Structure and function of hyperpolarization activated cation nonselective HCN pacemaker channels

The pacemaker channels have recently been identified at the molecular level by the cloning of the hyperpolarization-activated cation nonselective (HCN) gene family. These gene products generate non-specific cation currents (I_h) that underlie oscillatory electrical activities in brain and heart. In this thesis, I studied the structure and function of the HCN pacemaker channels.; I first tested the possibility of HCN1 and HCN2 subunit coassembly to form heteromultimers with unique properties. Coexpression of HCN1 and HCN2 yields I_h currents with novel voltage dependence, activation kinetics, and cAMP modulation, which cannot be reproduced by the simulated sums of independent populations of HCN1 and HCN2 homomers. This strongly suggests the formation of heteromeric channels with distinct properties. During this study, I also found that I_h is modulated by basal levels of cAMP in intact oocytes. Next, after studying a series of chimeric channels, I found that the cyclic nucleotide binding domain (CNBD) of the channel interacts with the transmembrane domain and the C-linker—a region linking the last transmembrane segment with the CNBD—to inhibit voltage gating. cAMP binding to the CNBD, meanwhile, relieves this inhibition due to an altered interaction between the CNBD and the C-linker. Finally, I characterized a novel mechanism for cAMP signaling in HCN channels based on allosteric coupling of voltage gating and cAMP binding. Due to this coupling, cAMP binds with much higher affinity to the open state of the channel than to the closed state. This allosteric model accounts for a slow component in HCN channel activation due to the slow binding of low levels of cAMP to open channels. A computer simulation of a thalamacortical relay neuron suggests that this allosteric model can explain thalamic spindling, which occurs during slow wave sleep. More generally, dynamic signaling through activity dependent changes in ligand affinity may be an important feature for a variety of channels dually gated by voltage and second messengers, such as Ca2+ activated BK channels, G protein modulated Ca 2+ channels, and cyclic nucleotide regulated HERG K+ channels.