Protein kinase A regulation of P2X4 receptors: Requirement for a specific motif in the C-terminus

AbstractThe P2X purinergic receptor sub-family of ligand-gated ion channels are subject to protein kinase modulation. We have previously demonstrated that P2X4R signaling can be positively regulated by increasing intracellular cAMP levels. The molecular mechanism underlying this effect was, however, unknown. The present study initially addressed whether protein kinase A (PKA) activation was required. Subsequently a mutational approach was utilized to determine which region of the receptor was required for this potentiation. In both DT-40 3KO and HEK-293 cells transiently expressing P2X4R, forskolin treatment enhanced ATP-mediated signaling. Specific PKA inhibitors prevented the forskolin-induced enhancement of ATP-mediated inward currents in P2X4R expressing HEK-293 cells. To define which region of the P2X4R was required for the potentiation, mutations were generated in the cytoplasmic C-terminal tail. It was determined that a limited region of the C-terminus, consisting of a non-canonical tyrosine based sorting motif, was required for the effects of PKA. Of note, this region does not harbor any recognizable PKA phosphorylation motifs, and no direct phosphorylation of P2X4R was detected, suggesting that PKA phosphorylation of an accessory protein interacts with the endocytosis motif in the C-terminus of the P2X4R. In support of this notion, using Total Internal Reflection Fluorescence Microscopy (TIRF)\ P2X4-EGFP was shown to accumulate at/near the plasma membrane following forskolin treatment. In addition, disrupting the endocytosis machinery using a dominant-negative dynamin construct also prevented the PKA-mediated enhancement of ATP-stimulated Ca2+ signals. Our results are consistent with a novel mechanism of P2XR regulation, whereby PKA activity, without directly phosphorylating P2X4R, markedly enhances ATP-stimulated P2X4R currents and hence cytosolic Ca2+ signals. This may occur at least in part, by altering the trafficking of a population of P2X4R present at the plasma membrane

How can we demonstrate the economic value of Precision Agriculture (PA) practices to New Zealand Agriculture service providers and arable farmers?

The amount of data collected has become a major challenge to the uptake of PA practices in New Zealand. There is a lack of clear value propositions around some PA practices, e.g. variable rate seeding (VRS). The importance of calibrating yield monitors, collecting yield data and mapping results has not been realised by farmers. The goal of the study is to provide economic evidence through yield data mining to encourage the adoption of PA

Redox regulation of type-I inositol trisphosphate receptors in intact mammalian cells.

A sensitization of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release is associated with oxidative stress in multiple cell types. These effects are thought to be mediated by alterations in the redox state of critical thiols in the IP3R, but this has not been directly demonstrated in intact cells. Here, we utilized a combination of gel-shift assays with MPEG-maleimides and LC-MS/MS to monitor the redox state of recombinant IP3R1 expressed in HEK293 cells. We found that under basal conditions, ∼5 of the 60 cysteines are oxidized in IP3R1. Cell treatment with 50 μm thimerosal altered gel shifts, indicating oxidation of ∼20 cysteines. By contrast, the shifts induced by 0.5 mm H2O2 or other oxidants were much smaller. Monitoring of biotin-maleimide attachment to IP3R1 by LC-MS/MS with 71% coverage of the receptor sequence revealed modification of two cytosolic (Cys-292 and Cys-1415) and two intraluminal cysteines (Cys-2496 and Cys-2533) under basal conditions. The thimerosal treatment modified an additional eleven cysteines, but only three (Cys-206, Cys-767, and Cys-1459) were consistently oxidized in multiple experiments. H2O2 also oxidized Cys-206 and additionally oxidized two residues not modified by thimerosal (Cys-214 and Cys-1397). Potentiation of IP3R channel function by oxidants was measured with cysteine variants transfected into a HEK293 IP3R triple-knockout cell line, indicating that the functionally relevant redox-sensitive cysteines are predominantly clustered within the N-terminal suppressor domain of IP3R. To our knowledge, this study is the first that has used proteomic methods to assess the redox state of individual thiols in IP3R in intact cells. © 2018 Joseph et al

The type 2 inositol 1,4,5-trisphosphate receptor, emerging functions for an intriguing Ca2+-release channel

AbstractThe inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) type 2 (IP3R2) is an intracellular Ca2+-release channel located on the endoplasmic reticulum (ER). IP3R2 is characterized by a high sensitivity to both IP3 and ATP and is biphasically regulated by Ca2+. Furthermore, IP3R2 is modulated by various protein kinases. In addition to its regulation by protein kinase A, IP3R2 forms a complex with adenylate cyclase 6 and is directly regulated by cAMP. Finally, in the ER, IP3R2 is less mobile than the other IP3R isoforms, while its functional properties appear dominant in heterotetramers. These properties make the IP3R2 a Ca2+ channel with exquisite properties for setting up intracellular Ca2+ signals with unique characteristics. IP3R2 plays a crucial role in the function of secretory cell types (e.g. pancreatic acinar cells, hepatocytes, salivary gland, eccrine sweat gland). In cardiac myocytes, the role of IP3R2 appears more complex, because, together with IP3R1, it is needed for normal cardiogenesis, while its aberrant activity is implicated in cardiac hypertrophy and arrhythmias. Most importantly, its high sensitivity to IP3 makes IP3R2 a target for anti-apoptotic proteins (e.g. Bcl-2) in B-cell cancers. Disrupting IP3R/Bcl-2 interaction therefore leads in those cells to increased Ca2+ release and apoptosis. Intriguingly, IP3R2 is not only implicated in apoptosis but also in the induction of senescence, another tumour-suppressive mechanism. These results were the first to unravel the physiological and pathophysiological role of IP3R2 and we anticipate that further progress will soon be made in understanding the function of IP3R2 in various tissues and organs

Integrating soil moisture measurements into pasture growth forecasting in New Zealand's hill country

Forecasting pasture growth in hill country landscapes requires information about soil water retention characteristics, which will help to quantify both water uptake, and its percolation below the root zone. Despite the importance of soil moisture data in pasture productivity predictions, current models use low-resolution estimates of water input into their soil water balance equations and plant growth simulations. As a result, they frequently fail to capture the spatial and temporal variability of soil moisture in hill country soils. Wireless Sensor Networks (WSN) are promising in-situ measurement systems for monitoring soil moisture dynamics with high temporal resolution in agricultural soils. This paper presents the deployment of a soil moisture sensing network, utilising WSN technology and multi-sensor probes, to monitor soil water changes over a hill country farm in the northern Wairarapa region of the North Island. Processed capacitance-based raw data was converted to volumetric water content by means of a factory calibration function to assess sensor accuracy and to calculate soil water storage within the pasture root zone. The derived volumetric soil moisture data was examined in terms of its dependence on the variability and influences of hill country landscape characteristics such as aspect. The integration of spatially distributed sensors and multi-depth soil moisture measurements from various hillslope positions showed that slope and aspect exerted a significant impact on soil moisture values. Furthermore, considerable differences were identified in soil water profile responses to significant rainfall events and subsequent soil water redistribution. Initial indications are that high-resolution time series of accurate multi-depth soil moisture measurements collected by a WSN are valuable for investigating root zone water movement. Sensor evaluation and data analysis suggest that these devices and their associated datasets are able to contribute to an improved understanding of drying and wetting cycles and soil moisture variability. Potentially, this will create an opportunity to generate improved pasture growth predictions in pastoral hill country environments