Voltage-activated potassium channels are membrane proteins that critically regulate electrical signaling in neurons and other excitable cells. Potassium channels are particularly important for determining the tendency of excitable cells to produce repetitive signaling. Normal repetitive signaling produces stimulus-frequency encoding, brain wave rhythms, and cardiac pacemaking; dysfunctions of rhythmic signaling lead to epilepsy, cardiac arrhythmia, and myotonia. The opening and closing of these potassium channels, which determines their effect on cellular electrical signaling, is accomplished by several separable but interrelated gating mechanisms. The outward rectifier K+ channels in this study have an activation gating process that opens the channel upon depolarization, and several distinguishable inactivation gating mechanisms. In the previous grant period a general strategy was developed and used to learn about the functional motions of the channel protein that correspond to gating. Genetically-altered channels with single cysteine residues substituted at specific locations were prepared, and these introduced cysteines served as targets for sulfydryl-specific modifying reagents and for metal ion binding. This work led to a strong hypothesis for channel gating, which is beautifully compatible with the recently reported structure of a bacterial K+ channel. This work will be extended to answer specific new questions about gating motions. Does the intracellular mouth of the channel open widely during channel gating? What is the relationship between the concerted opening of the pore and the independent movements of each subunit? How do the gating motions and the introduction of charged groups affect the binding of channel blockers and other drugs that act on the channel? A new class of modulator compounds capable of potentiating or inhibiting K+ channel activity has been discovered. They will be investigated to determine the mechanism and site of action. These are potentially a new category of therapeutic drugs. This work should provide fundamental new insights into channel gating that will help explain the physiological basis of repetitive signaling and its dysfunction. These insights will also increase the chances for rational development of drugs to control such disorders.
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