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Abstract.: Phosphoinositides(4,5)-bisphosphates [PI(4,5)P2] critically controls membrane excitability, the disruption of which leads to pathophysiological states. PI(4,5)P2 plays a primary role in regulating the conduction and gating properties of ion channels/transporters, through electrostatic and hydrophobic interactions that allow direct associations. In recent years, the development of many molecular tools have brought deep insights into the mechanisms underlying PI(4,5)P2-mediated regulation. This review summarizes the methods currently available to manipulate the cell membrane PI(4,5)P2 level including pharmacological interventions as well as newly designed molecular tools. We concisely introduce materials and experimental designs suitable for the study of PI(4,5)P2-mediated regulation of ion-conducting molecules, in order to assist researchers who are interested in this area. It is our further hope that the knowledge introduced in this review will help to promote our understanding about the pathology of diseases such as cardiac arrhythmias, bipolar disorders, and Alzheimer’s disease which are somehow associated with a disruption of PI(4,5)P2 metabolism. Keywords:: phosphoinositide, channel/transporter, PI-turnover, Ca2+ signal, membrane lipid

We investigated actions of uridine 5′-triphosphate (UTP) in rat aorta, cerebral and mesenteric arteries, and their single myocytes. UTP (≥10 μM) elicited an inward-rectifying current strongly reminiscent of activation of P2X1 receptor, and a similar current was also induced by α,β-methylene adenosine 5′-triphosphate (ATP) (≥100 nM). UTP desensitized α,β-methylene ATP–evoked current, and vice versa. The UTP-activated current was insensitive to G-protein modulators, TRPC3 inhibitors, or TRPC3 antibody, but was sensitive to P2-receptor inhibitors or P2X1-receptor antibody. Both UTP (1 mM) and α,β-methylene ATP (10 μM) elicited similar conductance single channel activities. UTP (≥10 μM) provoked a dose-dependent contraction of deendothelialized aortic ring preparation consisting of phasic and tonic components. Removal of extracellular Ca2+ or bath-applied 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) (30 μM) or nifedipine (10 μM) completely inhibited the phasic contraction while only partially reducing the tonic one. The tonic contraction was almost completely abolished by additional application of thapsigargin (2 μM). Similar biphasic rises in [Ca2+]i were also evoked by UTP in rat aortic myocytes. In contrast to the low expression of TRPC3, significant expression of P2X1 receptor was detected in all arteries by RT-PCR and immunoblotting, and its localization was limited to plasma membrane of myocytes as indicated by immunohistochemistry. These results suggest that UTP dually activates P2X1-like and P2Y receptors, but not TRPC3.[Supplementary materials: available only at http://dx.doi.org/10.1254/jphs.10281FP] Keywords:: uridine 5′-triphosphate (UTP), P2X receptor, TRPC3, non-selective cation channel

Stretch-activated cation channels (SACs) have been observed in many types of smooth muscle cells. However, the molecular identity and activation mechanisms of SACs remain poorly understood. We report that TRPM4-like cation channels are activated by membrane stretch in rat cerebral artery myocytes (CAMs). Negative pressure (≥20 mmHg, cell-attached mode) activated single channels (approximately 20 pS) in isolated CAMs. These channels were permeable to Na+ and Cs+ and inhibited by Gd3+ (30 µM) and DIDS (100 µM). The effect of negative pressure was abolished by membrane excision, but subsequent application of Ca2+ (>100 nM) to the intracellular side of the membrane restored single channel activity that was indistinguishable from SACs. Caffeine (5 mM), which depletes SR Ca2+-stores, first activated and then abolished SACs. Tetracaine (100 µM), a ryanodine receptor antagonist, inhibited SACs. Overexpression of hTRPM4B in HEK293 cells resulted in the appearance of cation channels that were activated by both negative pressure and Ca2+ and which had very similar biophysical and pharmacological properties as compared with SACs in CAMs. These studies indicate that TRPM4-like channels in CAMs can be activated by membrane stretch, possibly through ryanodine receptor activation, and this may contribute to the depolarization and concomitant vasoconstriction of intact cerebral arteries following mechanical stimulation. Keywords:: TRPM4, stretch, cation channel, cerebral artery, smooth muscle

We investigated the possible involvement of the melastatin family protein TRPM7 in Ca2+-mediated proliferative control of human retinoblastoma (RB) cells. The growth of RB cell was facilitated by elevating the extracellular Ca2+ concentration with a parallel increase in the magnitude of spontaneous Ca2+ influx. Under nystatin-perforated voltage-clamp, RB cells exhibited an outward-rectifying, spontaneous cation current (Ispont) having Ca2+/Mg2+-inhibited but -permeating properties. Various cation channel blockers inhibiting Ispont (Gd3+, La3+, LOE908, 2-APB) suppressed the spontaneous Ca2+ influx and decelerated the growth of RB cells with similar efficacies. Excision of the RB cell membrane (inside-out) into MgATP-free solution induced a 70pS single channel activity, which was effectively inhibited by millimolar concentrations of Mg2+ or MgATP. RT-PCR and immunocytochemical experiments revealed the expression of TRPM7 mRNA and protein in RB cells, and heterologous expression of TRPM7 in HEK293 cells reproduced the key features of Ispont. In contrast, elimination of this protein from RB cells by siRNA silencing markedly reduced Ispont density and the magnitude of spontaneous Ca2+ influx, which was paralleled by decreased TRPM7 immunoreactivity, decelerated cell proliferation, and retarded G1/S cell cycle progression. These results suggest a significant regulatory role of TRPM7 for RB cell proliferation as a spontaneously activated Ca2+ influx pathway. Keywords:: cell proliferation, basal Ca2+ influx, transient receptor potential protein, melastatin family

In both excitable and non-excitable cells, many chemical and physical stimuli elicit continuous Ca2+ influx through yet poorly understood pathways distinct from voltage-gated Ca2+ channels, leading to activation and modulation of various cellular functions. The molecular entities of these pathways have long been enigmatic, but important clues have been obtained from recent investigations on the Drosophila transient receptor potential (TRP) protein and its mammalian homologues. TRP proteins function as non-voltage-gated Ca2+ channels that are constitutively active or gated by a multitude of stimuli including light, pheromones, lipids, temperature, acid, osmolarity, and oxidative stress; and thus they may play divergent roles in cell pathophysiology. This short paper briefly overviews the current knowledge about these channels with a main focus on their possible linkage with in vivo function.