Navegando por Palavras-chave "Gating"

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    Investigação funcional do canal para potássio KIR2.6: novas perspectivas para o Gating do canal
    (Universidade Federal de São Paulo (UNIFESP), 2021) Paninka, Rolf Matias [UNIFESP]; Miranda Filho, Manoel de Arcisio [UNIFESP]; Universidade Federal de São Paulo
    The lipidic nature of the plasma membrane constitutes a barrier to the passage of electrically charged particles solvated in water. Ion channels are integral membrane proteins connecting the internal and external media of the cell allowing the passage of specific ions in a highly selective way. This work discusses a peculiar family of potassium channels called Kir, which stands for “Potassium inward rectifier”. Kir channels resemble a diode, allowing K+ to flow more easily into the cells, but limiting the flow in the reverse direction. Kir2.6 channel, encoded by the KCNJ18 gene, has been linked to a clinical condition called Thyrotoxic Periodic Paralysis (TPP). At the first part of the thesis, the direct DNA sequencing of two patients diagnosed with PPT, revealed two heterozygous mutations of the KCNJ18 gene: D252N and R386C, respectively. cDNAs of Kir2.6 wild-type (WT) channels and their mutants were transiently transfected to Embryonic Human Kidney cells (HEK293T) and analyzed by electrophysiology (voltage-clamp at whole-cell mode) and confocal laser scanning microscopy. D252N mutation showed a decrease in the current density of the channel in the order of 34%, when compared to the wild Kir2.6 (WT) channel. In addition, this mutation had a negative dominant effect when co-transfected in proportion of 50% with the Kir2.6 WT channel. Finally, D252N mutation demonstrated a substantial reduction (~ 51%) on membrane abundance of the channel compared to Kir2.6 WT channel. R386C mutation showed no significant change in comparation to Kir2.6 WT channel. At the second part of the thesis, the gating (the opening and closing mechanism) of the channel was investigated. For this purpose, two regions of the channel were selected so far not mentioned in the literature and, through a technique called alanine scanning, 20 amino acids of the channel-forming protein were systematically mutated by alanine. In doing so, we aim to study the individual contribution of each of these amino acids to the stability and functioning of the channel region located at the outer leaf of the plasma membrane.20 Using the same methodology used in the first part, nine amino acids were identified that proved to be critical for maintaining the conductive state of the channel: F102, W103, I105, A106, A158, V159, V162, and Q165. Six of them are in symmetrical positions in the first and second transmembrane segments of the channel-forming monomer: F102, I105, A106 are at the first segment and, A158, V159 and V162 are at the second segment. This suggests that these three pairs of amino acids may be important in maintaining the stability of the ion-conducting pore. In addition, the alignment of the total length of the protein between the Kir channel family (KCNJ1-KCNJ18) and the molecular analysis of distances between the amino acids at the pore region of the channel, showed possible interactions between amino acids F102 and F136, as well as between amino acids W103 and F130. The alanine mutation of the amino acids F130 and F136 revealed data consistent with this hypothesis, since the electrophysiological analysis showed total and partial suppression, respectively, of the channel activity. Our results suggest that these amino acids are important for maintaining the channel structure and the stability of the ion conducting pore and that there may be a mechanism for opening and closing the channel positioned in the most extracellular portion of the transmembrane domain of the channel.
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    Thyroid hormone activation by type2 deiodinase mediates exercise-induced peroxisome proliferator-activated receptor-gamma coactivator-1 alpha expression in skeletal muscle
    (Wiley-Blackwell, 2016) Bocco, Barbara Miranda Leite da Costa [UNIFESP]; Louzada, Ruy A. N.; Silvestre, Diego H. S.; Santos, Maria C. S.; Anne-Palmer, Elena; Rangel, Igor F.; Abdalla, Sherine; Ferreira, Andrea C.; Ribeiro, Miriam Oliveira; Gereben, Balazs; Carvalho, Denise P.; Bianco, Antonio Carlos; Werneck-de-Castro, João Pedro Saar; Universidade Federal de São Paulo (UNIFESP)
    Key points In skeletal muscle, physical exercise and thyroid hormone mediate the peroxisome proliferator-activated receptor- coactivator-1 (PGC-1a) expression that is crucial to skeletal muscle mitochondrial function. The expression of type 2 deiodinase (D2), which activates thyroid hormone in skeletal muscle is upregulated by acute treadmill exercise through a -adrenergic receptor-dependent mechanism. Pharmacological block of D2 or disruption of the Dio2 gene in skeletal muscle fibres impaired acute exercise-induced PGC-1a expression.Dio2 disruption also impaired muscle PGC-1a expression and mitochondrial citrate synthase activity in chronically exercised mice. Abstract: Thyroid hormone promotes expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1a), which mediates mitochondrial biogenesis and oxidative capacity in skeletal muscle (SKM). Skeletal myocytes express the type 2 deiodinase (D2), which generates 3,5,3′-triiodothyronine (T3), the active thyroid hormone. To test whether D2-generated T3 plays a role in exercise-induced PGC-1a expression, male rats and mice with SKM-specific Dio2 inactivation (SKM-D2KO or MYF5-D2KO) were studied. An acute treadmill exercise session (20 min at 70–75% of maximal aerobic capacity) increased D2 expression/activity (1.5- to 2.7-fold) as well as PGC-1a mRNA levels (1.5- to 5-fold) in rat soleus muscle and white gastrocnemius muscle and in mouse soleus muscle, which was prevented by pretreatment with 1 mg (100 g body weight)−1 propranolol or 6 mg (100 g body weight)−1 iopanoic acid (5.9- vs. 2.8-fold; P < 0.05), which blocks D2 activity . In the SKM-D2KO mice, acute treadmill exercise failed to induce PGC-1a fully in soleus muscle (1.9- vs. 2.8-fold; P < 0.05), and in primary SKM-D2KO myocytes there was only a limited PGC-1a response to 1 μm forskolin (2.2- vs. 1.3-fold; P < 0.05). Chronic exercise training (6 weeks) increased soleus muscle PGC-1a mRNA levels (∼25%) and the mitochondrial enzyme citrate synthase (∼20%). In contrast, PGC-1a expression did not change and citrate synthase decreased by ∼30% in SKM-D2KO mice. The soleus muscle PGC-1a response to chronic exercise was also blunted in MYF5-D2KO mice. In conclusion, acute treadmill exercise increases SKM D2 expression through a β-adrenergic receptor-dependent mechanism. The accelerated conversion of T4 to T3 within myocytes mediates part of the PGC-1a induction by treadmill exercise and its downstream effects on mitochondrial function.