Binding of the auxiliary subunit TRIP8b to HCN channels shifts the mode of action of cAMP

Abstract

Hyperpolarization-activated cyclic nucleotide-regulated cation (HCN) channels generate the hyperpolarization-activated cation current Ih present in many neurons. These channels are directly regulated by the binding of cAMP, which both shifts the voltage dependence of HCN channel opening to more positive potentials and increases maximal Ih at extreme negative voltages where voltage gating is complete. Here we report that the HCN channel brain-specific auxiliary subunit TRIP8b produces opposing actions on these two effects of cAMP. In the first action, TRIP8b inhibits the effect of cAMP to shift voltage gating, decreasing both the sensitivity of the channel to cAMP (K1/2) and the efficacy of cAMP (maximal voltage shift); conversely, cAMP binding inhibits these actions of TRIP8b. These mutually antagonistic actions are well described by a cyclic allosteric mechanism in which TRIP8b binding reduces the affinity of the channel for cAMP, with the affinity of the open state for cAMP being reduced to a greater extent than the cAMP affinity of the closed state. In a second apparently independent action, TRIP8b enhances the action of cAMP to increase maximal Ih. This latter effect cannot be explained by the cyclic allosteric model but results from a previously uncharacterized action of TRIP8b to reduce maximal current through the channel in the absence of cAMP. Because the binding of cAMP also antagonizes this second effect of TRIP8b, application of cAMP produces a larger increase in maximal Ih in the presence of TRIP8b than in its absence. These findings may provide a mechanistic explanation for the wide variability in the effects of modulatory transmitters on the voltage gating and maximal amplitude of Ih reported for different neurons in the brain.

Figure 1.

Effect of fusion of TRIP8b to HCN2 on relationship between [cAMP] and voltage dependence of channel gating. (A) Currents elicited by hyperpolarizing voltage steps in inside-out patches from oocytes expressing TRIP8b-HCN2 fusion channels or GFP-HCN2 channels in 0, 0.1, or 100 µM [cAMP]. The membrane was held at −40 mV for 0.5 s and then stepped for 3 s to test potentials from −70 to −140 mV in 10-mV decrements. (B and C) Normalized tail current G-V relationship for GFP-HCN2 (B) or TRIP8b-HCN2 (C) channels in the presence of 0, 0.1, or 100 µM [cAMP]. Fits of Boltzmann relation yield the following values for V_1/2 and slope with different [cAMP]. GFP-HCN2 0 cAMP: V_1/2 = −116.0 mV, s = 4.98; 0.1 cAMP: V_1/2 = −105.7 mV, s = 5.33; and 100 cAMP: V_1/2 = −96.9 mV, s = 4.65. TRIP8b-HCN2 0 cAMP: V_1/2 = −114.8mV, s = 4.05; 0.1 cAMP: V_1/2 = −113.3 mV, s = 4.91; and 100 cAMP: V_1/2 = −104.2 mV, s = 4.42. (D) ΔV_1/2 as a function of [cAMP] for GFP-HCN2, GFP-HCN2 + TRIP8b expressed as independent proteins, and TRIP8b-HCN2 fusion channels in inside-out patches. Solid lines show fits of Hill equation. Fits of the Hill equation yield GFP-HCN2: ΔV_max = 17.3 mV, K_1/2 = 0.08 µM, h = 1.43; GFP-HCN2 + TRIP8b: ΔV_max = 14.2 mV, K_1/2 = 0.19 µM, h = 0.83; and TRIP8b-HCN2: ΔV_max = 11.1 mV, K_1/2 = 3.42 µM, h = 0.79. Error bars indicate SEM.

Figure 2.

cAMP causes a larger increase in maximal current with TRIP8b-HCN2 and TRIP8b_core-HCN2 channels than with GFP-HCN2 channels. The membrane was held at −40 mV and then hyperpolarized to −140 mV with a 3-s test pulse, followed by a depolarizing pulse to −40 mV to measure the tail current. (A) Representative currents for GFP-HCN2, TRIP8b-HCN2, and TRIP8b_core-HCN2 channels before (black traces) and after (red traces) application of saturating concentrations of cAMP (100 µM for GFP-HCN2 and 1 mM for the other two channels) to inside-out patches. (B) Percent increase in maximal tail current amplitude in response to cAMP for GFP-HCN2, TRIP8b-HCN2, and TRIP8b_core-HCN2; error bars indicate SEM. Mean percent increases in maximal current ± SEM are as follows: GFP-HCN2: 37 ± 9% (n = 9); TRIP8b-HCN2: 83 ± 9% (n = 10); and TRIP8b_core-HCN2: 116 ± 18% (n = 9). Current amplitude increase with GFP-HCN2 by cAMP is significantly less than that seen with TRIP8b-HCN2 and TRIP8b_core-HCN2 (*, P < 0.05, ANOVA). There is no significant difference in current increase between the latter two constructs (P > 0.05, t test).

Figure 3.

TRIP8b core domain is necessary to antagonize action of cAMP on HCN2. (A) ΔV_1/2 as a function of [cAMP] for TRIP8b_ΔInt-HCN2 and TRIP8b_core-HCN2 channels in inside-out patches, compared with GFP-HCN2 and TRIP8b-HCN2 channels. Solid lines show fits of the Hill equation. Fits of the Hill equation yield TRIP8b_ΔInt-HCN2: ΔV_max = 18.2 mV, K_1/2 = 0.08 µM, h = 1.11; and TRIP8b_core-HCN2: ΔV_max = 13.3 mV, K_1/2 = 190 µM, h = 0.69. Note that TRIP8b_ΔInt, with internal deletion of 22 residues of the core domain, fails to alter cAMP dose–response relation. (B) V_1/2 values of various indicated constructs in the absence of cAMP show no significant differences (P > 0.05, ANOVA). Error bars indicate SEM.

Figure 4.

Direct application of TRIP8b_core polypeptide to inside-out patches suppresses HCN2 maximal current in the absence of cAMP. (A) Representative experiment showing effects of TRIP8b_core polypeptide on HCN2 currents in inside-out patches elicited by a hyperpolarization to −140 mV, either in the absence or presence of cAMP. The current recording protocol is described in Fig. 3. The internal bath solution contained control solution, 4 µM TRIP8b_core with no cAMP, 4 µM TRIP8b_core plus 100 µM cAMP, or 100 µM cAMP with no TRIP8b. (B) Dose–response curve for the percent reduction in current amplitude at extreme negative voltages as a function of TRIP8b_core polypeptide concentration (in the absence of cAMP). Error bars show SEM. (C) The binding activity of the HCN1 C-linker/CNBD (residues 390–611) with WT and mutant TRIP8b (constant region, exons 5–16) assessed using a yeast two-hybrid assay. For TRIP8b, the yellow square represents the conserved core region. In TRIP8b_core-mut, the WT EEEFE core residues are substituted by RRRAR. The red colors denote the TPR domains. Activity was detected by transactivation of a GFP reporter gene. “+++” indicates very strong fluorescence; “−” indicates no detectable fluorescence (see Santoro et al. [2011]). (D) Representative HCN2 currents before (black trace) and after (blue trace) the application of TRIP8b_core-mut polypeptide to inside-out patches. The current recording protocol is described in Fig. 3.

Figure 5.

Effects of TRIP8b_core polypeptide on action of cAMP on HCN2 channel voltage gating. (A) Representative experiment showing V_1/2 of HCN2 channels in inside-out patches with solutions containing the indicated [cAMP] in the absence or presence of 4 µM TRIP8b_core polypeptide. Open circles indicate V_1/2 in the absence of both cAMP and TRIP8b_core; closed circles indicate V_1/2 in the presence of 0.1 or 10 µM cAMP, with or without 4 µM TRIP8b_core polypeptide, as indicated. (B) Normalized G-V relationship for HCN2 channel tail currents in 0, 0.1, or 10 µM cAMP. (C) Normalized G-V relationship for HCN2 channel tail currents in the presence of 4 µM TRIP8b_core polypeptide plus 0, 0.1, or 10 µM cAMP. (D) Normalized G-V relationship for HCN2 tail currents in the presence of 4 µM TRIP8b_core-mut polypeptide plus 0, 0.1, or 10 µM cAMP. (E) Shift in V_1/2 as a function of [cAMP] in the absence (open circles) or presence (closed circles) of 4 µM TRIP8b_core polypeptide. Solid lines show fits of the Hill equation, which yield HCN2: ΔV_max = 18.1 mV, K_1/2 = 0.08 µM, h = 0.86; and HCN2 plus 4 µM TRIP8b_core: ΔV_max = 15.8 mV, K_1/2 = 5.64 µM, h = 0.86. (F) Percent decrease in ΔV_1/2 produced by the indicated concentrations of cAMP as a function of TRIP8b_core concentration (left ordinate). Fits of the Hill equation (solid lines) yield 0.1 µM cAMP: K_1/2 = 0.22 µM, h = 1.35, percent maximal decrease in ΔV_1/2 = 100%; 1 µM cAMP: K_1/2 = 1.57 µM, h = 1.69, percent maximal decrease in ΔV_1/2 = 97%; 10 µM cAMP: K_1/2 = 2.34 µM, h = 1.19, percent maximal decrease in ΔV_1/2 = 69%; and 100 µM cAMP: K_1/2 = 3.96 µM, h = 0.74, percent maximal decrease in ΔV_1/2 = 42%. The Hill fit to the TRIP8b dose–response curve for HCN2 maximal current reduction from Fig. 4 B is replicated here for comparison (red dashed line, right ordinate). Error bars indicate SEM.

Figure 6.

12-state allosteric model for regulation of HCN2 channel opening by voltage, cAMP, and TRIP8b. The vertical and front-back transitions of the cubic scheme represent the cAMP and TRIP8b binding reactions to the channel, respectively. The two horizontal transitions are the voltage-dependent activation step reflecting voltage sensor movement, followed by a voltage-independent opening step. The front face of the cube is the six-state cyclic allosteric model that represents the effects of voltage and cAMP on channel opening in the absence of bound TRIP8b (Zhou and Siegelbaum, 2007); the top face is a six-state cyclic allosteric model that represents the actions of voltage and TRIP8b binding on channel opening in the absence of bound cAMP. TRIP8b and cAMP can both bind to the channel at the same time, as represented by the back and bottom faces of the cube. Definition of states and ligands: C_R, unliganded closed channel with voltage sensor in the resting state; C_A, unliganded closed channel with voltage sensor in the activated state; O, unliganded channel in the open state; A, cAMP; T, TRIP8b. Definition of parameters: K_V, equilibrium constant for transition of closed channel transition between resting state and activated state; L, intrinsic equilibrium constant for channel opening; $K_C^A$ and $K_O^A,$ dissociation equilibrium constants for cAMP binding to closed and open states, respectively; $K_C^T$ and $K_O^T,$ dissociation equilibrium constants of TRIP8b binding to channel in closed and open states, respectively; $K_{TC}^A$ and $K_{TO}^A,$ dissociation equilibrium constants for cAMP binding to TRIP8b-bound channels in closed and open states, respectively; $K_{AC}^T$ and $K_{AO}^T,$ dissociation equilibrium constants for TRIP8b binding to cAMP-bound channels in closed and open states, respectively.

Figure 7.

ΔV_1/2 as a function of [cAMP] and [TRIP8b_core] polypeptide, fitted by the 12-state allosteric model. The TRIP8b_core polypeptide concentrations are indicated. The parameters of the front face of the model (corresponding to the six-state cyclic model) are adopted from Zhou and Siegelbaum (2007): L = 0.43, s = 4.4, $K_C^A$ = 0.844 µM, $K_O^A$ = 0.008 µM. The best fit of the model yields the following values for the other parameters: $K_C^T$ = 0.089 ± 0.027 µM, $K_O^T$ = 0.064 ± 0.010 µM, $K_{TC}^A$ = 33.58 ± 8.18 µM, $K_{TO}^A$ = 3.53 ± 1.33 µM. $K_{AC}^T$ and $K_{AO}^T$ are then derived from the other parameters: $K_{AC}^T$ = $K_{TC}^A·K_C^T/K_C^A$ = 3.54 µM and $K_{AO}^T$ = $K_{TO}^A·K_O^T/K_O^A$ = 28.24 µM. Error bars show SEM. See supplemental text for further details.

Figure 8.

Importance of C-terminal regions of HCN2 for the ability of TRIP8b to reduce maximal current. (A) Representative currents through HCN2 WT and mutant channels before (black traces) and after (blue traces) the application of 4 µM TRIP8b_core polypeptide to inside-out patches (no cAMP present). HCN2_ΔCNBD, truncation after residue V526 removing entire CNBD and all downstream residues; HCN2_R591E, point mutation of conserved arginine in CNBD required for high-affinity cAMP binding; HCN2_FPN, triple point mutation substituting FPN sequence for residues QEK in A′ helix of C-linker (residues 450–452 in HCN2). The recording protocol is described in Fig. 2. (B) Percent reduction in maximal tail current amplitude for HCN2 WT and mutant channels in response to 4 µM TRIP8b_core polypeptide. Error bars indicate SEM. Mean percent reductions ± SEM are as follows: HCN2, 43 ± 4% (n = 9); HCN2_ΔCNBD, −1.6 ± 2% (n = 3); HCN2_R591E, 48 ± 9% (n = 3); and HCN2_FPN, −1 ± 2% (n = 3).

Figure 9.

The effect of mutations in the HCN1 key CNBD residue R538 on TRIP8b binding. Western blot analysis shows binding of HCN1, HCN1_R538A, and HCN1_R538E mutants to WT TRIP8b(1b-2) assessed by coimmunoprecipitation from Xenopus oocyte extracts coinjected with TRIP8b and HCN1 cRNA. The top row shows the HCN1 input signal using an HCN1 antibody. The middle row shows the TRIP8b input signal using an anti-TRIP8b antibody. The bottom row shows the amount of HCN1 protein coimmunoprecipitated with the TRIP8b antibody (Western blot probed using HCN1 antibody). Note that exposure times are directly comparable along each row but not down each column. Individual bands have been cut from intact gel pictures and aligned to allow direct comparison of intensities for WT and mutant constructs.