William Harvey Heart Centre, Barts & The London School of Medicine & Dentistry, Charterhouse Square, London EC1M 6BQ, UK.
Biochem J. 2012 May 1;443(3):635-42. doi: 10.1042/BJ20111912.
The nonsense mutations R518X-KCNQ1 and Q530X-KCNQ1 cause LQT1 (long-QT syndrome type 1) and result in a complete loss of I(Ks) channel function. In the present study we attempted to rescue the function of these mutants, in HEK (human embryonic kidney)-293 cells, by promoting readthrough of their PTCs (premature termination codons) using the pharmacological agents G-418, gentamicin and PTC124. Gentamicin and G-418 acted to promote full-length channel protein expression from R518X at 100 μM and from Q530X at 1 mM. In contrast, PTC124 did not, at any dose tested, induce readthrough of either mutant. G-418 (1 mM) treatment also acted to significantly (P<0.05) increase current density and peak-tail current density, at +80 mV for R518X, but not Q530X, to 58±11% and 82±17% of the wild-type level respectively. However, the biophysical properties of the currents produced from R518X, while similar, were not identical with wild-type as the voltage-dependence of activation was significantly (P<0.05) shifted by +25 mV. Overall, these findings indicate that although functional rescue of LQT1 nonsense mutations is possible, it is dependent on the degree of readthrough achieved and the effect on channel function of the amino acid substituted for the PTC. Such considerations will determine the success of future therapies.
无义突变 R518X-KCNQ1 和 Q530X-KCNQ1 导致 LQT1（长 QT 综合征 1 型），并导致 I(Ks) 通道功能完全丧失。在本研究中，我们试图通过使用药物 G-418、庆大霉素和 PTC124 促进其 PTC（过早终止密码子）的通读，来挽救这些突变体在 HEK（人胚肾）-293 细胞中的功能。庆大霉素和 G-418 在 100μM 时可促进全长通道蛋白从 R518X 表达，在 1mM 时可促进全长通道蛋白从 Q530X 表达。相比之下，在测试的任何剂量下，PTC124 均不能诱导任一突变体的通读。G-418（1mM）处理还显著（P<0.05）增加了 R518X 在 +80 mV 时的电流密度和峰尾电流密度，分别达到野生型水平的 58±11%和 82±17%，但 Q530X 则不然。然而，来自 R518X 的电流的生物物理特性虽然相似，但与野生型并不完全相同，因为激活的电压依赖性显著（P<0.05）偏移了+25 mV。总体而言，这些发现表明，尽管可以实现 LQT1 无义突变的功能挽救，但这取决于实现的通读程度以及对 PTC 取代的氨基酸对通道功能的影响。这些考虑因素将决定未来治疗的成功。
