Mechanism for the inhibition of the cAMP dependence of HCN ion channels by the auxiliary subunit TRIP8b

Abstract

TRIP8b, an accessory subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels, alters both the cell surface expression and cyclic nucleotide dependence of these channels. However, the mechanism by which TRIP8b exerts these dual effects is still poorly understood. In addition to binding to the carboxyl-terminal tripeptide of HCN channels, TRIP8b also binds directly to the cyclic nucleotide-binding domain (CNBD). That interaction, which requires a small central portion of TRIP8b termed TRIP8b_core, is both necessary and sufficient for reducing the cAMP-dependent regulation of HCN channels. Here, using fluorescence anisotropy, we report that TRIP8b binding to the CNBD of HCN2 channels decreases the apparent affinity of cAMP for the CNBD. We explored two possible mechanisms for this inhibition. A noncompetitive mechanism in which TRIP8b inhibits the conformational change of the CNBD associated with cAMP regulation and a competitive mechanism in which TRIP8b and cAMP compete for the same binding site. To test these two mechanisms, we used a combination of fluorescence anisotropy, biolayer interferometry, and double electron-electron resonance spectroscopy. Fitting these models to our fluorescence anisotropy binding data revealed that, surprisingly, the TRIP8b-dependent reduction of cAMP binding to the CNBD can largely be explained by partial competition between TRIP8b and cAMP. On the basis of these findings, we propose that TRIP8b competes with a portion of the cAMP-binding site or distorts the binding site by making interactions with the binding pocket, thus acting predominantly as a competitive antagonist that inhibits the cyclic-nucleotide dependence of HCN channels.

Keywords: cyclic nucleotide; electron paramagnetic resonance (EPR); fluorescence anisotropy; inhibition mechanism; ion channel; protein-protein interaction.

Figure 1.

TRIP8b inhibits the cAMP dependence of HCN2 channels. A, schematic cartoon showing the major domains of TRIP8b. The orange rectangle represents the variable domain 1a. The yellow rectangle represents a conserved region that is absolutely conserved in all orthologs of TRIP8b. The light green hexagons represent the individual TPRs that make up the TPR domain. B, cartoon showing TRIP8b interacting with the carboxyl-terminal region of HCN channels. The structure of the C-linker/CNBD is adapted from PDB 1Q43 (30). C, representative current traces elicited by stepping the voltage to hyperpolarized potentials between −70 and −140 mV in inside-out patches from oocytes expressing HCN2 channels. The top panel shows currents in the absence of ligand, the middle panel is in the presence of 1 μm cAMP, and the bottom panel is in the presence of both 1 μm cAMP and 10 μm TRIP8b.

Figure 2.

TRIP8b and cAMP both bind to HCN2-CNBDxt. A, fluorescence anisotropy measurements of a fluorescent analog of cAMP (8-fluo-cAMP) plotted versus the total concentration of HCN2-CNBDxt (n = 3). These data were fit with Equation 2 to give an apparent binding affinity of 324 ± 88 nm. B, fluorescence anisotropy measurements of a bimane-labeled TRIP8b_core plotted versus the total concentration of HCN2-CNBDxt (n = 3). These data were fit with Equation 2 to give an apparent binding affinity of 8.4 ± 4.1 μm. The data are plotted as means ± S.E.

Figure 3.

Biolayer interferometry reveals TRIP8b_core affinity for HCN2-CNBDxt. A, cartoon representation of biolayer interferometry experiments. The colored rectangle represents the optical probe, and the green is the layer coated with Ni-NTA. The probes are loaded with HCN2-CNBDxt (blue ovals). The probe is then dipped into a well of a 96-well plate containing TRIP8b_core (yellow rectangles). TRIP8b_core binding kinetics are then measured. The probe is then moved to a well with only buffer, and TRIP8b_core unbinding kinetics are measured. B, binding curves that show the shift in interference pattern of light as a function of time. This shift is directly related to the change in thickness of the optical layer, which is in turn related to the binding of TRIP8b_core. The concentration of TRIP8b_core in the wells is indicated. C, the boxed regions from B are shown at an expanded time scale. These data are fit with Equation 3 revealing the on and off rates of TRIP8b binding. The average rates were k_on = 1 × 10⁵±0.16 × 10⁵ m⁻¹ s⁻¹ and k_off = 0.72 ± 0.14 s⁻¹ (n = 3). D, plot of k_obs versus the concentration of TRIP8b (n = 3). The slope of the linear fit to Equation 4 gives an estimate of k_on of 1.12 × 10⁵ m⁻¹ s⁻¹, and the vertical intercept estimates a k_off of 0.73 s⁻¹. These data are consistent with the global fits to the data in B and C. The data are plotted as means ± S.E.

Figure 4.

Data and models for TRIP8b inhibition of the cAMP dependence of HCN channels. A, normalized fluorescence anisotropy of 8-fluo-cAMP as a function of the total concentration of HCN2-CNBDxt in the presence of different concentrations of TRIP8b_core ranging from 0 to 250 μm (n = 4). The data are plotted as means ± S.E. and fit with Equation 2. B, a noncompetitive model of TRIP8b inhibition of HCN channels where TRIP8b and cAMP can both bind (state H_TC), but once TRIP8b is bound, the conformational change associated with HCN2-CNBDxt activation is inhibited by a factor y. Grayed out states represent largely unpopulated states. C, a partially competitive model of TRIP8b inhibition of HCN channels where binding of TRIP8b inhibits binding of cAMP and vice versa by a factor of x. Again, grayed out states represent largely unpopulated states.

Figure 5.

DEER reveals the equilibrium constant for the conformational change in the CNBD when bound to cAMP. A, HCN2-CNBDxt structure with spin-label rotamers at a position on the β-roll (S563C) and the C-helix (A624C). Rotameric models of MTSL attached to HCN2-CNBDxt were obtained using MMM (15). B, distance distributions of HCN2-CNBDxt S563C/A624C in the absence (blue) and presence (red) of 1 mm 8-fluo-cAMP.

Figure 6.

Data for cAMP binding to HCN2-CNBDxt in the presence of TRIP8b are best fit with a partial competition model. A, normalized fluorescence anisotropy data for 8-fluo-cAMP binding to HCN2-CNBDxt at difference concentrations of TRIP8b_core fit with the noncompetitive model at the top. In this example, y = 10,000, making the H_TCA state essentially unpopulated. The fits are very poor. For the noncompetitive model, K_C = 0.745 μm, K_T = 7.34 μm, L = 1.3, and y = 10,000. B, normalized fluorescence anisotropy data for 8-fluo-cAMP binding to HCN2-CNBDxt fit with the partially competitive model at the top. Here, x = 100 resulted in a good fit to the data. For the competitive model, K_C = 0.745 μm, K_T = 7.34 μm, L = 1.3, and x = 100. The data are plotted as means ± S.E.

Figure 7.

DEER data support the partial competition hypothesis. A, model predictions for the fraction of active CNBD molecules with 25 μm HCN2-CNBDxt and 1 mm cAMP as a function of TRIP8b concentration for both the noncompetitive and competitive models. For both models, K_C = 0.745 μm, K_T = 7.34 μm, and L = 1.3. For the noncompetitive model y = 100, and for the competitive model x = 100. The noncompetitive model (blue line) shows that the probability of finding the CNBD in the active state is strongly dependent on TRIP8b concentration. However, the partially competitive model (red line) shows that the probability of finding the CNBD in the active state is only weakly dependent on TRIP8b concentration. The vertical dotted line marks 300 μm TRIP8b_core. B, distance distributions of HCN2-CNBDxt S563C/A624C (blue), with 300 μm TRIP8b_core (cyan), with 1 mm 8-fluo-cAMP (red), and with 1 mm 8-fluo-cAMP and 300 μm TRIP8b_core (green).