Dictyostelium myosin heavy chain kinase A (MHCK A) and mammalian transient receptor potential melastatin-related 7 (TRPM7) are two divergent members of a family of atypical protein kinases called the alpha kinases. The crystal structures of the alpha-kinase domains of MHCK A (A-CAT, residues 552-841) and mouse TRPM7 (TRPM7-CAT, residues 1548-1862) are very similar. In both cases a C-terminal tail (C-tail) sequence (A-CAT, residues 806-841 and TRPM7-CAT, residues 1819-1862) is missing from the crystal structure. Here I show that the unstructured C-tail is required for the catalytic activity of A-CAT and TRPM7-CAT. Truncation of the C-tail of A-CAT to residue 823 decreased kinase activity by ~98% and ATPase activity by ~97%. Truncation of the C-tail of TRPM7-CAT to residue 1827 decreased kinase activity by ~97% and ATPase activity by ~58%. Ligation of the C-tail sequence of MHCK B (residues 326-354) to A-CAT-802 (residues 552-802), fully rescued kinase activity. Alignment of the C-tail sequences of MHCK A-D revealed a conserved Gly-Thr-hydrophobic motif. Previous work has shown that in A-CAT, the conserved threonine (T825) is a site of autophosphorylation. Mutation of the T825 to alanine reduced A-CAT kinase and ATPase activities by 97%, whereas mutation to serine decreased kinase and ATPase rates by 85% and 60%, respectively. This result is consistent with the finding that A-CAT strongly prefers to phosphorylate threonine residues. Surprisingly, mutation of T825 to glutamic acid reduced kinase activity by ~93% and ATPase activity by ~96%. This result suggests that glutamic acid does not properly mimic phosphothreonine in this situation, or that the free hydroxyl group of T825 is required for the catalytic activity of A-CAT. Mutation of T825 to alanine or glutamic acid in full-length MHCK A reduced kinase activity by ~90% and ATPase activity by ~40%. Further studies are required to determine if the C-tail of TRPM7-CAT also contains an essential threonine residue. / Thesis (Master, Biochemistry) -- Queen's University, 2010-02-11 11:23:04.195

Ion channels are proteins that facilitate ion diffusion across cell membrane. Nevertheless, various groups of ion channels can act as surface receptors and play important roles in signal transduction. Transient Receptor Potential Melastatin 7 (TRPM7) ion channel has been implicated in diverse cellular functions including actomyosin cytoskeletal remodeling and anoxic neuronal death. However the mechanisms behind TRPM7’s physiological roles remain undetermined. TRPM7 possesses unusually long intracellular domains and a functional C-terminal alpha kinase domain that may contribute to regulation of channel activity and signal transduction. We therefore identified proteins that interact with TRPM7 C-terminus. Pull-down assays coupled with LC-MS/MS revealed that cytoskeletal proteins (actin and tubulin) and synaptic vesicle proteins (VAMP2 and SNAP25) associate with the TRPM7. In addition, we further found that TRPM7 does not directly bind microtubules or single dimeric tubulin subunits. Thus one or more microtubule binding proteins is involved in the association between TRPM7 and microtubules.

Ion channels are proteins that facilitate ion diffusion across cell membrane. Nevertheless, various groups of ion channels can act as surface receptors and play important roles in signal transduction. Transient Receptor Potential Melastatin 7 (TRPM7) ion channel has been implicated in diverse cellular functions including actomyosin cytoskeletal remodeling and anoxic neuronal death. However the mechanisms behind TRPM7’s physiological roles remain undetermined. TRPM7 possesses unusually long intracellular domains and a functional C-terminal alpha kinase domain that may contribute to regulation of channel activity and signal transduction. We therefore identified proteins that interact with TRPM7 C-terminus. Pull-down assays coupled with LC-MS/MS revealed that cytoskeletal proteins (actin and tubulin) and synaptic vesicle proteins (VAMP2 and SNAP25) associate with the TRPM7. In addition, we further found that TRPM7 does not directly bind microtubules or single dimeric tubulin subunits. Thus one or more microtubule binding proteins is involved in the association between TRPM7 and microtubules.

The Transient Receptor Potential, Melastatin-like 7 (TRPM7) protein is composed of a long amino terminus, 6 trans-membrane domains, and a carboxy terminal α-kinase domain; TRPM7 tetramers form non-selective cation channels with unusual permeability to Mg2+. TRPM7 is thought to be expressed in all cell types, and studies conducted primarily on cultured cells have implicated TRPM7 in cellular functions that include cell adhesion, synaptic vesicle release, kidney cation balance, differentiation, survival, and cellular magnesium homeostasis. The full extent of its physiological functions in vivo remains elusive because mouse TRPM7 homozygous null mutants die at embryonic stages. By contrast, zebrafish homozygous for hypomorphic alleles of trpm7 survive for two weeks post fertilization, making it possible to study the physiological consequences of Trpm7 deficiency in a living organism. My work primarily utilizes homozygous animals carrying the trpm7b508 allele, which we suspect encodes a non-functional protein for three reasons. This protein variant is predicted to lack a kinase domain, patch clamp studies fail to detect current, and morpholino knockdown of Trpm7 yields a similar phenotype. Herein, we explore the mechanisms behind each of three phenotypes in trpm7b508 homozygous embryos, i.e., trpm7 mutants. First, we find that cell death of embryonic melanophores in trpm7 mutants is not by apoptosis, and it is dependent upon melanin synthesis and the ion channel Trpm2. Second, we show that paralysis of trpm7 mutants is rescued by surgical opening of the circulatory system to surrounding media, implying that paralysis results from an organismal ion imbalance. Third, we report a variety of findings supporting the model that abnormally low levels of spontaneous swimming in larval trpm7 mutants results from reduced dopamine signaling. We find that specific populations of catecholaminergic neurons are reduced in mutants relative to their unaffected siblings, and that mutants are sensitized to the neurotoxic effects of 1-Methyl-4-phenylpyridinium iodide (MPP+). Together, these results suggest that Trpm7 has a role in ameliorating the toxic effects of reactive oxygen species in certain populations of melanophores and neurons. These findings advance understanding of the function of TRPM7 during embryonic development, and may have relevance to the gene-environment interaction behind certain neurodegenerative conditions.

Transient Receptor Potential ‘Melastatin’ 7 (TRPM7) is a ubiquitously expressed, non-selective divalent cation channel implicated in diverse cellular functions including actomyosin cytoskeletal remodeling, magnesium homeostasis, and anoxic neuronal death. The present study investigates the role of TRPM7 in modulating neuronal morphology and regulating neuronal cytoskeletal dynamics after anoxia. Overexpression of GFP-tagged TRPM7 in neuronal cultures caused a stunted morphology with fewer neurite branches than controls, suggesting that TRPM7 regulates the neuronal cytoskeleton during dendritic outgrowth. I have discovered that TRPM7 may regulate morphology via activation of cofilin-1 (an actin binding protein). I found that TRPM7-dependent cofilin activation during anoxia mediated neuronal death. Overall my work reveals a novel link between anoxia-induced TRPM7 activity and cofilin activation, which likely contributes to neurodegeneration after ischemia.

Transient Receptor Potential ‘Melastatin’ 7 (TRPM7) is a ubiquitously expressed, non-selective divalent cation channel implicated in diverse cellular functions including actomyosin cytoskeletal remodeling, magnesium homeostasis, and anoxic neuronal death. The present study investigates the role of TRPM7 in modulating neuronal morphology and regulating neuronal cytoskeletal dynamics after anoxia. Overexpression of GFP-tagged TRPM7 in neuronal cultures caused a stunted morphology with fewer neurite branches than controls, suggesting that TRPM7 regulates the neuronal cytoskeleton during dendritic outgrowth. I have discovered that TRPM7 may regulate morphology via activation of cofilin-1 (an actin binding protein). I found that TRPM7-dependent cofilin activation during anoxia mediated neuronal death. Overall my work reveals a novel link between anoxia-induced TRPM7 activity and cofilin activation, which likely contributes to neurodegeneration after ischemia.

A resistência dos tecidos à ação da insulina é uma das principais complicações do excesso de peso. O aumento da gordura corporal, decorrente do consumo excessivo de nutrientes, é acompanhado por um quadro de inflamação crônica de baixa intensidade que está relacionado com a fisiopatologia da resistência à insulina. O magnésio (Mg) é um mineral envolvido com diversos processos fisiológicos e bioquímicos, especialmente aqueles relacionados ao metabolismo energético e ao controle glicêmico. Apesar de a deficiência deste mineral estar relacionada com condições pré-diabéticas, não está claro se a inadequação dietética promove alterações na sensibilidade à insulina e/ou se condições de resistência à insulina causam distúrbios na homeostase de Mg. O objetivo deste trabalho foi investigar os efeitos da restrição dietética de Mg e sua associação com o excesso de lipídios sobre a homeostase do mineral e a sensibilidade à insulina. Ratos Wistar, machos, com peso entre 97-123 g, permaneceram em gaiolas individuais por 24 semanas. Os animais receberam rações normolipídicas (CON, 7% de lipídios) ou hiperlipídicas (HL, 32% de lipídios), adequadas (CON e HL Mg; 500 mg de Mg/kg de ração; n = 6 para cada grupo) ou com restrição de Mg (Mg[50] e HL Mg[50]; 50 mg de Mg/kg de ração; n = 6 para cada grupo). O consumo da dieta HL promoveu maior acúmulo de tecido adiposo e maior ganho de peso corporal (p < 0,05). Os animais que consumiram rações com restrição de Mg apresentaram hipomagnesemia (p<0,01), menor excreção urinária (p < 0,01) e fecal (p < 0,001) de Mg e menor concentração óssea desse mineral (p < 0,001). No entanto, não foram observadas alterações no Mg muscular (p > 0,05). O grupo HL Mg[50] apresentou maior concentração de Mg no eritrócito quando comparado aos outros grupos. A restrição dietética de Mg, isoladamente, não promoveu alterações na sensibilidade à insulina (avaliada pelo teste de tolerância à insulina). Quando associada à dieta hiperlipídica, resultou em aumento da glicemia de jejum e em redução da sensibilidade à insulina, após 16 semanas (p < 0,01). Em nível molecular, a fosforilação da proteína quinase B (Akt) no músculo e no fígado foi significantemente menor no grupo HL Mg[50] (p < 0,05). A restrição dietética de Mg induziu ao aumento do conteúdo proteico dos canais TRPM6 e TRPM7 no rim, independentemente da sensibilidade à insulina. Os resultados deste estudo apontam que a deficiência de Mg tende a agravar as repercussões metabólicas do consumo de dietas hiperlipídicas na sensibilidade à insulina e que a resistência à insulina altera a compartimentalização do Mg. / Insulin resistance is one of the main complications of overweight. Increase body fat, due to excessive consumption of nutrients is accompanied by a chronic low-grade inflammation related to insulin resistance pathophysiology. Magnesium (Mg) is a mineral involved in many physiological and biochemical processes, especially those related to energy metabolism and glycemic control. Although Mg deficiency is related to pre-diabetic conditions, it is unclear whether dietary inadequacy promotes changes in insulin sensitivity and/or if conditions of insulin resistance cause disturbances in Mg homeostasis. This work aimed to investigate the effects of dietary Mg restriction and its association with high-fat diet on mineral homeostasis and insulin sensitivity. Male Wistar rat (97-123 g) remained in individual cages for 24 weeks. Animals received normolipid diet (CON, 7% lipid) or high-fat diet (HF, 32% lipid), adequate (CON and HF, 500 mg Mg / kg diet, n = 6 for each group) or Mg restricted (Mg[50] and HF Mg[50], 50 mg of Mg / kg of diet, n = 6 for each group). High-fat diet promoted a greater adipose tissue excess and body weight gain (p<0.05). Animals with Mg restricted diet had hypomagnesemia (p<0.01), lower Mg urinary (p<0.01) and faecal loss (p<0.001) and lower bone Mg concentration (p<0.001). However, no changes were observed in muscle Mg (p>0.05). HF Mg[50] group presented higher concentration of erythrocyte Mg when compared to the other groups. Singly, dietary Mg restriction did not induce changes in insulin sensitivity (as assessed by the insulin tolerance test). When associated with high-fat diet, dietary Mg restriction resulted in higher fasting glycemia and lower insulin sensitivity after 16 weeks (p<0.01). At the molecular level, protein kinase B (Akt) phosphorylation in muscle and liver was significantly lower in HFMg [50] group (p<0.05). Dietary Mg restriction induced increased protein content of renal TRPM6 and TRPM7 channels, regardless of insulin sensitivity. The results of this study indicate that Mg deficiency worsens metabolic effects of high-fat diet on insulin sensitivity. In addition, insulin resistance changes Mg compartmentalization.

La mucoviscidose est une maladie causée par des mutations du gène cftr entraînant des défauts importants de la protéine CFTR. La mutation la plus fréquente (F508del) est caractérisée par un repliement incorrect conduisant à la rétention de la protéine dans le RE.L’accumulation de CFTR-F508del dans le RE, l’inflammation et les infections vont déclencher un stress du RE dans les cellules épithéliales ainsi que l’UPR. Cette dernière est une réponse adaptative déclenchée par le stress du RE et permet de rétablir l’homéostasie de ce compartiment. L’UPR est constituée de trois voies majeures dont l’une d’entre elles est activée dans les cellules exprimant un CFTR-F508del. Il s’agit de la voie ATF6 qui est de plus responsable de la répression transcriptionnelle du CFTR, ce qui en fait une cible thérapeutique potentielle. Nous avons montré que son inhibition conduit à l’amélioration de la fonction duCFTR-F508del et à l’augmentation de sa présence à la membrane des cellules.Nous nous sommes également intéressés au Mg2+ et au TRPM7, le régulateur principal de la [Mg2+]i dans les cellules. Nous avons émis l’hypothèse que TRPM7 était en partie responsable de l’activation d’ATF6 dans les cellules exprimant un CFTR-F508del. Le but de cette seconde partie du projet était donc tout d’abord d’étudier la relation existante entre le Mg2+, TRPM7 et le CFTR. Nous avons montré qu’il existait des différences de [Mg2+]i selon le type de mutation du CFTR exprimé par les cellules. Ces différences sont en partie dues à un défaut d’activation de TRPM7, lui-même probablement lié à un défaut du CFTR. En augmentant l’activité de TRPM7 par du Naltriben, nous avons pu montrer un effet potentialisant sur leCFTR-G551D. / Cystic fibrosis is caused by mutations in the cftr gene resulting in several defaults on the CFTR protein. The most frequent mutation is F508del which is characterized by an incorrect folding causing its retention within the ER. CFTR-F508del protein accumulation in the ER, inflammation and infections will trigger the ER stress in epithelial cells, as well as UPR. UPR constitutes an adaptive response of the ER in order to restore ER’s homeostasis. UPR consists in three major pathways. Among them, one is activated in cells expressing CFTR-F508del protein. The ATF6 pathway of UPR is responsible of the transcriptional repression of CFTR, which makes of it a potential therapeutic target. We showed that the inhibition of ATF6 leads to the improvement of CFTR-508del function, as well as its increased presence in the cellular membrane. We were also interested in Mg2+ and TRPM7, the main regulator of [Mg2+]i. We suspected that TRPM7 is, at least in part, responsible for the activation of ATF6 in cells expressing the mutant CFTR-F508del. Thus, the second part of my work was focused on the study of the relationship between Mg2+, TRPM7 and CFTR. We showed the existence of [Mg2+]I differences according to CFTR mutant expressed in cells. These differences are the result of an altered TRPM7 activation, probably in link with the mutated CFTR’s malfunction. We proved that increasing TRPM7 activity by Naltriben treatment potentiates CFTR-G551D.

A resistência dos tecidos à ação da insulina é uma das principais complicações do excesso de peso. O aumento da gordura corporal, decorrente do consumo excessivo de nutrientes, é acompanhado por um quadro de inflamação crônica de baixa intensidade que está relacionado com a fisiopatologia da resistência à insulina. O magnésio (Mg) é um mineral envolvido com diversos processos fisiológicos e bioquímicos, especialmente aqueles relacionados ao metabolismo energético e ao controle glicêmico. Apesar de a deficiência deste mineral estar relacionada com condições pré-diabéticas, não está claro se a inadequação dietética promove alterações na sensibilidade à insulina e/ou se condições de resistência à insulina causam distúrbios na homeostase de Mg. O objetivo deste trabalho foi investigar os efeitos da restrição dietética de Mg e sua associação com o excesso de lipídios sobre a homeostase do mineral e a sensibilidade à insulina. Ratos Wistar, machos, com peso entre 97-123 g, permaneceram em gaiolas individuais por 24 semanas. Os animais receberam rações normolipídicas (CON, 7% de lipídios) ou hiperlipídicas (HL, 32% de lipídios), adequadas (CON e HL Mg; 500 mg de Mg/kg de ração; n = 6 para cada grupo) ou com restrição de Mg (Mg[50] e HL Mg[50]; 50 mg de Mg/kg de ração; n = 6 para cada grupo). O consumo da dieta HL promoveu maior acúmulo de tecido adiposo e maior ganho de peso corporal (p < 0,05). Os animais que consumiram rações com restrição de Mg apresentaram hipomagnesemia (p<0,01), menor excreção urinária (p < 0,01) e fecal (p < 0,001) de Mg e menor concentração óssea desse mineral (p < 0,001). No entanto, não foram observadas alterações no Mg muscular (p > 0,05). O grupo HL Mg[50] apresentou maior concentração de Mg no eritrócito quando comparado aos outros grupos. A restrição dietética de Mg, isoladamente, não promoveu alterações na sensibilidade à insulina (avaliada pelo teste de tolerância à insulina). Quando associada à dieta hiperlipídica, resultou em aumento da glicemia de jejum e em redução da sensibilidade à insulina, após 16 semanas (p < 0,01). Em nível molecular, a fosforilação da proteína quinase B (Akt) no músculo e no fígado foi significantemente menor no grupo HL Mg[50] (p < 0,05). A restrição dietética de Mg induziu ao aumento do conteúdo proteico dos canais TRPM6 e TRPM7 no rim, independentemente da sensibilidade à insulina. Os resultados deste estudo apontam que a deficiência de Mg tende a agravar as repercussões metabólicas do consumo de dietas hiperlipídicas na sensibilidade à insulina e que a resistência à insulina altera a compartimentalização do Mg. / Insulin resistance is one of the main complications of overweight. Increase body fat, due to excessive consumption of nutrients is accompanied by a chronic low-grade inflammation related to insulin resistance pathophysiology. Magnesium (Mg) is a mineral involved in many physiological and biochemical processes, especially those related to energy metabolism and glycemic control. Although Mg deficiency is related to pre-diabetic conditions, it is unclear whether dietary inadequacy promotes changes in insulin sensitivity and/or if conditions of insulin resistance cause disturbances in Mg homeostasis. This work aimed to investigate the effects of dietary Mg restriction and its association with high-fat diet on mineral homeostasis and insulin sensitivity. Male Wistar rat (97-123 g) remained in individual cages for 24 weeks. Animals received normolipid diet (CON, 7% lipid) or high-fat diet (HF, 32% lipid), adequate (CON and HF, 500 mg Mg / kg diet, n = 6 for each group) or Mg restricted (Mg[50] and HF Mg[50], 50 mg of Mg / kg of diet, n = 6 for each group). High-fat diet promoted a greater adipose tissue excess and body weight gain (p<0.05). Animals with Mg restricted diet had hypomagnesemia (p<0.01), lower Mg urinary (p<0.01) and faecal loss (p<0.001) and lower bone Mg concentration (p<0.001). However, no changes were observed in muscle Mg (p>0.05). HF Mg[50] group presented higher concentration of erythrocyte Mg when compared to the other groups. Singly, dietary Mg restriction did not induce changes in insulin sensitivity (as assessed by the insulin tolerance test). When associated with high-fat diet, dietary Mg restriction resulted in higher fasting glycemia and lower insulin sensitivity after 16 weeks (p<0.01). At the molecular level, protein kinase B (Akt) phosphorylation in muscle and liver was significantly lower in HFMg [50] group (p<0.05). Dietary Mg restriction induced increased protein content of renal TRPM6 and TRPM7 channels, regardless of insulin sensitivity. The results of this study indicate that Mg deficiency worsens metabolic effects of high-fat diet on insulin sensitivity. In addition, insulin resistance changes Mg compartmentalization.

Transient Receptor Potential Melastatin 7 (TRPM7) is a ubiquitously expressed divalent cation channel implicated as a key regulator of neuronal cell death in stroke. Our research group has previously shown that TRPM7 dependent cytoskeletal regulation particularly via cofilin mediates neuronal death in oxygen glucose deprivation (in vitro stroke model). LIMK1 phosphorylation was also shown to decrease downstream of TRPM7 activation during anoxia. In the present study we investigated the effects of TRPM7 activation during anoxia, on three regulators of LIMK and cofilin; Rho-associated kinase 2 (ROCK2), P-21 activated kinase 3 (PAK3) and Slingshot family phosphatase 1 (SSH1). Our findings suggest that PAK3 activity is downregulated during OGD through TRPM7 independent mechanisms. However, SSH1 activity appears to be regulated downstream of TRPM7 in a manner that is consistent with LIMK and cofilin regulation. Overall, our work suggests that SSH1 is a new link between anoxia-induced TRPM7activity and cofilin hyperactivation.