Large conductance Ca-activated K-channels [BK(Ca) or Slo K(Ca) channels] are activated by membrane depolarization and intracellular Ca. BK(Ca) channels participate in the regulation of neurotransmitter release from synaptic terminals. In neurons and other tissues, BK(Ca) channels modulate cellular responses to a rise in intracellular Ca. A molecular understanding of how this process occurs may be used to guide rational drug development. BK(Ca) channels are recognized as a drug target that could be exploited in therapy of cerebral ischemia, stroke, brain trauma, neurodegeneration, epilepsy, hypertension, and other conditions. BK(Ca) channels differ from voltage-activated K channels by the presence of a unique intracellular C-terminal domain of about 800 residues that has been implicated in Ca-sensing, channel activation, and diverse interactions with regulatory proteins. The long-term goal of this research is to understand how this essential part of the BK(Ca) channel functions in cellular physiology.
The specific aims are to: 1. Investigate the structure and function of the Slo K(Ca) channel protein and an intracellular domain that binds Ca and Kunitz protease inhibitors. Test the hypothesis that an intracellular C-terminal domain of the Slo K(Ca) channel is homologous or structurally related to the trypsin family of serine proteases (SerP) by electrophysiological assay of mutations and chimeras. Test the hypothesis that the inhibitor binding site is involved in beta subunit-mediated inactivation. Express the SerP-like domain of the Slo K(Ca) channel as a soluble protein suitable for functional and crystallographic analysis. 2. Analyze Ca binding to the C-terminal region of Slo K(Ca) channels using qualitative assays such as 45Ca autoradiography followed by quantitative Ca-binding titrations of isolated protein domains. Test the hypothesis that a conserved Asp-rich sequence motif called the Ca-bowl located near the C-terminus of the Slo K(Ca) channel directly mediates Ca-binding responsible for channel activation. 3. Analyze the function of intracellular domains of Slo K(Ca) and Slack K channel proteins. Based on the hypothesis that activation involves the interaction of Core and Tail domains, define the minimum functional unit of the BK(Ca) Tail required for channel activation. Develop a reconstitution assay to study the interaction between soluble Tail domain and the membrane-bound Core domain. Test whether the Slack K channel involves a Core/Tail domain interaction. Determine the molecular basis of insensitivity to charybdotoxin of certain BK(Ca) channels in mouse neurons. Determine whether oxygen sensitivity of Slo K(Ca) channels in central neurons is regulated by beta-subunits, Slo Core or Slo Tail domains.
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