Novel C-terminus frameshift mutation, 1122fs/147, of HERG in LQT2: additional amino acids generated by frameshift cause accelerated inactivation.
Novel C-terminus frameshift mutation, 1122fs/147, of HERG in LQT2: additional amino acids generated by frameshift cause accelerated inactivation.
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DOI：
10.1016/j.yjmcc.2004.09.010
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发表时间：
2004-12
影响因子：
5
通讯作者：
T. Sasano;K. Ueda;Minako Orikabe;Y. Hirano;S. Kawano;M. Yasunami;M. Isobe;A. Kimura;M. Hiraoka
中科院分区：
文献类型：
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作者：
T. Sasano;K. Ueda;Minako Orikabe;Y. Hirano;S. Kawano;M. Yasunami;M. Isobe;A. Kimura;M. Hiraoka
人类ether-a-go-go相关基因（HERG）C端区域的功能除了参与运输外，还没有得到很好的表征。为了进一步了解C-末端区域的作用，我们进行了一个新的移码突变（1122 fs/147）的功能分析，确定在日本长QT综合征2（LQT 2）患者谁曾反复发作syncep.MethodsWild型（WT）和突变HERG质粒转染到人胚肾（HEK-293）细胞，全细胞电流记录的膜片钳技术。共聚焦显微镜检查使用绿色荧光蛋白标记的N-末端的HERG.ResultsThe突变体1122 fs/147单独可以表达电流的通道蛋白的膜分布，但减少了74%的控制密度。WT和1122 fs/147的共表达未观察到显性负效应。激活和失活时间常数无变化，失活时间比WT加快1122 fs/147，稳态失活曲线V1/2负移11 mV。与WT相比，1123 stop突变体的电流密度降低了49%，并且在稳态失活中没有显示出变化。共聚焦显微镜显示，在1122 fs/147和1123 stop突变体的细胞表面上的蛋白质表达减少相比，野生型。ConclusionFrameshift突变在C-末端区域与额外的147个氨基酸引起的HERG通道的功能丧失。由额外的147个氨基酸和运输缺陷诱导的稳态失活的负移有助于降低电流幅度1122 fs/147。
ObjectiveThe function of the C-terminus region of the human ether-a-go-go related gene (HERG) has not been well characterized except for its involvement in trafficking. To understand further the role of C-terminus region, we performed a functional analysis of a novel frameshift mutation (1122fs/147) identified in a Japanese long QT syndrome 2 (LQT2) patient who had recurrent episodes of syncope.MethodsWild type (WT) and mutant HERG plasmids were transfected into human embryonic kidney (HEK-293) cells, and whole-cell current was recorded by the patch-clamp technique. Confocal microscopy was performed to examine the membrane distribution of channel protein using a green fluorescent protein tagged to the N-terminus of HERG.ResultsThe mutant 1122fs/147 alone could express current, but reduced density by 74% of control. No dominant negative effect was noted with co-expression of WT and 1122fs/147. Activation and deactivation time constants were not changed, while inactivation was accelerated in 1122fs/147 compared to WT, and V1/2of steady-state inactivation curve shifted by 11 mV in the negative direction. Current density of 1123stop mutant revealed 49% reduction compared to WT and showed no shift in steady-state inactivation. Confocal microscopy revealed reduced protein expression on the cell surface both in 1122fs/147 and 1123stop mutants compared to WT.ConclusionFrameshift mutation at the C-terminus region with additional 147 amino acids evoked a loss of function of the HERG channel. A negative shift in steady-state inactivation induced by the additional 147 amino acids and trafficking defect contribute to a reduced current amplitude of 1122fs/147.