Ions, second messengers, and anesthetics: Deconvolving the mechanistic actions of regulators of HCN pacemaker channel function

Hyperpolarization opens HCN-encoded (hyperpolarization-activated, cyclic nucleotide sensitive) IH pacemaker channels by promoting inward exposure of the four S4 voltage sensors thus stabilizing a dilated arrangement of a helical bundle formed from the cytoplasmic ends of the S6 pore lining helices. Once open, the channels carry an inward, depolarizing current. Direct interaction of cAMP with a gating ring (formed from sequences that lie immediately distal to the S6 activation gate) and of acidic lipids with undetermined regions of the channels, favors channel activation and opening. These properties result in IH functioning as a voltage and ligand-modulated regulator of excitability at subthreshold potentials. Block by intracellular Mg controls the inhibitory effect of the channels at threshold and above. The roles of IH in numerous physiological and pathological conditions (including pain and epilepsy) suggest the HCN channels may represent an interesting therapeutic target, but exploitation of this will require development of isoform selective antagonists. Intriguingly, the intravenous general anesthetic propofol (2,6-diisopropylphenol) selectively inhibits the gating of HCN1 channels. As an antagonist of HCN1 channels could represent a novel, cardiac sparing agent to treat peripheral neuropathic pain or epilepsy, we have explored the basis of propofol's selective action on these channels. These studies reveal that the anesthetic acts directly on HCN1 channels via the membrane embedded core domain rather than through modification of second messenger cascades. Modeling of the equilibrium and kinetic behavior of HCN1 channels and chimeric channels, formed from HCN1 and the relatively propofol insensitive HCN4 isoform, reveals that (1) gating of the channels is best described by models in which closed and open states communicate via a voltage-independent reaction with no significant equilibrium occupancy of a deactivated open state at non-permissive voltages, (2) propofol modifies gating by preferentially associating with closed-resting and closed-activated states but a low affinity interaction with the activated open state shapes the effect of the drug under physiological conditions, (3) the energetics of the opening isomerization and interactions of the open pore with permeant ions shape how the channel-drug interaction affects the activation of the channel and the loss of the maximal current due to a decline in the maximal open probability. These results illuminate the mechanism of HCN channel gating and provide a framework that will facilitate development of HCN1 isoform specific propofol derivates that have altered pharmacological properties and therapeutic potentials. In addition to determining the mechanism of action of propofol, we have used the models we have developed to further our understanding of the mechanism of inhibition of the HCN channels by the endogenous regulator, Mg. We show that cAMP binding to the gating ring enhances not only channel opening but also the kinetics of Mg block. Our results reveal this effect arises not as a result of the ability of the ligand to increase the channel's maximal open probability but via acceleration of the blocking reaction itself. Complexity in the kinetics of Mg block in the absence of cAMP indicates that the cAMP-sensitivity arises because the gating ring and pore of open, but unliganded HCN channels adopt more than one conformation. This observation is consistent with the hypothesis that motions within the gating ring and the pore are not strictly coupled. The finding that cAMP can impart a second messenger sensitivity to rectification of open HCN channels suggests a novel mechanism by which rapid control of these erstwhile "slow" channels may influence cellular excitability. Together, our results reveal a complex set of interactions between permeant and impermeant ion binding in the channel selectivity filter, the cyclic nucleotide gating ring and a pharmacologically relevant exogenous ligand, the anesthetic, propofol.