Kv Channel Interacting Proteins (KChIPs) as calcium sensors for Kv4 channels

钾通道控制着不同细胞类型的静息膜电位和膜兴奋，这反映在大量的钾通道类型和亚家族中。电压门控钾通道（Kv通道），特别是Kv 4亚家族的钾通道，在心肌细胞复极化和神经元树突树中兴奋的受控传播中起关键作用。心肌细胞膜和神经元树突中的Kv 4通道与Kv通道相互作用蛋白（KChIP）形成复合物。KChIP属于钙结合EF-手蛋白的神经元钙传感器（NCS）家族。在心肌细胞和神经元中，细胞质钙的局部波动对正常细胞功能和可塑性至关重要。然而，细胞质钙升高超过正常范围可能导致病理生理过程，并最终导致细胞死亡。从KChIP被发现为Kv 4通道的特异性β-亚基的时候起，Kv 4通道的钙传感器的作用就归因于KChIP，这是基于它们属于NCS蛋白家族的事实。在文献中确实有关于这种作用的暗示。然而，尚未对其进行详细研究，并且仍未证实KChIP的钙依赖性构象变化是否影响Kv 4/KChIP复合物的形成，或者在现有的Kv 4/KChIP复合物中，KChIP的钙依赖性构象变化是否直接传递到Kv 4通道。因此，本研究的目的是从分子水平阐明KChIPs在生理和病理生理钙波动过程中作为Kv 4通道钙传感器的作用。用表位标记的Kv 4 α-和KChIP β-亚基进行荧光测量，以及在不同细胞质钙浓度存在下对Kv 4/KChIP介导的电流进行电生理学测量，结合使用具有突变钙结合位点的KChIP，应该提供关于这个问题的有价值的信息。

Potassium channels control the resting membrane potential and membrane excitation in very different cell types, as reflected by the huge number of potassium channel types and subfamilies. Voltage-gated potassium channels (Kv channels), particularly those of the Kv4 subfamily, are critically involved in cardiomyocyte repolarisation and the controlled spread of excitation in the dendritic trees of neurons. The Kv4 channels in the membrane of cardiomyocytes and neuronal dendrites form complexes with Kv Channel Interacting Proteins (KChIPs). KChIPs belong to the Neuronal Calcium Sensor (NCS) family of calcium-binding EF-hand proteins. Both in cardiomyocytes and in neurons local fluctuations in cytoplasmic calcium are central to normal cell function and plasticity. However, a rise in cytoplasmic calcium above the normal range may lead to pathophysiological processes and eventually to cell death. From the time when the KChIPs were discovered as being specific β-subunits of Kv4 channels, the role of a calcium sensor for Kv4 channels has been attributed to the KChIPs, based on the fact that they belong to the NCS protein family. Hints on such a role do exist in the literature. However, it has not been studied in detail and it remains unproven whether calcium-dependent conformational changes of the KChIPs influence Kv4/KChIP complex formation or whether in an existing Kv4/KChIP complex calcium-dependent conformational changes of the KChIPs are directly transmitted to the Kv4 channel. Therefore, the aim of the present research proposal is the molecular elucidation of the role of KChIPs as calcium sensors for Kv4 channels during physiologic and pathophysiologic calcium fluctuations. Fluorescence measurements with epitope-tagged Kv4 α- und KChIP β-subunits as well as electrophysiological measurements of Kv4/KChIP-mediated currents in the presence of different cytoplasmic calcium concentrations, combined with the use of KChIPs with mutated calcium binding sites, should provide valuable information regarding this issue.