STRUCTURAL BASIS OF ION CHANNEL OPENING

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Ion channels play a crucial role in the physiology of the nervous system. By forming ion specific pores that open and close stochastically, they control membrane potential and ion concentrations both inside and outside the cell. They play an important role in excitability, neuromodulation, and neurotransmission. The long term goal is to explain the behavior of ion channels in terms of their molecular structure. This is important for understanding the molecular basis of many nervous system disorders, including epilepsy, depression, neurotoxicity, and learning and memory deficits. The behavior of ion channels is controlled by voltage, ligands or G-proteins, which determine the time the channel is open. This proposal addresses the following key questions: what is the mechanism by which ion channels open and close, how is this behavior regulated ? Drk1, a delayed rectifier K+ channel cloned from rat brain, is used as a model ion channel. The functional channel consists of four identical subunits each containing six putative transmembrane segments (S1-S6), that surround a central aqueous pore. A beta-hairpin loop region between S5 and S6 forms the pore. Membrane potential is sensed by the positively charged S4 segment, the movement of which control open/close behavior in way that is not understood. Preliminary results on heteromeric channels and subconductance levels suggest that the individual subunits play a key role in channel opening and permeation. Also, two glutamate residues flanking S5, which are unique for K+ channels, are shown to be involved in stabilizing the open state. These preliminary data, together with the structural assignment of both the voltage sensor (S4) and the pore region (S5-S6), set the stage for addressing basic questions concerning the structural basis of the open/close mechanism and its regulation by voltage. The specific aims of this project are: (i) localize regions of the channel that are critically involved in channel opening and closing, (ii) investigate how the voltage sensor is coupled to the open/close mechanism, (iii) determine what the role of the individual subunits is in voltage sensing and channel opening. Site-directed mutants (point mutations and chimaeras) of drk1 will be constructed and expressed in Xenopus oocytes. The single channel behavior of the mutants will be studied using patch clamp techniques. An important tool will be the study of heteromeric channels, obtained by tandem constructs or co-injection of different cRNAs.