Ion channel currents are the fundamental units of electrical activity in most organisms. In our nervous system, K+ and Ca2+ channels are critical, and their regulation provides a way to directly control neuronal excitability and release of neurotransmitter at synapses. The modulations are crucial to basic nervous function and their understanding should contribute to novel modes of medical interventions for a range of disorders involving the brain, nerves and muscles. We focus primarily on the family of Kv7 (KCNQ/M-type) K+ channels that underlies several neuronal K+ currents and on Cav2.2 (N-type) Ca2+ channels. In particular, we seek to elucidate the molecular mechanisms of several modulatory pathways that act on these types of ion channels. We use both a heterologous system in which cDNA clones of the channels, receptors and signaling molecules are expressed in mammalian tissue-culture cells, and a preparation of primary sympathetic neurons. Two pathways that we study have in common receptors coupled to the Gq/11 family of G proteins, the activation of phospholipase C, and hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). Both Kv7 and Cav2.2 channels have emerged as being regulated by PIP2, and in this project we will investigate how PIP2 interacts with the channels, the site(s) of the interaction, and the parts of the channel proteins that mediate the regulation of gating. Stimulation of certain raises intracellular Ca2+ and acts on Kv7 channels in concert with calmodulin (CaM). We will further investigate the molecular mechanism by which Ca2+/CaM acts. Other Gq/11-coupled receptors do not raise intracellular Ca2+ and act by depleting the membrane of PIP2. Using a variety of approaches, we will probe this specificity in receptor action. The tools to be used include patch-clamp electrophysiology, molecular biology, biochemistry, and advanced imaging techniques. The molecules and signaling pathways that we study have broad relevance to human health and disease. Kv7 channels play a dominant role in regulating excitability of neurons, and their regulation likely underlies changes in emotional state, memory and regulation of body organs. Dysfunctional Kv7 channels cause specific epileptic syndromes. The modulation of Cav2.2 channels regulates release of neurotransmitter which is the basic signal at synapses between neurons. Thus, our research should provide the basis for the development of novel modes of therapeutic intervention for a variety of nervous diseases. Lay summary: This project studies how ion channels, which mediate the electricity in nerve cells, are regulated. We investigate these signals at the level of the molecule and the individual living cell using biophysical and molecular tools. We seek to understand this regulation of the nervous system that underlies the complex phenomena of human thought, emotion and behavior. Our findings may help to alleviate the many diseases of mood, motion and consciousness that are disorders of nervous function.
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