Dynamic modulation of HCN4 and ASICs

University Of Colorado Denver, Aurora CO

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Abstract

Project Summary Ion channels are precisely tuned molecular machines that control the flow of ions across the plasma membrane. They can sense a wide array of stimuli including voltage, pH, neurotransmitters, temperature, and mechanical force. The function of these proteins depends critically on an array of factors that can regulate their activity including the composition of the lipid membrane, protein interacting partners, post-translational modifications, and assembly with different subunits. Our lab focuses on two families of ion channels: Acid-sensing ion channels (ASICs) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Acid-sensing ion channels (ASICs) are critical sensors of extracellular pH that contribute to excitability in cells in both the central and peripheral nervous system. ASICs are trimeric sodium selective ion channels that can assemble as both homo- and heterotrimers and their precise properties are governed by the channel composition. They have important roles in cell death post ischemia as well as in pain sensing. HCN channels are unusual voltage-gated channels that respond to cellular hyperpolarization and the direct binding of the cyclic nucleotide cAMP. HCN channels are important determinants of cellular excitability in a neurons and cardiac pacemaker cells. Both families of channels have emerged as potential drug targets for pathophysiological conditions like stroke and pain. Our research seeks to understand mechanisms of channel function and regulation for both families of channels. This grant outlines three ongoing lines of research being conducted in the lab. 1) We are seeking to understand the structural determinants of lipid regulation of ASIC3. We have shown that ASIC3 is potentiated by at least three classes of single acyl chain lipids: polyunsaturated fatty acids (PUFAs), lysophosphatidyl cholines (LPC), and N- acyl amino acids (NAAAs). The effects of these lipids can be strong enough to activate the channel in the absence of acidification suggesting ASIC3 may also serve as a lipid sensor. Our goal is to understand how lipids bind to ASIC3 and how those interactions alter channel function. 2) We have developed a novel single molecule photobleaching technique to determine ion channel stoichiometry. We are using this new approach to look at how ASICs that are expressed in dorsal root ganglion cells (ASIC1a, ASIC2b, ASIC3) assemble into heteromeric channels. 3) Finally, we are studying how unique structural elements in HCN4 make it particularly sensitive to modulation by ions, small molecules, and protein binding partners. The site of this isoform specific difference in HCN4 is likely the C-linker which couples the cyclic nucleotide binding domain to the pore of the channel. By understanding the sites of regulation for both families of channels we believe we can uncover novel sites for the development of drugs that target each of these families of ion channels.