucleotide diphosphates, and this is characteristic of the behavior of Kir6.1. [17][18][19] Finally, more direct studies of this issue with the Kir6.1 and SUR2 knockout mice and our own work in a human primary pulmonary artery smooth muscle cell line are also consistent with the native vascular channel being composed of Kir6.1/SUR2B. 20 -22 However, little is known of the molecular mechanisms by which protein kinases and more specifically PKA modulate the vascular K ATP channel. We investigate this problem here using biochemical and electrophysiological approaches. We ask whether channel modulation occurs with the cloned channel isoforms, whether this is mediated by direct phosphorylation of the protein complex, and whic...

Our data revealed that channel trafficking may contribute to the pathogenesis of LQT1.

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