Genetic dissection of the mechanisms and functions of post-transcriptional calcium channel modifications in Drosophila

电压门控钙通道(VGCC)控制着神经元功能的多个关键方面，包括突触前终末的突触小泡释放、树突突触后电位的放大以及神经元体细胞转录的活性依赖控制。哺乳动物VGCCα1亚基由3个家族10个基因编码，分别为Cav1、Cav2和Cav3。每个通道在生物物理特性、定位、药理特性和功能方面都不同。无论如何，VGCC的基本功能超过了可用的基因，但VGCC的功能多样性可以通过成孔α1亚基与辅助亚基的选择性剪接和共组装来显著增加。然而，由此产生的对VGCC生物物理性质和亚细胞定位的影响以及功能后果还不完全清楚。我们将在果蝇遗传模型系统中将电和光生理学工具与分子生物学相结合来解决这些问题。在果蝇中，每个脊椎动物VGCC家族由一个基因代表：Dmca1D与Cav1同源，Dmca1A或Cacophony与Cav2同源，DmαG与Cav3同源。这导致与附属亚基共同组装的组合不到10个，而哺乳动物的组合超过100个。在上一次资助期间，我们在果蝇运动神经元的不同亚区鉴定了特定的VGCC的发育和急性功能。接下来，我们开始解开果蝇HVA通道与辅助亚基相互作用的功能代码，证明了Cav2同源杂音与不同辅助亚基的配对影响了通道生物物理特性和亚神经元定位的不同方面。通过差异拼接进一步增加了通道功能多样性。事实上，果蝇Cav2通道产生的电流具有明显不同的激活电压和动力学，我们已经开发了工具来测试交替异构体选择的潜在作用。基于这些发现，我们现在将测试替代剪接(目标1)和配对与辅助亚基(目标2)如何调整生物物理性质和果蝇Cav2通道的定位。然后，我们将通过探测运动神经元的兴奋性和输入输出操作，以及行为过程中的放电模式，来测试在细胞和行为水平上产生的功能后果(目标3)。我们期待对产生功能性VGCC多样性的机制有新的见解，这与特定的神经元操作和健康的大脑功能相关。

Voltage gated calcium channels (VGCCs) control multiple pivotal aspects of neuronal function, including synaptic vesicle release from presynaptic terminals, the amplification of postsynaptic potentials in dendrites, and activity dependent control of transcription in neuronal somata. Mammalian VGCC α1 subunits are encoded by 10 genes that are classified in 3 families, Cav1, Cav2, and Cav3. Each channel differs with respect to biophysical properties, localization, pharmacological profile, and thus function. Regardless, the essential functions of VGCC outnumber the available genes, but VGCC functional diversity can be dramatically increased by alternative splicing and co-assembly of the pore forming alpha1 subunit with accessory subunits. However, the resulting effects on VGCC biophysical properties and Sub cellular localization as well as the functional consequences are incompletely understood. We will combine electro- and optophysiological tools with molecular biology in the Drosophila genetic model system to address these questions. In Drosophila, each vertebrate VGCC family is represented by one gene: Dmca1D is homologous to Cav1, Dmca1A or cacophony to Cav2, andDmαG to Cav3. This results in less than 10 combinations for coassembly with accessory subunits, as opposed to more than 100 in mammals. During the last funding period, we have identifieddevelopmental and acute functions of specific VGCCs in different subneuronalcompartments of Drosophila motoneurons. We next started unraveling the functional code of Drosophila HVA channel interactions with accessory subunits by demonstrating that pairing of the Cav2 homolog cacophony with different accessory subunits affects distinctaspects of channel biophysical properties and sub-neuronal localization. Channel functional diversity is further increased by differential splicing. In fact Drosophila Cav2 channels produce currents with markedly different activation voltages and kinetics, andwe have developed the tools to test the underlying roles of alternativeisoform selection. Based on these findings we will now test how alternative splicing (aim 1) and pairing with accessory subunits (aim 2) tune the biophysical properties and the localization of DrosophilaCav2 channels. We will then test the resulting functional consequences on the cellular and behavioral levels by probing motoneuron excitability and input-output operations as well as firing patterns during behavior (aim 3). We expect novel insights into themechanisms that generate functional VGCC diversity that is relevant for specific neuronal operations and healthy brain function.