Genetic Studies of the Synapse

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DESCRIPTION (provided by applicant): The long-term objectives are to understand how individual potassium (K+) channels regulate neuronal excitability, using a combination of physiological and molecular approaches starting with positional cloning of the first voltage-gated K+ channel (with six transmembrane segments per subunit) in 1987 and expression cloning of one of the first two inwardly rectifying K+ channels (with two TM segments per subunit) in 1993. It is now well known that the 6-TM Kv channels and 2-TM Kir channels constitute two very large families of structurally related K* channels. The health relatedness of the project is evident from the fact that K+ channels are linked to human diseases of the brain, ear, heart, muscle and other organs. Indeed, K+ channel blockers and openers have been developed for pharmaceutical purposes, for the treatment of epilepsies, stroke, migraine, arrhythmias, diabetes, hypertension, neuropathic pain, and anxiety-related disorders. Having used the simpler 2-TM Kir channels to develop new methodologies, such as the unbiased approach of relying on yeast screens of randomly mutagenized K+ channels to deduce how TM helices are arranged in a K+ channel-an approach validated by the excellent agreement between predictions made in 1999 based on yeast studies of mammalian Kir2.1 channels and the bacterial KirBad.1 structure reported in 2003, we plan to apply these new methods to study how the 6 TM helices are arranged in a Kv channel. With a long-standing interest in the question how neurons control the number and location of their K+ channels, thereby allowing these channels to fulfill their physiological functions, we will make use of the conceptual and technological advances made in recent studies of channel trafficking and targeting, and pursue the following questions: (1) How might the Kv1 channel protein level be regulated in cultured hippocampal neurons and cortical neurons? Preliminary studies using a translation reporter we developed have identified one signaling pathway for this regulation. (2) What mediates the axonal targeting of Kv1 channels? We will pursue new leads obtained with the assays we have worked out to quantify the relative abundance of K+ channels on the surface of axons versus dendrites of cultured hippocampal neurons.