AIM To study the membrane trafficking modulation of ROMK1 channel by ubiquitination. METHODS we immunoprecipitated protein with ROMK antibody from rat renal cortex and outer medulla, and apply western blot with ubiqui...AIM To study the membrane trafficking modulation of ROMK1 channel by ubiquitination. METHODS we immunoprecipitated protein with ROMK antibody from rat renal cortex and outer medulla, and apply western blot with ubiquitin antibody to detect that ROMK protein is being ubiquitination under physiological condition in vivo. Furthermore, we used His-antibody and ubiquitin antibodies to recongnize proteins in the cotransfected HEK293 cells with hemagglutinin-ubiquitin and His- tagged ROMK1 constructs, respectively. After all intracellular lysine (Lys) residues of ROMK1 had been individually mutated to arginine (Arg), two -electrode voltage clamp was used to measure the ROMK1 channel activity in Xenopus oocytes. To detect the effect of ubiquitin on ROMK1, we used Western blotting technique to search the difference on total protein expression levelspus oocytes.展开更多
WNK（with no lysine（k））激酶是一类新型丝氨酸／苏氨酸蛋白激酶亚型。WNK家族的两种突变型会导致以高血压、高血钾、代谢性酸中毒为临床特征的Ⅱ型假性低醛固酮血症（PHAⅡ）。这些临床症状表明WNK对肾脏远曲小管主要的钾离子转运...WNK（with no lysine（k））激酶是一类新型丝氨酸／苏氨酸蛋白激酶亚型。WNK家族的两种突变型会导致以高血压、高血钾、代谢性酸中毒为临床特征的Ⅱ型假性低醛固酮血症（PHAⅡ）。这些临床症状表明WNK对肾脏远曲小管主要的钾离子转运体包括肾外部髓质钾通道（ROMK）和钙激活钾通道（MaxiKorBK）可能有调节作用。最新研究表明，WNK4激酶不仅抑制ROMK通道功能，并且PHAⅡ型突变的WNK4激酶能够增强对非洲爪蟾卵的ROMK通道功能的抑制。目标：为确定WNK激酶是否会影响MaxiK通道功能，我们研究稳定表达MaxiK通道α亚基的HEK293细胞（HEK293-MaxiK）中的WNK4激酶对MaxiK通道功能的影响。方法：用CD4、WNK4野生型或者WNK4激酶失活变异体型D321A分别转染HEK293-MaxiK，2至4天后用细胞粘附式膜片钳记录单离子通道的活动。展开更多
文摘AIM To study the membrane trafficking modulation of ROMK1 channel by ubiquitination. METHODS we immunoprecipitated protein with ROMK antibody from rat renal cortex and outer medulla, and apply western blot with ubiquitin antibody to detect that ROMK protein is being ubiquitination under physiological condition in vivo. Furthermore, we used His-antibody and ubiquitin antibodies to recongnize proteins in the cotransfected HEK293 cells with hemagglutinin-ubiquitin and His- tagged ROMK1 constructs, respectively. After all intracellular lysine (Lys) residues of ROMK1 had been individually mutated to arginine (Arg), two -electrode voltage clamp was used to measure the ROMK1 channel activity in Xenopus oocytes. To detect the effect of ubiquitin on ROMK1, we used Western blotting technique to search the difference on total protein expression levelspus oocytes.
文摘WNK（with no lysine（k））激酶是一类新型丝氨酸／苏氨酸蛋白激酶亚型。WNK家族的两种突变型会导致以高血压、高血钾、代谢性酸中毒为临床特征的Ⅱ型假性低醛固酮血症（PHAⅡ）。这些临床症状表明WNK对肾脏远曲小管主要的钾离子转运体包括肾外部髓质钾通道（ROMK）和钙激活钾通道（MaxiKorBK）可能有调节作用。最新研究表明，WNK4激酶不仅抑制ROMK通道功能，并且PHAⅡ型突变的WNK4激酶能够增强对非洲爪蟾卵的ROMK通道功能的抑制。目标：为确定WNK激酶是否会影响MaxiK通道功能，我们研究稳定表达MaxiK通道α亚基的HEK293细胞（HEK293-MaxiK）中的WNK4激酶对MaxiK通道功能的影响。方法：用CD4、WNK4野生型或者WNK4激酶失活变异体型D321A分别转染HEK293-MaxiK，2至4天后用细胞粘附式膜片钳记录单离子通道的活动。
