Functions of the C terminal domain of inwardly rectifying K+ channel

内向整流钾离子通道广泛存在于心肌细胞和神经细胞中，具有维持静息电导、起搏、抑制突触和神经元放电等多种细胞功能。IRK 1是经典的内向型钾离子通道之一，其C端有一个长的、由约250个氨基酸组成的结构域，其功能尚未完全阐明，为了研究IRK 1的功能，我们将IRK 1和GFP基因转染PC 12细胞，并用神经生长因子（NGF）诱导其向神经细胞分化。用同样的基因转染培养的胎鼠海马神经元。Western blotting检测到PC 12细胞中有表达，但在培养的海马神经元中无表达。我们通过免疫组织化学方法检测了IRK 1基因产物在转染的PC 12细胞中的定位和分布。免疫反应性似乎主要定位于质膜，在较低程度上，在其他细胞器，这是不能明确证实，因为表达水平低。然后，我们用腺病毒载体将IRK 1基因导入细胞。将IRK 1基因插入腺病毒载体中，构建重组腺病毒DNA，转染HEK 293细胞，制备重组病毒颗粒。重组病毒颗粒的滴度为1.5 × 105 pfu/mL。我们用病毒颗粒在不同MOI感染PC 12细胞和培养的海马神经元，但由于病毒滴度低和病毒溶液的高毒性，不能获得高水平的表达。我们尝试纯化病毒颗粒以去除毒性并获得高滴度的病毒。但在纯化过程中病毒滴度迅速下降并失活。我们推测插入的IRK 1基因本身可能抑制重组病毒的活性。

The inwardly rectifying K+ channels have been found in a variety of cell types including cardiac myocytes and neuronal cells, and play diverse cellular functions such as maintenance of the resting conductance, pacemaker activity, synaptic inhibition, and neuronal firing rates. IRK1, one of the classical inwardly retifying K+ channel, has long C terminal domain comprised of 〜250 amino acidsand the function of the domain has not been elucidated.To analyze the function, we transfected PC12 cells with IRK1 and GFP genes and treated with NGF to differentiate to neuronal cells. Cultured hippocampal neurons from rat fetus were also transfected with the same genes. The gene products could be detected by Western blotting in the PC12 cells but not in the clutured hippocampal neurons. We examined the localization and distribution of the IRK1 gene products by immunohistochemistry in the transfected PC12 cells. The immunoreactivity appeared to be localized mainly in plasma membrane and to a lower extent, in other organelles, which could not been confirmed clearly because of the low expression levels. Then, we used adenovirus vector to introduce IRK1 gene to the cells. IRK1 gene was inserted into the adenovirus vector and the created recombinant adenovirus DNA was used to transfect HEK293 cells to make the recombinant virus particles. The titer of the recombinant virus particles were 〜5x105 pfu/mL.. We used the virus particles to infect PC12 cells and cultured hippocampal neurons at various MOI, but could not get high expression levels owing to low virus titer and high toxicity of the virus solution. We tried to purify the virus particles to remove the toxicity and to get virus with high titer. However, the virus titer was decreased quickly and inactivated during the purification. We suppose the inserted IRK1 gene itself may inhibit the recombinant virus activity.