Molecular Properties of Voltage-Sensitive Calcium Channels

描述（由申请人提供）：学习和记忆的丧失是衰老和神经退行性疾病中最使人衰弱的方面之一，然而我们不了解这些关键大脑过程的基本机制，我们不能有效地干预这些缺陷。学习和记忆主要发生在突触上。突触前钙（Cav2.1）通道启动大脑中大多数突触的神经递质释放。这些通道的活性受到一个大的信号蛋白复合物的严格调节，包括钙调蛋白和相关的钙传感器蛋白。Katz和Miledi在20世纪60年代的经典著作首次描述了短期突触促进和抑郁。这些形式的短期突触可塑性形成了突触后对冲击突触前末端的动作电位序列的反应，从而编码动作电位频率和模式中包含的信息，并将其传递给突触后细胞。突触的突触前侧短期突触可塑性的机制仍然知之甚少。我们最近的研究表明Ca通道调节是短期突触可塑性的一个重要组成部分。在细胞培养中对单个颈上神经节神经元转染Cav2.1通道的研究表明，钙/钙调蛋白和其他钙传感器蛋白阻止Cav2.1通道活性的促进和失活的突变，可以阻断短期突触促进和突触抑制的快速阶段。基于这些结果，我们假设钙调蛋白和钙传感器蛋白对Cav2.1通道的调节是海马突触短期突触可塑性的重要因素，这种形式的突触可塑性对空间学习和记忆很重要。我们将在分子水平上解决这一假设，基于Rosetta结构建模、化学交联和高分辨率质谱，开发Cav2.1通道和CaS蛋白相互作用域的高分辨率分子模型。我们将使用最近开发的敲入小鼠品系在功能和行为水平上解决这一假设，其中cam依赖性Cav2.1通道促进所需的iq样基序已发生突变，以防止通道活性的促进（Cav2.1/IM-AA小鼠）。我们将在CA3神经元突触前可塑性缺失的野生型和IM-AA突变小鼠海马切片中，确定Cav2.1通道在神经回路短期突触可塑性中的调节作用。我们将探讨Cav2.1通道和短期突触可塑性在环境依赖恐惧条件反射缺失的野生型和IM-AA突变小鼠空间学习和记忆中的作用。我们利用这种独特的小鼠模型进行的实验将为海马神经元短期突触前可塑性的机制及其在空间学习和记忆中的作用提供新的见解。这一信息将对理解空间学习和记忆在衰老和疾病中的失败至关重要。

DESCRIPTION (provided by applicant): Failure of learning and memory is one of the most debilitating aspects of aging and neurodegenerative disease, yet we do not understand the basic mechanisms of these crucial brain processes and we cannot intervene effectively in these deficits. Learning and memory takes place primarily at synapses. Presynaptic calcium (Cav2.1) channels initiate neurotransmitter release at most synapses in the brain. The activity of these channels is tightly regulated by a large complex of signaling proteins, including calmodulin and related calcium sensor proteins. The classic work of Katz and Miledi in the 1960's first described short-term synaptic facilitation and depression. These forms of short-term synaptic plasticity shape the postsynaptic response to trains of action potentials impinging on the presynaptic terminal and thereby encode information contained in the frequency and pattern of action potentials for transmission to the postsynaptic cell. The mechanisms that underlie short-term synaptic plasticity on the presynaptic side of the synapse remain poorly understood. Our recent work has implicated Ca channel regulation as an important component of short-term synaptic plasticity. Studies of Cav2.1 channels transfected in individual superior cervical ganglion neurons in cell culture showed that both short-term synaptic facilitation and the rapid phase of synaptic depression are blocked by mutations that prevent facilitation and inactivation of Cav2.1 channel activity by calcium/calmodulin and other calcium sensor proteins. Based on these results, we hypothesize that regulation of Cav2.1 channels by calmodulin and calcium sensor proteins is an important contributor to short-term synaptic plasticity at synapses in the hippocampus and that this form of synaptic plasticity is important for spatial learning and memory. We will address this hypothesis at the molecular level by developing a high-resolution molecular model for the interacting domains of Cav2.1 channels and CaS proteins based on Rosetta structural modeling, chemical crosslinking, and high-resolution mass spectrometry. We will address this hypothesis at the functional and behavioral levels using a recently developed knock-in mouse line in which the IQ-like motif that is required for CaM-dependent facilitation of Cav2.1 channels has been mutated to prevent facilitation of channel activity (Cav2.1/IM-AA mice). We will determine the role of regulation of Cav2.1 channels in short-term synaptic plasticity of neural circuits in hippocampal slices from wild-type and IM-AA mutant mice, which are deficient in presynaptic plasticity in the nerve terminals of CA3 neurons. We will explore the role of regulation of Cav2.1 channels and short-term synaptic plasticity in spatial learning and memory in wild-type and IM-AA mutant mice, which are deficient in context-dependent fear conditioning. Our experiments with this unique mouse model will give new insights into the mechanism of short-term presynaptic plasticity in hippocampal neurons and its role in spatial learning and memory. This information will be essential to understanding of failure of spatial learning and memory in aging and disease.