State-Dependent cAMP Binding to Functioning HCN Channels Studied by Patch-Clamp Fluorometry

Abstract

One major goal of ion channel research is to delineate the molecular events from the detection of the stimuli to the movement of channel gates. For ligand-gated channels, it is challenging to separate ligand binding from channel gating. Here we studied the cyclic adenosine monophosphate (cAMP)-dependent gating in hyperpolarization-activated cAMP-regulated (HCN) channel by simultaneously recording channel opening and ligand binding, using the patch-clamp fluorometry technique with a unique fluorescent cAMP analog that fluoresces strongly in the hydrophobic binding pocket and exerts regulatory effects on HCN channels similar to those imposed by cAMP. Corresponding to voltage-dependent channel activation, we observed a robust, close-to-threefold increase in ligand binding, which was more pronounced at subsaturating ligand concentrations than higher concentrations. This observation supported the cyclic allosteric models and indicated that protein allostery can be implemented through differentiating ligand binding affinities between resting and active states. The kinetics of ligand binding largely matched channel activation. However, during channel deactivation, ligand unbinding was slower than channel closing, suggesting a delayed response to membrane potential by the ligand binding machinery. Our results provide what we believe to be new insights into the cAMP-dependent gating in HCN channel and the interpretation of protein allostery for general ligand-gated channels and receptors.

Tetramerization Dynamics of C-terminal Domain Underlies Isoform-specific cAMP Gating in Hyperpolarization-activated Cyclic Nucleotide-gated Channels

“…Our data suggest that cAMP-induced tetramerization, which is mediated by the C-linker, forces HCN1 CB into a high affinity state that in turn stabilizes the tetramer. This finding is in line with the reports of state-dependent affinity of HCN channels for their ligand (30,32) and agrees with the cyclic allosteric model proposed for HCN channels that postulates the coexistence of the protein in resting and active states with different affinities for the ligand (30,33,34). Furthermore, the presence of a stable tetrameric structure agrees with the finding that cAMP traps channels in an open state that kinetically affects the depolarization-dependent deactivation (35).…”

“…As a negative control, we prepared the mutant protein R538E HCN1 CB . Mutation of this invariant amino acid (Arg-591 in HCN2 and Arg-669 in HCN4) that interacts with the exocyclic oxygen of the cAMP phosphate (29) reduces the affinity of HCN CNBD for the ligand by more than 3 orders of magnitude (7,27,30). Fig.…”

A KcsA/MloK1 Chimeric Ion Channel Has Lipid-dependent Ligand-binding Energetics

“…Negative binding cooperativity has previously been suggested for full-length CNG and HCN eukaryotic channels based on patch clamp fluorometry experiments (11)(12)(13)(14). Similarly, binding curves for cAMP to the isolated CNBDs of both HCN2 and HCN4 suggest negative cooperativity, with the first cAMP molecule binding with a 10-fold higher affinity than the subsequent molecules (48).…”

“…Interestingly, because the binding process itself occurs in a domain located outside of the membrane, the observed increase in cAMP binding affinity to lipid-embedded channels relative to detergent-solubilized channels suggests that binding induces a conformational change in the transmembrane portion of the protein, which is in direct contact with lipids. Thus, the nanodisc and detergent environments may favor different conformations of the channel with different cAMP affinities (14).…”

“…Current understanding largely derives from electrophysiological experiments on cells heterologously expressing these channels (1,10). Recent technical advances in confocal patch clamp fluorometry permit concomitant ligand-binding measurements and current recordings in the same membrane patch, allowing for detailed analysis of binding cooperativity and activation states triggered by partial ligand binding (11)(12)(13)(14). For example, Kusch et al (12) provide evidence that HCN2 channels possess alternating cooperative and noncooperative ligand binding behaviors (favoring two or four bound ligands rather than one or three) and are fully activated upon binding of two ligands.…”