STE2/SCG1-dependent inhibition of STE4-induced growth arrest by mutant STE4ΔC6 in the yeast pheromone response pathway

AbstractThe yeast pheromone response pathway involves the activation of a heterotrimeric G protein composed by SCGI (α) (also GPA1), STE4 (β), and STE18 (γ) subunits by the pheromone-activated receptors STE2 and STE3 in a and α cells, respectively. Upon exchange of bound GDP for GTP in the SCG1 subunit, the release of STE4/STE18 dimer occurs which, in turn causes activation of downstream effectors leading growth arrest and mating competence. Over-expression of STE4 also leads to growth arrest in a STE18 dependent manner. Removal of 6 amino acids from the C-terminus of STE4 rendered a subunit incapable of downstream signalling but still able to interact with STE18. This ΔC6 mutant acts as a dominant negative because it blocks the growth arresting effect obtained by over-expression of STE4. The inhibitory effect of STE4ΔC6 is dependent on the presence of the SCG1 subunit in a STE2 but not ste2 background. Inhibition of the growth arresting effect of STE4 by the ΔC6 mutant is not due to competition at the effector site, but rather involves an intrinsic activity of STE2 that is dependent on SCG1

Store-operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells.

KEY POINTS: Depletion of Ca(2+) stores activates store-operated channels (SOCs), which mediate Ca(2+) entry pathways that regulate cellular processes such as contraction, proliferation and gene expression. In vascular smooth muscle cells (VSMCs), stimulation of SOCs composed of canonical transient receptor potential channel 1 (TRPC1) proteins requires G protein α q subunit (Gαq)/phospholipase C (PLC)β1/protein kinase C (PKC) activity. We studied the role of stromal interaction molecule 1 (STIM1) in coupling store depletion to this activation pathway using patch clamp recording, GFP-PLCδ1-PH imaging and co-localization techniques. Store-operated TRPC1 channel and PLCβ1 activities were inhibited by STIM1 short hairpin RNA (shRNA) and absent in TRPC1(-/-) cells, and store-operated PKC phosphorylation of TRPC1 was inhibited by STIM1 shRNA. Store depletion induced interactions between STIM1 and TRPC1, Gαq and PLCβ1, which required STIM1 and TRPC1. Similar effects were produced with noradrenaline. These findings identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, where store-operated STIM1-TRPC1 interactions stimulate Gαq/PLCβ1/PKC activity to induce channel gating. ABSTRACT: In vascular smooth muscle cells (VSMCs), stimulation of canonical transient receptor potential channel 1 (TRPC1) protein-based store-operated channels (SOCs) mediates Ca(2+) entry pathways that regulate contractility, proliferation and migration. It is therefore important to understand how these channels are activated. Studies have shown that stimulation of TRPC1-based SOCs requires G protein α q subunit (Gαq)/phospholipase C (PLC)β1 activities and protein kinase C (PKC) phosphorylation, although it is unclear how store depletion stimulates this gating pathway. The present study examines this issue by focusing on the role of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum Ca(2+) sensor. Store-operated TRPC1 channel activity was inhibited by TRPC1 and STIM1 antibodies and STIM1 short hairpin RNA (shRNA) in wild-type VSMCs, and was absent in TRPC1(-/-) VSMCs. Store-operated PKC phosphorylation of TRPC1 was reduced by knockdown of STIM1. Moreover, store-operated PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH was reduced by STIM1 shRNA and absent in TRPC1(-/-) cells. Immunocytochemistry, co-immunoprecipitation and proximity ligation assays revealed that store depletion activated STIM1 translocation from within the cell to the plasma membrane (PM) where it formed STIM1-TRPC1 complexes, which then associated with Gαq and PLCβ1. Noradrenaline also evoked TRPC1 channel activity and associations between TRPC1, STIM1, Gαq and PLCβ1, which were inhibited by STIM1 knockdown. Effects of N-terminal and C-terminal STIM1 antibodies on TRPC1-based SOCs and STIM1 staining suggest that channel activation may involve insertion of STIM1 into the PM. The findings of the present study identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, in which store-operated STIM1-TRPC1 interactions stimulate PLCβ1 activity to induce PKC phosphorylation of TRPC1 and channel gating

Differential effects of inhibitory G protein isoforms on G protein-gated inwardly rectifying K+ currents in adult murine atria.

G protein-gated inwardly rectifying K+ (GIRK) channels are the major inwardly rectifying K+ currents in cardiac atrial myocytes and an important determinant of atrial electrophysiology. Inhibitory G protein α-subunits can both mediate activation via acetylcholine but can also suppress basal currents in the absence of agonist. We studied this phenomenon using whole cell patch clamping in murine atria from mice with global genetic deletion of Gαi2, combined deletion of Gαi1/Gαi3, and littermate controls. We found that mice with deletion of Gαi2 had increased basal and agonist-activated currents, particularly in the right atria while in contrast those with Gαi1/Gαi3 deletion had reduced currents. Mice with global genetic deletion of Gαi2 had decreased action potential duration. Tissue preparations of the left atria studied with a multielectrode array from Gαi2 knockout mice showed a shorter effective refractory period, with no change in conduction velocity, than littermate controls. Transcriptional studies revealed increased expression of GIRK channel subunit genes in Gαi2 knockout mice. Thus different G protein isoforms have differential effects on GIRK channel behavior and paradoxically Gαi2 act to increase basal and agonist-activated GIRK currents. Deletion of Gαi2 is potentially proarrhythmic in the atria.We thank the British Heart Foundation (RG/15/15/31742) and the Intramural Research Program of the NIH (project Z01ES101643) for funding this research. D.M. was supported by a grant from la Fédération Française de Cardiologie

Evidence that Orai1 does not contribute to store-operated TRPC1 channels in vascular smooth muscle cells.

Ca(2+)-permeable store-operated channels (SOCs) mediate Ca(2+) entry pathways which are involved in many cellular functions such as contraction, growth, and proliferation. Prototypical SOCs are formed of Orai1 proteins and are activated by the endo/sarcoplasmic reticulum Ca(2+) sensor stromal interaction molecule 1 (STIM1). There is considerable debate about whether canonical transient receptor potential 1 (TRPC1) proteins also form store-operated channels (SOCs), and if they do, is Orai1 involved. We recently showed that stimulation of TRPC1-based SOCs involves store depletion inducing STIM1-evoked Gαq/PLCβ1 activity in contractile vascular smooth muscle cells (VSMCs). Therefore the present work investigates the role of Orai1 in activation of TRPC1-based SOCs in freshly isolated mesenteric artery VSMCs from wild-type (WT) and Orai1(-/-) mice. Store-operated whole-cell and single channel currents recorded from WT and Orai1(-/-) VSMCs had similar properties, with relatively linear current-voltage relationships, reversal potentials of about +20mV, unitary conductances of about 2pS, and inhibition by anti-TRPC1 and anti-STIM1 antibodies. In Orai1(-/-) VSMCs, store depletion induced PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH, which was prevented by knockdown of STIM1. In addition, in Orai1(-/-) VSMCs, store depletion induced translocation of STIM1 from within the cell to the plasma membrane where it formed STIM1-TRPC1 interactions at discrete puncta-like sites. These findings indicate that activation of TRPC1-based SOCs through a STIM1-activated PLCβ1 pathway are likely to occur independently of Orai1 proteins, providing evidence that TRPC1 channels form genuine SOCs in VSMCs with a contractile phenotype

Long lasting facilitation of the rabbit cardiac Ca2+ channel: correlation with the coupling efficiency between charge movement and pore opening

AbstractFacilitation of Ca2+ entry into cells enhances Ca2+-activated events such as transmitter release and stimulation of second messenger systems. We have found a long lasting prepulse facilitation (up to 3-fold) of the cardiac Ca2+ channel α1C+β1b that lasts for tens of seconds without altering current kinetics. The voltage- and time-dependence of the installation of facilitation was characterized as well as the time- and use-dependence of the decay of facilitation. The degree of facilitation was correlated with the coupling efficiency between the charge movement and pore opening channels that were poorly coupled prior to facilitation exhibited the largest facilitation

Differential regulation of lipopolysaccharide and Gram-positive bacteria induced cytokine and chemokine production in splenocytes by Gαi proteins

AbstractHeterotrimeric Gi proteins play a role in lipopolysaccharide (LPS) and Staphylococcus aureus (SA) activated signaling leading to inflammatory mediator production. We hypothesized that genetic deletion of Gi proteins would alter cytokine and chemokine production induced by LPS and SA. LPS- and heat killed SA-induced cytokine and chemokine production in splenocytes from wild type (WT), Gαi2 (−/−) or Gαi1/3 (−/−) mice were investigated. LPS- or SA-induced production of TNFα, IL-6, IFNγ, IL-12, IL-17, GM-CSF, MIP-1α, MCP-1, MIG and IP-10 were significantly increased (1.2 to 33 fold, p<0.05) in splenocytes harvested from Gαi2(−/−) mice compared with WT mice. The effect of Gαi protein depletion was remarkably isoform specific. In splenocytes from Gαi1/3 (−/−) mice relative to WT mice, SA-induced IL-6, IFNγ, GM-CSF, and IP-10 levels were decreased (59% to 86%, p<0.05), whereas other LPS- or SA-stimulated cytokines and chemokines were not different relative to WT mice. LPS- and SA-induced production of KC were unchanged in both groups of the genetic deficient mice. Splenocytes from both Gαi2 (−/−) and Gαi1/3 (−/−) mice did not exhibit changes in TLR2 and TLR4 expression. Also analysis of splenic cellular composition by flow cytometry demonstrated an increase in splenic macrophages and reduced CD4 T cells in both Gαi2 (−/−) and Gαi1/3 (−/−) mice relative to WT mice. The disparate response of splenocytes from the Gαi2 (−/−) relative to Gαi1/3 (−/−) mice therefore cannot be attributed to major differences in spleen cellular composition. These data demonstrate that Gi2 and Gi1/3 proteins are both involved and differentially regulate splenocyte inflammatory cytokine and chemokine production in a highly Gi isoform specific manner in response to LPS and Gram-positive microbial stimuli

Mathematical modelling of polyamine metabolism in bloodstream-form trypanosoma brucei: An application to drug target identification

© 2013 Gu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedThis article has been made available through the Brunel Open Access Publishing Fund.We present the first computational kinetic model of polyamine metabolism in bloodstream-form Trypanosoma brucei, the causative agent of human African trypanosomiasis. We systematically extracted the polyamine pathway from the complete metabolic network while still maintaining the predictive capability of the pathway. The kinetic model is constructed on the basis of information gleaned from the experimental biology literature and defined as a set of ordinary differential equations. We applied Michaelis-Menten kinetics featuring regulatory factors to describe enzymatic activities that are well defined. Uncharacterised enzyme kinetics were approximated and justified with available physiological properties of the system. Optimisation-based dynamic simulations were performed to train the model with experimental data and inconsistent predictions prompted an iterative procedure of model refinement. Good agreement between simulation results and measured data reported in various experimental conditions shows that the model has good applicability in spite of there being gaps in the required data. With this kinetic model, the relative importance of the individual pathway enzymes was assessed. We observed that, at low-to-moderate levels of inhibition, enzymes catalysing reactions of de novo AdoMet (MAT) and ornithine production (OrnPt) have more efficient inhibitory effect on total trypanothione content in comparison to other enzymes in the pathway. In our model, prozyme and TSHSyn (the production catalyst of total trypanothione) were also found to exhibit potent control on total trypanothione content but only when they were strongly inhibited. Different chemotherapeutic strategies against T. brucei were investigated using this model and interruption of polyamine synthesis via joint inhibition of MAT or OrnPt together with other polyamine enzymes was identified as an optimal therapeutic strategy.The work was carried out under a PhD programme partly funded by Prof. Ray Welland, School of Computing Science, University of Glasgo

Determinants of the voltage dependence of G protein modulation within calcium channel β subunits

CaVβ subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3) domain and a guanylate kinase-like (GK) domain with an intervening HOOK domain. We have shown in a previous study that, although Gβγ-mediated inhibitory modulation of CaV2.2 channels did not require the interaction of a CaVβ subunit with the CaVα1 subunit, when such interaction was prevented by a mutation in the α1 subunit, G protein modulation could not be removed by a large depolarization and showed voltage-independent properties (Leroy et al., J Neurosci 25:6984–6996, 2005). In this study, we have investigated the ability of mutant and truncated CaVβ subunits to support voltage-dependent G protein modulation in order to determine the minimal domain of the CaVβ subunit that is required for this process. We have coexpressed the CaVβ subunit constructs with CaV2.2 and α2δ-2, studied modulation by the activation of the dopamine D2 receptor, and also examined basal tonic modulation. Our main finding is that the CaVβ subunit GK domains, from either β1b or β2, are sufficient to restore voltage dependence to G protein modulation. We also found that the removal of the variable HOOK region from β2a promotes tonic voltage-dependent G protein modulation. We propose that the absence of the HOOK region enhances Gβγ binding affinity, leading to greater tonic modulation by basal levels of Gβγ. This tonic modulation requires the presence of an SH3 domain, as tonic modulation is not supported by any of the CaVβ subunit GK domains alone

TRPC6 channel translocation into phagosomal membrane augments phagosomal function

Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H(+) into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired.Fil: Riazanski, Vladimir. University of Chicago; Estados UnidosFil: Gabdoulkhakova, Aida G.. University of Chicago; Estados UnidosFil: Boynton, Lin S.. University of Chicago; Estados UnidosFil: Eguchi, Raphael R.. University of Chicago; Estados UnidosFil: Deriy, Ludmila V.. University of Chicago; Estados UnidosFil: Hogarth, D. Kyle. University of Chicago; Estados UnidosFil: Loaëc, Nadège. ManRos Therapeutics; FranciaFil: Oumata, Nassima. ManRos Therapeutics; FranciaFil: Galons, Hervé. Universite de Paris; FranciaFil: Brown, Mary E.. University of Chicago; Estados UnidosFil: Shevchenko, Pavel. University of Chicago; Estados UnidosFil: Gallan, Alexander J.. University of Chicago; Estados UnidosFil: Yoo, Sang Gune. University of Chicago; Estados UnidosFil: Naren, Anjaparavanda P.. Cincinnati Children’s Hospital Medical Center; Estados UnidosFil: Villereal, Mitchel L.. University of Chicago; Estados UnidosFil: Beacham, Daniel W.. Thermo Scientific; Estados UnidosFil: Bindokas, Vytautas P.. University of Chicago; Estados UnidosFil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Meijer, Laurent. ManRos Therapeutics; FranciaFil: Nelson, Deborah J.. University of Chicago; Estados Unido

Differences in Physiological Responses to Cardiopulmonary Exercise Testing in Adults With and Without Type 1 Diabetes: A Pooled Analysis

OBJECTIVE To investigate physiological responses to cardiopulmonary exercise (CPX) testing in adults with type 1 diabetes compared with age-, sex-, and BMI-matched control participants without type 1 diabetes.RESEARCH DESIGN AND METHODS We compared results from CPX tests on a cycle ergometer in individuals with type 1 diabetes and control participants without type 1 diabetes. Parameters were peak and threshold variables of VO2, heart rate, and power output. Differences between groups were investigated through restricted maximum likelihood modeling and post hoc tests. Differences between groups were explained by stepwise linear regressions (P < 0.05).RESULTS Among 303 individuals with type 1 diabetes (age 33 [interquartile range 22; 43] years, 93 females, BMI 23.6 [22; 26] kg/m2, HbA1c 6.9% [6.2; 7.7%] [52 (44; 61) mmol/mol]), VO2peak (32.55 [26.49; 38.72] vs. 42.67 ± 10.44 mL/kg/min), peak heart rate (179 [170; 187] vs. 184 [175; 191] beats/min), and peak power (216 [171; 253] vs. 245 [200; 300] W) were lower compared with 308 control participants without type 1 diabetes (all P < 0.001). Individuals with type 1 diabetes displayed an impaired degree and direction of the heart rate-to-performance curve compared with control participants without type 1 diabetes (0.07 [−0.75; 1.09] vs. 0.66 [−0.28; 1.45]; P < 0.001). None of the exercise physiological responses were associated with HbA1c in individuals with type 1 diabetes.CONCLUSIONS Individuals with type 1 diabetes show altered responses to CPX testing, which cannot be explained by HbA1c. Intriguingly, the participants in our cohort were people with recent-onset type 1 diabetes; heart rate dynamics were altered during CPX testing