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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.