Hyperpolarization-activated nonselective cation channels (HCNs), also known as pacemaker channels, play a crucial role in producing rhythmic firing in electrically excitable cells such as heart cells and neurons. Their activity is modulated by cyclic AMP, which binds directly to the principal channel subunits to alter the channel gating. This cAMP effect is known to be centrally important in regulation of the heartbeat, regulation of the sleeping versus waking patterns apparent in EEG, and no doubt in many other cellular processes. We are primarily interested in the molecular mechanisms responsible for HCN gating. The HCN's are members of the 6TM voltage-gated channel family, with similarities both to voltage-gated IC (Ky) channels and to cyclic nucleotide gated (CNG) channels. These two channel classes share a common architecture but apparently use different mechanisms for their principal gating. How do HCN's work? Their voltage-dependence is reversed from that of normal Ky gating and there are some mechanistic similarities to both the Ky and CNG gating. Furthermore, a cyclic nucleotide binding domain (like that in CNG's) has important but subtle effects on gating. We will use a sea urchin HCN, with more substantial effects of cAMP, as a route to understanding the mammalian channels. Our first goal is to produce a kinetic description of the HCN currents that will serve as a basis for understanding their role in cellular electrophysiology and pacemaking behavior. We will incorporate the effect of both voltage and cAMP; in doing so, we will also test specific molecular mechanisms. Second, we will investigate the mechanisms of gating, using cysteine mutagenesis, metal binding, and chemical modification to test where the gates are and specifically if the gating induced by cAMP and by voltage arises from two separate mechanisms. This is particularly interesting because we have already found that the pharmacology of the channel is highly dependent on gating. Finally, we will test specific kinetic and physical mechanisms by which cAMP (and the cyclic nucleotide binding domain) alter gating of the channel.
