The long-term goal of this project is to understand the mechanisms underlying gating of voltage-dependent channels. Voltage-dependent channels are the basis of electrical activity in neurons and other excitable cells, and many diseases involve abnormalities in ion channel behavior (e. g., epilepsy). Specifically, we will focus on two channels: T-type calcium channels, and Kv-type delayed rectifier potassium channels. We will use di- and trivalent cation blockers of T-channels as probes for the channel pore. We will investigate the voltage- and state-dependence of channel block. We will ask whether blocking ions can enter and/or exit the pore when the channel is closed, or when it is inactivated. In essence, this asks whether the gates of the channel are on the intracellular side of the selectivity filter, on the extracellular side, or both. Regarding Kv channels, we will begin to analyze the structural basis of a recently characterized form of slow inactivation, called U-type inactivation, that occurs primarily from """"""""partially activated"""""""" closed states along the pathway for channel activation. Using the Shaker K channel (with the N-terminal region deleted to remove fast inactivation), we will examine whether mutations affect C-type inactivation, U-type inactivation, or both. We will examine mutations at two positions: T449, a site near the outer mouth of the pore that strongly affects both slow inactivation and TEA block; and P475, where (in the Kv2.1 channel) mutations greatly enhance U-type inactivation. We will also examine the effects of mutations at the corresponding sites in the Kv2.1 channel, where we have found the wild-type channel to exhibit purely U-type inactivation. These studies will primarily use whole-cell voltage-clamp recording from cloned channels expressed in a mammalian cell line, HEK 293 cells. An important feature of our analysis is the use of kinetic models of channel gating both as empirical descriptions of our results, and as working hypotheses regarding the molecular mechanisms of channel gating. We expect our results to provide important information regarding the location of the gates that regulate ion flow, for different gating processes: activation, fast inactivation (for T-channels), and two forms of slow inactivation (Kv channels).
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