On the roles of Mg in the activation of G proteins

In this review, we highlight the evolution of our knowledge about the way Mg2+ participates in the activation of heterotrimeric G proteins, beginning with its requirement in hormonal stimulation of fat cell adenylyl cyclase (1969) and ending with knowledge that incorporates information obtained from site directed mutagenesis and examination of the crystal structures of G proteins (2010). Our current view is that, as it seeks to fill its octahedral coordination shell, Mg acts as a keystone locking the G protein-α subunits into a conformation in which Gα dissociates from the Gβγ dimer, is competent in regulating effectors, and acquires GTPase activity. The latter is the result of moving the backbone carbonyl group of the Mg-coordinating threonine into a location in space that positions the hydrolytic water so as to facilitate the water's nucleophilic attack that leads to hydrolysis of the link between the β and γ phosphates of guanosine triphosphate (GTP). The role of the backbone carbonyl group of the Mg-coordinating threonine is equi-hierarchical with a similar and long-recognized role of the Switch II glutamine δ amide carbonyl group. Disruption of either leads to loss of GTPase activity.Fil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Public Healt Service. National Institute Of Health. Laboratorio Of Developmental Neurobiology; Estados UnidosFil: Zurita, Adolfo Ramón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Public Healt Service. National Institute Of Health. Laboratorio Of Developmental Neurobiology; Estados Unido

From GTP and G proteins to TRPC channels: a personal account

By serendipity and good fortune, as a postdoctoral fellow in 1967, I landed at the right place at the right time, as I was allowed to investigate the mechanism by which hormones activate the enzyme adenylyl cyclase (then adenyl cyclase) in Martin Rodbell’s Laboratory at the NIH in Bethesda, Maryland. The work uncovered first, the existence of receptors separate from the enzyme and then, the existence of transduction mechanisms requiring guanosine-5′-triphosphate (GTP) and Mg2+. With my laboratory colleagues first and postdoctoral fellows after leaving NIH, I participated in the development of the field “signal transduction by G proteins,” uncovered by molecular cloning several G-protein-coupled receptors (GPCRs) and became interested in both the molecular makeup of voltage-gated Ca channels and Ca2+ homeostasis downstream of activation of phospholipase C (PLC) by the Gq/11 signaling pathway. We were able to confirm the hypothesis that there would be mammalian homologues of the Drosophila “transient receptor potential” channel and discovered the existence of six of the seven mammalian genes, now called transient receptor potential canonical (TRPC) channels. In the present article, I summarize from a bird’s eye view of what I feel were key findings along this path, not only from my laboratory but also from many others, that allowed for the present knowledge of cell signaling involving G proteins to evolve. Towards the end, I summarize roles of TRPC channels in health and disease.Fil: Birnbaumer, Lutz. 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; Argentina. National Institute of Environmental Research; Estados Unido

TRPC1 regulates calcium-activated chloride channels in salivary gland cells

Calcium-activated chloride channel (CaCC) plays an important role in modulating epithelial secretion. It has been suggested that in salivary tissues, sustained fluid secretion is dependent on Ca2+ influx that activates ion channels such as CaCC to initiate Cl- efflux. However direct evidence as well as the molecular identity of the Ca2+ channel responsible for activating CaCC in salivary tissues is not yet identified. Here we provide evidence that in human salivary cells, an outward rectifying Cl- current was activated by increasing [Ca2+]i, which was inhibited by the addition of pharmacological agents niflumic acid (NFA), an antagonist of CaCC, or T16Ainh-A01, a specific TMEM16a inhibitor. Addition of thapsigargin (Tg), that induces store-depletion and activates TRPC1-mediated Ca2+ entry, potentiated the Cl- current, which was inhibited by the addition of a non-specific TRPC channel blocker SKF96365 or removal of external Ca2+. Stimulation with Tg also increased plasma membrane expression of TMEM16a protein, which was also dependent on Ca2+ entry. Importantly, in salivary cells, TRPC1 silencing, but not that of TRPC3, inhibited CaCC especially upon store depletion. Moreover, primary acinar cells isolated from submandibular gland also showed outward rectifying Cl- currents upon increasing [Ca2+]i. These Cl- currents were again potentiated with the addition of Tg, but inhibited in the presence of T16Ainh-A01. Finally, acinar cells isolated from the submandibular glands of TRPC1 knockout mice showed significant inhibition of the outward Cl- currents without decreasing TMEM16a expression. Together the data suggests that Ca2+ entry via the TRPC1 channels is essential for the activation of CaCC.Fil: Sun, Yuyang. University Of North Dakota; Estados UnidosFil: Birnbaumer, Lutz. National Institutes of Health; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Singh, Brij B.. University Of North Dakota; Estados Unido

Reduced Necrosis and Content of Apoptotic M1 Macrophages in Advanced Atherosclerotic Plaques of Mice With Macrophage-Specific Loss of Trpc3

In previous work we reported that ApoeKO mice transplanted with bone marrow cells deficient in the Transient Receptor Potential Canonical 3 (TRPC3) channel have reduced necrosis and number of apoptotic macrophages in advanced atherosclerotic plaques. Also, in vitro studies with polarized macrophages derived from mice with macrophage-specific loss of TRPC3 showed that M1, but not M2 macrophages, deficient in Trpc3 are less susceptible to ER stress-induced apoptosis than Trpc3 expressing cells. The questions remained (a) whether the plaque phenotype in transplanted mice resulted from a genuine effect of Trpc3 on macrophages, and (b) whether the reduced necrosis and macrophage apoptosis in plaques of these mice was a manifestation of the selective effect of TRPC3 on apoptosis of M1 macrophages previously observed in vitro. Here, we addressed these questions using Ldlr knockout (Ldlr−/−) mice with macrophage-specific loss of Trpc3 (MacTrpc3−/−/Ldlr−/− → Ldlr−/−). Compared to controls, we observed decreased plaque necrosis and number of apoptotic macrophages in MacTrpc3−/−/Ldlr−/− → Ldlr−/− mice. Immunohistochemical analysis revealed a reduction in apoptotic M1, but not apoptotic M2 macrophages. These findings confirm an effect of TRPC3 on plaque necrosis and support the notion that this is likely a reflection of the reduced susceptibility of Trpc3-deficient M1 macrophages to apoptosis.Fil: Solanki, Sumeet. University of Toledo; Estados UnidosFil: Dube, Prabhatchandra R.. University of Toledo; Estados UnidosFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Vazquez, Guillermo. University of Toledo; Estados Unido

Critical role of canonical transient receptor potential channel 7 in initiation of seizures

Status epilepticus (SE) is a life-threatening disease that has been recognized since antiquity but still causes over 50,000 deaths annually in the United States. The prevailing view on the pathophysiology of SE is that it is sustained by a loss of normal inhibitory mechanisms of neuronal activity. However, the early process leading to the initiation of SE is not well understood. Here, we show that, as seen in electroencephalograms, SE induced by the muscarinic agonist pilocarpine in mice is preceded by a specific increase in the gamma wave, and genetic ablation of canonical transient receptor potential channel (TRPC) 7 significantly reduces this pilocarpine-induced increase of gamma wave activity, preventing the occurrence of SE. At the cellular level, TRPC7 plays a critical role in the generation of spontaneous epileptiform burst firing in cornu ammonis (CA) 3 pyramidal neurons in brain slices. At the synaptic level, TRPC7 plays a significant role in the long-term potentiation at the CA3 recurrent collateral synapses and Schaffer collateral-CA1 synapses, but not at the mossy fiber-CA3 synapses. Taken together, our data suggest that epileptiform burst firing generated in the CA3 region by activity-dependent enhancement of recurrent collateral synapses may be an early event in the initiation process of SE and that TRPC7 plays a critical role in this cellular event. Our findings reveal that TRPC7 is intimately involved in the initiation of seizures both in vitro and in vivo. To our knowledge, this contribution to initiation of seizures is the first identified functional role for the TRPC7 ion channel.Fil: Phelan, K. D.. University of Arkansas for Medical Sciences; Estados UnidosFil: Shwe, U. T.. University of Arkansas for Medical Sciences; Estados UnidosFil: Abramowitz, J.. National Institute of Environmental Health Sciences; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences; Estados UnidosFil: Zheng, F.. University of Arkansas for Medical Sciences; Estados Unido

Deletion of diacylglycerol-responsive TRPC genes attenuates diabetic nephropathy by inhibiting activation of the TGFβ1 signaling pathway

TRPC6 plays a critical role in proteinuric kidney diseases, and TRPC3 is involved in tubulointerstitialdamage and renal fibrosis in obstructed kidneys. Podocyte loss is a characteristic event in diabetic nephropathy(DN). The aim of this study was to examine whether deletion of the closely related diacylglycerol (DAG)-responsiveTRPCs in mice (TRPC3/6/7-/-) affects diabetes-induced renal dysfunction and podocyte loss. We compared urinevolume, kidney hypertrophy, glomerular enlargement, albuminuria and podocyte loss between wild type (WT) andTRPC3/6/7-/- diabetic mice. Finally, we examined whether the TGFβ1 signaling pathway is changed in diabetic WTand TRPC3/6/7-/- mice. TRPC6 protein in the renal cortex was increased in WT diabetic mice. High glucose (HG)treatment increased TRPC6 expression in human podocytes. TRPC3 protein, however, was not altered in eitherdiabetic mice or HG-treated human podocytes. Although diabetic WT and TRPC3/6/7-/- mice had similar levels ofhyperglycemia, the TRPC3/6/7-/- diabetic mice showed less polyuria, kidney hypertrophy, glomerular enlargement,albuminuria, and had lost less podocytes compared with WT diabetic mice. In addition, we observed decreasedexpression of anti-apoptotic Bcl2 and increased expression of pro-apoptotic cleaved caspase 3 in WT diabetic mice,but such changes were not significant in TRPC3/6/7-/- diabetic mice. Western blot and immunohistochemistry revealedthat TGFβ1, p-Smad2/3, and fibronectin were upregulated in WT diabetic mice; however, expression of thesesignaling molecules was not changed in TRPC3/6/7-/- diabetic mice. In conclusion, deletion of DAG-responsiveTRPCs attenuates diabetic renal injury via inhibiting the upregulation of TGFβ1 signaling in diabetic kidneys.Fil: Liu, Benju. Huazhong University of Science and Technology; ChinaFil: He, Xiju. Huazhong University of Science and Technology; ChinaFil: Li, Shoutian. Yangtze University; ChinaFil: Xu, Benke. Yangtze University; ChinaFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Liao, Yanhong. Huazhong University of Science and Technology; Chin

TRPV4, TRPC1, and TRPP2 assemble to form a flow-sensitive heteromeric channel

Transient receptor potential (TRP) channels, a superfamily of ion channels, can be divided into 7 subfamilies, including TRPV, TRPC, TRPP, and 4 others. Functional TRP channels are tetrameric complexes consisting of 4 pore-forming subunits. The purpose of this study was to explore the heteromerization of TRP subunits crossing different TRP subfamilies. Two-step coimmunoprecipitation (co-IP) and fluorescence resonance energy transfer (FRET) were used to determine the interaction of the different TRP subunits. Patch-clamp and cytosolic Ca2+ measurements were used to determine the functional role of the ion channels in flow conditions. The analysis demonstrated the formation of a heteromeric TRPV4-C1-P2 complex in primary cultured rat mesenteric artery endothelial cells (MAECs) and HEK293 cells that were cotransfected with TRPV4, TRPC1, and TRPP2. In functional experiments, pore-dead mutants for each of these 3 TRP isoforms nearly abolished the flow-induced cation currents and Ca2+ increase, suggesting that all 3 TRPs contribute to the ion permeation pore of the channels. We identified the first heteromeric TRP channels composed of subunits from 3 different TRP subfamilies. Functionally, this heteromeric TRPV4- C1-P2 channel mediates the flow-induced Ca2+ increase in native vascular endothelial cells.-Du, J., Ma, X., Shen, B., Huang, Y., Birnbaumer, L., Yao, X. TRPV4, TRPC1, and TRPP2 assemble to form a flowsensitive heteromeric channel.Fil: Du, Juan. Chinese University Of Hong Kong; Hong Kong. Anhui Medical University; ChinaFil: Ma, Xin. Chinese University Of Hong Kong; Hong KongFil: Shen, Bing. Chinese University Of Hong Kong; Hong Kong. Anhui Medical University; ChinaFil: Huang, Yu. Chinese University Of Hong Kong; Hong KongFil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. National Institutes of Health; Estados UnidosFil: Yao, Xiaoqiang. Chinese University Of Hong Kong; Hong Kon

Deep Transcriptomic Profiling of M1 Macrophages Lacking Trpc3

In previous studies using mice with macrophage-specific loss of TRPC3 we found a significant, selective effect of TRPC3 on the biology of M1, or inflammatory macrophages. Whereas activation of some components of the unfolded protein response and the pro-apoptotic mediators CamkII and Stat1 was impaired in Trpc3-deficient M1 cells, gathering insight about other molecular signatures within macrophages that might be affected by Trpc3 expression requires an alternative approach. In the present study we conducted RNA-seq analysis to interrogate the transcriptome of M1 macrophages derived from mice with macrophage-specific loss of TRPC3 and their littermate controls. We identified 160 significantly differentially expressed genes between the two groups, of which 62 were upregulated and 98 downregulated in control vs. Trpc3-deficient M1 macrophages. Gene ontology analysis revealed enrichment in processes associated to cellular movement and lipid signaling, whereas the enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways included networks for calcium signaling and cell adhesion molecules, among others. This is the first deep transcriptomic analysis of macrophages in the context of Trpc3 deficiency and the data presented constitutes a unique resource to further explore functions of TRPC3 in macrophage biology.Fil: Kumarasamy, Sivarajan. University of Toledo; Estados UnidosFil: Solanki, Sumeet. University of Toledo; Estados UnidosFil: Atolagbe, Oluwatomisin T.. University of Toledo; Estados UnidosFil: Joe, Bina. University of Toledo; Estados UnidosFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Vazquez, Guillermo. University of Toledo; Estados Unido

Increased size and cellularity of advanced atherosclerotic lesions in mice with endothelial overexpression of the human TRPC3 channel

In previous in vitro studies, we showed that Transient Receptor Potential Canonical 3 (TRPC3), a calcium-permeable, nonselective cation channel endowed with high constitutive function, is an obligatory component of the inflammatory signaling that controls expression of the vascular cell adhesion molecule-1 (VCAM-1) and monocyte adhesion to coronary artery endothelial cells. Also, TRPC3 expression in these cells was found to be up-regulated by proatherogenic factors, which enhanced inflammation and VCAM-1 expression. However, it remained to be determined whether these in vitro findings were of relevance to atherosclerotic lesion development in vivo. To answer this important question in the present work, we generated mice with endothelial-specific overexpression of human TRPC3 in an Apoe knockout background (TgEST3ApoeKO) and examined lesions in the aortic sinus following 10 and 16 wk on a high-fat diet. No significant differences were found in size or complexity of early stage lesions (10 wk). However, advanced plaques (16 wk) from TgEST3ApoeKO mice exhibited a significant increase in size and macrophage content compared with nontransgenic littermate controls. Remarkably, this change was correlated with increased VCAM-1 and phospho-IkBα immunoreactivity along the endothelial lining of lesions from transgenic animals compared with controls. These findings validate the in vivo relevance of previous in vitro findings and represent, to our knowledge, the first in vivo evidence for a proatherogenic role of endothelial TRPC3.Fil: Smedlund, Kathryn B.. University of Toledo College of Medicine; Estados UnidosFil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences; Estados Unidos. Comisión Nacional de Investigación Científica y Tecnológica; ChileFil: Vazquez, Guillermo. University of Toledo College of Medicine; Estados Unido

RNA-seq analysis reveals TRPC genes to impact an unexpected number of metabolic and regulatory pathways

The seven-member transient receptor potential canonical genes (TRPC1-7) encode cation channels linked to several human diseases. There is little understanding of the participation of each TRPC in each pathology, considering functional redundancy. Also, most of the inhibitors available are not specifc. Thus, we developed mice that lack all of the TRPCs and performed a transcriptome analysis in eight tissues. The aim of this research was to address the impact of the absence of all TRPC channels on gene expression. We obtained a total of 4305 diferentially expressed genes (DEGs) in at least one tissue where spleen showed the highest number of DEGs (1371). Just 21 genes were modifed in all the tissues. Performing a pathway enrichment analysis, we found that many important signaling pathways were modifed in more than one tissue, including PI3K (phosphatidylinositol 3-kinase/protein kinase-B) signaling pathway, cytokine-cytokine receptor interaction, extracellular matrix (ECM)-receptor interaction and circadian rhythms. We describe for the frst time the changes at the transcriptome level due to the lack of all TRPC proteins in a mouse model and provide a starting point to understand the function of TRPC channels and their possible roles in pathologies.Fil: Formoso, Karina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Susperreguy, Sebastian. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Freichel, Marc. Universität Heidelberg; AlemaniaFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin