BK Channel B4 Subunit in the Dentate Gyrus and Seizures

描述(申请人提供)：钾通道具有降低兴奋性的作用，在控制癫痫发作中起着关键作用。大电导钙激活钾通道(BK型通道)具有调节膜兴奋性的特殊作用，这种作用与钙内流相一致。以前发现，形成孔道的亚基与新的辅助亚基的异源共表达可产生具有所谓神经元“II型BK通道”特性的BK电流。？4亚基使人对伊比利亚毒素阻断具有抵抗力，降低了门控动力学，降低了开放几率。此外，？4亚基可能使神经元对依赖PKA的磷酸化反应敏感。 我们已经培育出了基因敲除小鼠。我们的初步数据提供了直接证据，证明BK通道与第4亚基组装在II型BK通道的基础上。在齿状回(DG)，敲除细胞更容易兴奋，并支持高频放电。最后，脑电记录显示，4基因敲除的小鼠表现出非惊厥性部分癫痫发作。4基因敲除小鼠可能是第一个非惊厥性颞叶癫痫的遗传模型。对这些小鼠的研究将使我们有一个独特的机会来了解离子通道属性的变化，这些变化可能是这类癫痫的基础。 我们感兴趣的是了解a/？4亚单位BK通道如何调节DG细胞的输入/输出特性，从而有助于抵抗DG的同步性癫痫样活动。我们的工作假设是，β4亚基下调BK通道，导致钙内流增加，并通过SK通道的招募而降低兴奋性。我们有三个目标。1)利用DG细胞的膜片钳/切片记录，了解膜特性的变化导致基因敲除小鼠AP放电增加。2)确定4亚基如何调节DG内BK通道对PKA依赖的磷酸化的敏感性，从而调节代谢性谷氨酸受体的兴奋性。3)利用免疫组织化学和电生理记录技术，确定4亚基在神经末梢的亚细胞定位及其在苔藓纤维终末的神经传递中的作用。这些研究将提供对一个相对不具特征的BK通道亚型的第一个了解，并在癫痫的一个新的遗传模型的背景下进一步了解该通道的功能。

DESCRIPTION (provided by applicant): Potassium channels have the role of reducing excitability and are pivotal in controlling seizures. The large conductance calcium-activated potassium channels (BK-type channels) have the specialized role of regulating membrane excitability that is coincident with calcium influx. Previously, heterologous co- expression of the pore-forming a subunit with the novel ¿4 accessory subunit was found to produce BK currents with properties of so called neuronal "type II BK channels". The ¿4 subunit confers resistance to iberiotoxin block, slow gating kinetics, and a reduced open probability. In addition, the ¿4 subunit may confer sensitivity to pKA dependent phosphorylation in neurons. We have generated ¿4 gene knockout mice. Our preliminary data provides direct evidence that BK channel assembly with the ¿4 subunit underlies type II BK channels. In the dentate gyrus (DG), knockout cells are more excitable and support high frequency firing. Finally, EEG recording demonstrate that the ¿4 knockout mice exhibit non-convulsive partial seizures. The ¿4 knockout mice may be the first genetic model for non-convulsive temporal lobe epilepsy. Investigation of these mice will allow us the unique opportunity to have a biophysical understanding of the changes in ion channel properties that may underlie this class of epilepsy. Our interest is to understand how a/¿4 subunit BK channels regulate the input/output properties of DG cells, and thereby contribute to resistance of synchronized epileptiform activity that is a property of the DG. Our working hypothesis is that beta4 subunits down-regulates BK channels, resulting in increased calcium influx and reduced excitability by recruitment of SK channels. We have 3 aims. 1) Utilizing patch clamp/slice recordings from DG cells, understand the change in membrane properties that result in increased AP firing in the knockout mice. 2) Determine how the (¿4 subunit modulates sensitivity of BK channels to pKA dependent phosphorylation in the DG, and thereby regulates excitability by metabotropic glutamate receptors 3) Utilize immunohistochemistry and electrophysiological recording techniques to determine the subcellular localization of the ¿4 subunit in nerve terminals and its' contribution to neurotransmission in the mossy fiber terminals. These studies will provide the first understanding of a relatively uncharacterized BK channel subtype and further our understanding of this channels' function in the context of a novel genetic model for epilepsy.