Falk T, Kilani R K, Strazdas L A, Borders R S, Steidl J V, Yool A J, Sherman S J
Department of Physiology, The University of Arizona, 1501 North Campbell Avenue, Tucson, AZ 85724, USA.
Neuroscience. 2003;120(2):387-404. doi: 10.1016/s0306-4522(03)00044-7.
The rapidly inactivating A-type K+ current (IA) is prominent in hippocampal neurons; and the speed of its inactivation may regulate electrical excitability. The auxiliary K+ channel subunit Kvbeta 1.1 confers fast inactivation to Shaker-related channels and is postulated to affect IA. Whole-cell patch clamp recordings of rat hippocampal pyramidal neurons in primary culture showed a developmental decrease in the time constant of inactivation (tau(in)) of voltage-gated K+ currents: 17.9+/-1.5 ms in young neurons (5-7 days in vitro; n=53, mean+/-S.E.M.); 9.9+/-1.0 ms in mature neurons (12-15 days in vitro; n=72, mean+/-S.E.M., P<0.01). During the same developmental time, the level of Kvbeta 1.1 transcript increased more than two-fold in vitro and in vivo, determined by semi-quantitative reverse transcriptase-polymerase chain reaction for hippocampus. The hypothesis that up-regulation of Kvbeta 1.1 led to the changes in tau(in) was tested in vitro, using antisense knockdown. Kvbeta 1.1-specific antisense DNA was introduced with a modified herpes virus co-expressing enhanced green fluorescent protein and knockdown of Kvbeta 1.1 was verified by immunocytochemistry. Following transduction with the antisense virus, mature neurons reverted to tau(in) values characteristic of young neurons: 18.3+/-2.4 ms (n=20). The effect of antisense knockdown on electrical excitability was tested using current-clamp protocols to induce repetitive firing. Treatment with the antisense virus increased the interspike interval over a range of membrane depolarization (baseline membrane potential=-40 to +20 mV). This effect was most pronounced at -40 mV, where the ISI of the first pair of action potentials was nearly doubled. These data indicate that Kvbeta 1.1 contributes to the developmental control of IA in hippocampal neurons and that the magnitude of effect is sufficient to regulate electrical excitability. Viral-mediated antisense knockdown of Kvbeta 1.1 is capable of decreasing the electrical excitability of post-mitotic hippocampal neurons, suggesting this approach has applicability to gene therapy of neurological diseases associated with hyperexcitability.
快速失活的A 型钾电流（IA）在海马神经元中很突出；其失活速度可能调节电兴奋性。辅助性钾通道亚基Kvbeta 1.1赋予与Shaker相关通道快速失活特性，并推测其会影响IA。对原代培养的大鼠海马锥体神经元进行全细胞膜片钳记录显示，电压门控钾电流的失活时间常数（tau(in)）在发育过程中降低：幼龄神经元（体外培养5 - 7天；n = 53，平均值±标准误）为17.9±1.5毫秒；成熟神经元（体外培养12 - 15天；n = 72，平均值±标准误，P < 0.01）为9.9±1.0毫秒。在相同的发育时期，通过对海马进行半定量逆转录聚合酶链反应测定，Kvbeta 1.1转录本水平在体外和体内均增加了两倍多。使用反义敲低技术在体外对Kvbeta 1.1上调导致tau(in)变化这一假说进行了验证。通过一种共表达增强型绿色荧光蛋白的改良疱疹病毒导入Kvbeta 1.1特异性反义DNA，并通过免疫细胞化学验证Kvbeta 1.1的敲低。用反义病毒转导后，成熟神经元恢复到幼龄神经元特有的tau(in)值：18.3±2.4毫秒（n = 20）。使用电流钳记录方案诱导重复放电来测试反义敲低对电兴奋性的影响。用反义病毒处理在一系列膜去极化（基线膜电位 = -40至 +20毫伏）范围内增加了峰间间隔。这种效应在 -40毫伏时最为明显，此时第一对动作电位的峰间间隔几乎翻倍。这些数据表明，Kvbeta 1.1参与了海马神经元IA的发育调控，且其作用强度足以调节电兴奋性。病毒介导的Kvbeta 1.1反义敲低能够降低有丝分裂后海马神经元的电兴奋性，表明这种方法适用于与兴奋性过高相关的神经疾病的基因治疗。
