Li Li, Rojas Asheebo, Wu Jianping, Jiang Chun
Department of Biology, Georgia State University, 24 Peachtree Center Avenue, Atlanta, Georgia 30302-4010, USA.
Endocrinology. 2004 Sep;145(9):4408-14. doi: 10.1210/en.2004-0285. Epub 2004 May 27.
The ATP-sensitive K+ (KATP) channel, composed of Kir6.2 and sulfonylurea receptor (SUR1), in pancreatic beta-cells is believed to serve as a metabolic sensor regulating insulin secretion according to glucose levels. Thus, genetic disruption of Kir6.2 expression may impair KATP channel function in glucose sensing and insulin secretion. Here we show evidence obtained from functional genetic assays supporting this hypothesis. To avoid adaptive cellular mechanisms in transgenic preparations, we designed a hammerhead ribozyme that specifically targeted the Kir6.2 mRNA at serine 78. The Kir6.2-ribozyme was constructed in an adenoviral vector and expressed in insulin-secreting RINm5F cells. Both RT-PCR and Northern blot analyses showed that Kir6.2 transcripts were significantly reduced with a Kir6.2-ribozyme treatment. Whole-cell patch-clamp studies indicated that the Kir6.2-ribozyme treatment lowered KATP channel density by 66%. In response to higher glucose challenge, insulin release from the RINm5F cells dropped by approximately 20% in a transfection dose of 0.7 multiplicity of infection, and by 30-40% in a dose of 2.7 multiplicity of infection. These results therefore indicate that KATP channels play an important role in glucose sensing and insulin secretion, and ribozyme Kir6.2-gene targeting is an effective approach for selective inhibition of functional expression of KATP channels.
胰腺β细胞中由Kir6.2和磺脲类受体（SUR1）组成的ATP敏感性钾通道（KATP通道）被认为是一种代谢传感器，可根据葡萄糖水平调节胰岛素分泌。因此，Kir6.2表达的基因破坏可能会损害KATP通道在葡萄糖感应和胰岛素分泌中的功能。在此，我们展示了从功能基因检测中获得的证据来支持这一假设。为了避免转基因制剂中的适应性细胞机制，我们设计了一种锤头状核酶，它特异性地靶向丝氨酸78处的Kir6.2 mRNA。Kir6.2核酶构建在腺病毒载体中，并在胰岛素分泌性RINm5F细胞中表达。RT-PCR和Northern印迹分析均表明，Kir6.2核酶处理后Kir6.2转录本显著减少。全细胞膜片钳研究表明，Kir6.2核酶处理使KATP通道密度降低了66%。在更高葡萄糖刺激下，RINm5F细胞的胰岛素释放量在感染复数为0.7的转染剂量下下降了约20%，在感染复数为2.7的剂量下下降了30%-40%。因此，这些结果表明KATP通道在葡萄糖感应和胰岛素分泌中起重要作用，核酶靶向Kir6.2基因是选择性抑制KATP通道功能表达的有效方法。
