Investigation of Mechanisms of Volatile Anesthetic Action by the Use of Gene Expression Techniques

在这项研究的第一年，我们研究了挥发性麻醉剂异氟醚和七氟烷对KvLQT1和水貂共表达的克隆I_lt；Ks和gt；的影响。将体外转录自正常大鼠心脏cDNA的KvLQT1mRNA注入卵母细胞，加入或不加入水貂mRNAs，可产生电流。采用双电极电压钳记录技术，观察了异氟醚(0-1.5MAC)和七氟烷(0-1.5MAC)对KvLQT1和KvLQT1电流的影响。在2-S去极化至+40 mV后，异氟醚和七氟醚使I_1、Ks和Gt；和KvLQT_1电流呈剂量依赖性地降低。被试的两种挥发性麻醉剂均可加速I_lt；Ks&Gt；和KvLQT1电流的失活。我们的结论是，挥发性麻醉药对克隆的I_t；Ks&gt；的显著抑制作用可能是临床上观察到麻醉药延长心室复极(Q-T间期)的部分原因。在接下来的一年里，我们从大鼠心脏克隆了另一个截短的LCNQ1剪接变体。从TCNQT1的缺失序列来看，大鼠这一部分的基因组结构可能与人和小鼠的不同。在过去的一年里，我们研究了由β；2a&gt；或β_&lt；2c&gt；亚基重组的Ca^&lt；2+&gt；通道的单通道特性，并与单纯通道的特性进行了比较。与β_2a&>亚基相反，长时间关闭在钙通道中占主导地位，β^；2c&>亚基和天然通道。含β_2c&gt；亚基的钙通道的单通道特性与天然通道无明显差异。这些发现表明，β_&lt；2c&gt；亚基是大鼠心脏中具有功能的β亚基之一。

In the first year of this investigation, we investigated the effects of the volatile anesthetics isoflurane and sevoflurane on cloned I_<Ks> coexpressed by KvLQT1 and minK. Currents were induced following injection into oocytes of KvLQT1 mRNA with or without minK mRNA, which were transcribed in vitro from cDNAs of normal rats hearts. A Two-electrode voltage-clamp recording technique was used to investigate the effects of isoflurane (0-1.5 MAC) and sevoflurane (0-1.5 MAC) on I_<Ks> (KvLQT1 with mink) and KvLQT1 alone currents. Following a 2-s depolarization to +40 mV, isoflurane and sevoflurane caused potency-dependent reductions in I_<Ks> and KvLQT1 currents. Both of the volatile anesthetics tested accelerated the deactivation of I_<Ks> and KvLQT1 currents. We conclude that the significant inhibitory effect of volatile anesthetics on the cloned I_<Ks > may partly contribute to the clinical observations of the prolongation of the ventricular repolarization (Q-T interval) by the anesthetics. In the following year, we cloned another truncated splice variant of LCNQ1 from rat heart. Judging from the deleted sequence of the TCNQT1, the genomic structure of rat in this portion might be different from those of human and mouse. In the last year, we investigated the single-channel properties of the Ca^<2+> channels reconstituted with β_<2a> or β_<2c> subunit, and compared them with the properties of naive channel. In contrast to β_<2a> subunit, long-lasting closings were dominant in the Ca^<2+> channel with β^<2c> subunit and the native channel. The single-channel properties of Ca^<2+> channel with β_<2c> subunit were indistinguishable from those of native channel. Those findings suggest that β_<2c> subunit is one of the functional β subunits in the rat heart.