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

John R Bankston,Hannah A Deberg,Stefan Stoll,William N Zagotta

doi:10.1074/jbc.m117.800722

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 TRIP8bcore, 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.

Highlights

Electrical activity in cells throughout the body requires the precisely regulated opening and closing of ion channels
The last is a carboxyl-terminal series of six tetratricopeptide repeats (TPRs) repeats that binds to the terminal amino acids at the carboxyl terminus of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (Fig. 1B)
We have previously shown that this fragment of HCN2 is able to bind cAMP and undergo the conformational change associated with cyclic nucleotide binding [10]

Summary

Results

TRIP8b is a cytosolic protein composed of three primary domains (Fig. 1A). The first is the variable N-terminal domain that is the site of alternative splicing. The Kd value (koff/kon) of 6.5 ␮M is in good agreement with the global fit calculation, as well as the anisotropy measurement These data establish the rates of the binding and unbinding of TRIP8bcore to HCN2-CNBDxt and demonstrate that the fluorophore labeling of TRIP8bcore did not significantly affect binding to HCN2-CNBDxt. Functional studies have shown that TRIP8b reduces the apparent affinity of cAMP for activation of HCN channels [9, 10]. The normalized anisotropy data of 8-fluocAMP binding in the absence of Trip8bcore were fit using Equation 2 and produced a Kd of 324 nM This number reflects the apparent affinity of cAMP for HCN2-CNBDxt. To determine KC, we can use this apparent affinity, as well as the equilibrium constant for the activation conformational change, and solve the equation KC ϭ Kd*(1 ϩ L). These experiments were done using 8-fluocAMP to maintain consistency in the experiments throughout this study; supplemental Fig. S2 shows that qualitatively the same pattern is seen when using cAMP

Discussion

Experimental procedures

C Fraction cAMP bound ϭ 1 Ϫ