In response to membrane potential depolarization, voltage-dependent potassium channels undergo a series of conformational changes from a non-conducting state (closed) to an activated (conducting) 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. This understanding encompasses two interrelated processes, the protein rearrangements that lead to channel opening and the energy transduction events that convert external stimuli (voltage, ligand binding, etc) into protein motion. Specifically we will address the following key questions: What are the molecular entities determining channel activity? How energy (in the form of specific ligand binding or transmembrane electric field) is transduced into protein motion? How different parts of the channel interact to define open channel activity? We plan to study these problems by combining site-directed spin labeling/EPR spectroscopy and electrophysiological methods with classical biochemical and molecular biological procedures. This particular strategy has proven very successful over the previous application period, leading to direct structural determinations of KcsA, the Streptomyces K+ channel, the types of molecular movements underlying its gating mechanism and structural information on the role of the selectivity filter in gating. We intend to continue these structure-function studies while extending them using new experimental approaches like Double Quantum Resonance FT-EPR. In addition, we will focus our attention on a newly characterized six-transmembrane segment (6TM) channel from Methanococcus janschii (which we have named KchV-O). This channel contains a bona fide S4 segment and is ideally suited to study the structure and dynamics of the voltage-sensing domain and voltage-dependent gating mechanisms. 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.
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