In response to membrane potential depolarization, voltage-dependent channels undergo a series of conformational changes from a non-conducting state (closed) to an activated (conducting), finally stabilizing in a non-conducting inactivated state. K+ channel function has been associated with such basic cellular functions as the regulation of electrical activity, signal transduction and osmotic balance. In higher organisms, K+ channel dysfunction may lead to uncontrolled periods of electrical hyperexcitability, like epileptic episodes, myotonia and cardiac arrhythmia. Consequently, efforts to understand K+ channel structure function and dynamics relate directly to human health and disease. The continuing long-term goal of this project is to further understand the molecular mechanisms of gating in voltage-dependent channels, by focusing on the analysis of K+ channel gating in prokaryotic and eukaryotic systems. Specifically we will address the following key questions: What are the atomic structures of the key conformations that determine channel activity? This question will be answered for membrane embedded systems as well as those ordered in a lattice. What are the molecular bases of voltage-dependent gating? We will be testing the hypothesis that a specific sliding helix movement (the one click motion) can explain charge translocation in certain voltage sensors, but perhaps not others. The more charge a sensor translocates, the larger the number of clicks its sensor needs to move. And how different parts of the channel interact to define open channel activity? We plan to study these problems by combining spectroscopic techniques (EPR and NMR), X-ray crystallography electrophysiological and computational methods. We intend to continue these structure-function studies by investigating a wealth of biochemically-defined systems from KcsA and KvAP, to the Shaker voltage sensor and the hyperpolarization- activated channel from Methanococcus janschii (MVP). In addition, we will focus our attention on the voltage- sensing domain from the Ciona intestinalis-Voltage-Sensor-containing Phosphatase (Ci-VSP) aiming to improve the resolution of our recent crystals structures. Finally, we will examine the structure of the human voltage-dependent proton channel Hv1 in membranes though an extensive site-directed spin labeling analysis and computational modeling. This proposal should open new experimental avenues that will contribute to our understanding of biologically important events such as electrical signaling, signal transduction and ion channel gating.

R01GM057846

1998-08-01

2020-01-31