Molecular mechanisms of voltage-gated ion channels

描述(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/收缩耦合。电压门控通道功能异常与许多神经系统疾病有关，如癫痫、发作性共济失调和周期性瘫痪。对这些通道的正常和病理功能的了解可能会导致一些神经疾病的治疗方法的发展。电压激活的离子通道是通过细胞膜上电压的变化来激活的。当膜去极化时，经典的电压激活钾通道开放。这已经被证明是由于固有电压传感器S4的向外移动，它触发了打开通道的构象变化。然而，S4的运动如何耦合到通道的打开并不被理解。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开放；即，当膜超极化时，HCN通道开放。我们假设S4也是HCN通道中的电压传感器，但S4运动和通道开放之间的耦合涉及到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。S4的移动将使用半胱氨酸特异的荧光探针或不透膜的半胱氨酸试剂在半胱氨酸取代的通道中进行测量。这些测量将被用来确定S4如何在不同的通道中运动，并将进一步加深我们对这两类通道开放的不同机制的理解。

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.