Molecular mechanisms of voltage-gated ion channels

描述（由申请人提供）：电压门控离子通道参与神经冲动传播、突触传递、肌肉动作电位和兴奋/收缩偶联。电压门控通道的功能异常与许多神经系统疾病有关，例如癫痫、发作性共济失调和周期性麻痹。了解这些通道的正常和病理功能可能会导致许多神经系统疾病的治疗方法的发展。电压激活的离子通道通过细胞膜上电压的变化而激活。当细胞膜去极化时，经典的电压激活钾通道打开。这已被证明是由于一个内在的电压传感器，S4，它触发了构象变化，打开通道的向外移动。然而，S4的运动如何与通道的开口耦合尚不清楚。最近，一类新的电压激活离子通道被克隆：超极化激活的环核苷酸门控离子通道（HCN通道）。这些通道还包含一个假定的电压传感器S4。令人惊讶的是，HCN通道以与电压激活的钾通道相反的极性打开;也就是说，HCN通道在膜超极化时打开。我们假设S4也是HCN通道中的电压传感器，但是S4运动和通道开放之间的耦合涉及与电压激活的钾通道不同的机制。该项目的目的是确定S4是否是HCN通道中的电压传感器，并将这些通道中的S4运动与电压激活钾通道中的S4运动进行比较和对比。将使用半胱氨酸特异性荧光探针或膜不可渗透半胱氨酸试剂在半胱氨酸取代通道中测量S4的移动。这些测量将用于确定S4如何在不同的通道中移动，并将进一步加深我们对这两类通道的不同开放机制的理解。

DESCRIPTION (provided by applicant): Voltage-gated ion channels are involved in nerve impulse propagation, synaptic transmission, muscle action potentials, and excitation/contraction coupling. Abnormal function of voltage-gated channels has been implicated in many neurological diseases, for example, epilepsy, episodic ataxia, and periodic paralyses. An understanding of the normal and pathological function of these channels could lead to the development of treatments for a number of neurological diseases. Voltage-activated ion channels are activated by changes in the voltage across the cell membrane. The classic voltage-activated potassium channels open when the membrane is depolarized. This has been shown to be due to the outward movement of an intrinsic voltage sensor, S4, which triggers the conformational changes that open the channel. However, how the movement of S4 is coupled to the opening of the channel is not understood. Recently, a new class of voltage-activated ion channels was cloned: hyperpolarization-activated cyclic nucleotide-gated ion channels (HCN channels). These channels also contain a putative voltage sensor, S4. Surprisingly, the HCN channels open with the opposite polarity from the voltage-activated potassium channels; that is, the HCN channels open when the membrane is hyperpolarized. We hypothesize that S4 is also the voltage sensor in the HCN channels, but that the coupling between S4 movement and the opening of the channel involves a different mechanism than in voltage-activated potassium channels. The aim of the proposed project is to determine whether S4 is the voltage sensor in the HCN channels and to compare and contrast the S4 movement in these channels with the S4 movement in voltage-activated potassium channels. The movement of S4 will be measured in cysteine-substituted channels using cysteine-specific fluorescent probes or membrane-impermeable cysteine reagents. These measurements will be used to determine how S4 moves in the different channels and will further our understanding of the different mechanisms of opening of these two classes of channels.