Elimination of the fast transient in superior cervical ganglion neurons with expression of KV4.2W362F:: Molecular dissection of IA
Elimination of the fast transient in superior cervical ganglion neurons with expression of KV4.2W362F:: Molecular dissection of IA
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DOI：
10.1523/jneurosci.20-14-05191.2000
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发表时间：
2000-07-15
影响因子：
5.3
通讯作者：
Nerbonne, JM
中科院分区：
文献类型：
--
作者：
Malin, SA;Nerbonne, JM
电生理学和分子研究表明，在哺乳动物神经元中，电压门控K+电流和这些通道下的亚单位具有相当大的异质性。然而，目前对天然K+电流和克隆亚基之间的关系知之甚少。在本实验中，我们利用分子遗传学的方法来确定交感神经元快速瞬时外向钾电流I-Af的分子相关性，并探讨I-Af在形成动作电位波形和控制重复放电模式中的功能作用。利用基因枪法，将编码显性负性突变体Kv4.2α亚基(Kv4.2W362F)和增强型绿色荧光蛋白(EGFP)的cDNA导入体外培养的大鼠交感神经元。从EGFP阳性细胞获得的全细胞电压钳记录显示，在表达Kv4.2W362F的细胞中，I-Af被选择性地消除，这表明在交感神经元中，Kv4α亚基是I-Af的基础。此外，在过度表达野生型Kv4.2的细胞中，I-Af密度显著增加。在表达Kv4.2W362F的细胞中，输入电阻增加，动作电位产生的(电流)阈值降低，表明I-Af在兴奋性调节中起关键作用。Kv4.2W362F的表达和I-Af的消除也改变了在长时间注入去极化电流时观察到的重复放电模式的分布。野生型颈上神经节由时相神经元、适应神经元和紧张性放电神经元组成。I-Af的消除通过将时相细胞转移到适应的放电模式来增加适应细胞的百分比，而增加I-Af的密度减少了适应细胞的数量。
Electrophysiological and molecular studies have revealed considerable heterogeneity in voltage-gated K+ currents and in the subunits that underlie these channels in mammalian neurons. At present, however, the relationship between native K+ currents and cloned subunits is poorly understood. In the experiments here, a molecular genetic approach was exploited to define the molecular correlate of the fast transient outward K+ current, I-Af, in sympathetic neurons and to explore the functional role of I-Af in shaping action potential waveforms and controlling repetitive firing patterns. Using the biolistic gene gun, cDNAs encoding a dominant negative mutant Kv4.2 alpha-subunit (Kv4.2W362F) and enhanced green fluorescent protein (EGFP) were introduced into rat sympathetic neurons in vitro. Whole-cell voltage-clamp recordings obtained from EGFP-positive cells revealed that I-Af is selectively eliminated in cells expressing Kv4.2W362F, demonstrating that Kv4 alpha-subunits underlie I-Af in sympathetic neurons. In addition, I-Af density is increased significantly in cells overexpressing wild-type Kv4.2. In cells expressing Kv4.2W362F, input resistances are increased and (current) thresholds for action potential generation are decreased, demonstrating that I-Af plays a pivotal role in regulating excitability. Expression of Kv4.2W362F and elimination of I-Af also alters the distribution of repetitive firing patterns observed in response to a prolonged injection of depolarizing current. The wild-type superior cervical ganglion is composed of phasic, adapting, and tonic firing neurons. Elimination of I-Af increases the percentage of adapting cells by shifting phasic cells to the adapting firing pattern, and increased I-Af density reduces the number of adapting cells.