cAMP binding to closed pacemaker ion channels is cooperative

Abstract

The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides. Here, we show positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes by following the binding of individual fluorescence-labeled cAMP molecules. Kinetic modeling reveals that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in closed HCN2 channels not pre-activated by voltage is already cooperative. Hence, cooperativity is not causally linked to channel activation by voltage. Our analysis also shows that single-molecule binding measurements at equilibrium can quantify cooperativity in ligand binding to receptors in native membranes.

Keywords: HCN-channels; TIRF; cAMP; cooperativity; single molecule.

Fig. 1.

Generation of supported membrane sheets. (A) Supported membrane sheets were generated by using cultured HEK293 cells stably transfected with eGFPmHCN2 and sticking the membrane to cover slips. Coverslips were put upside down onto plasma-cleaned cover slides and pushed down by 5.3 kPa. Coverslips were then removed, leaving the top membrane on the cover slide while the intracellular part was accessible for solutions. (B) TIRF measurements of fluorescent cAMP derivatives and eGFP tags attached to HCN2 channels were performed on supported membranes. Only ligands near the membrane are excited due to the decay of the laser power in the evanescent field. (C) eGFP (Left) and f1cAMP (Right) TIRF recordings of a supported membrane. Individual signals were fitted by a 2D-Gaussian function to ensure evaluation of single molecules.

Fig. 2.

Recording of binding events and data processing. (A) Raw data traces were smoothened by the Chung–Kennedy Filter. Filtered traces were idealized to the number of bound ligands by the DISC algorithm (41). (B) The proportion of observed states was compared to the expected proportion of non-cooperative binding calculated by Eq. 2. Error bars show SEM. *indicate significant differences (one sample t test, P < 0.05).

Fig. 3.

Kinetic and equilibrium constants calculated for a simple binding reaction. (A) Simplest model with four consecutive binding steps. (B–D) Comparison of present to previously published constants. (B) Microscopic binding rate constants k_on and (C) microscopic dissociation rate constants k_off. (D) Association constants K_A of individual binding steps. (E) Dwell times of isolated single-liganded states at 0.1 µM f1cAMP fitted by a mono- or biexponential function.

Fig. 4.

Favored model to describe the single binding events. (A) Scheme of model 13. The model consists of four individual binding steps of which each has an own transition to a flip state. In a flip state, additional binding is possible. Due to extremely small rate constants between the non-liganded and the single-liganded flip state, this transition is excluded. Symbols describe subunit properties. (B) Equilibrium association constants (K_A) for binding of ligands in the non-flip (red) and flip state (purple) The affinity constant of a hypothetical binding to the empty flip state was calculated (gray), assuming microscopic reversibility (see text). (C) Equilibrium constants for switching into the flip state depending on the state of receptor occupation. All error bars show SEM.