Spectroscopic and Biochemical Studies of TRIP8b Regulation of HCN Channels

Abstract

TRIP8b, an accessory subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, alters both cell surface expression and cyclic nucleotide dependence of these channels. The mechanism through which TRIP8b exerts these dual effects is still poorly understood. Besides binding the terminal three residues of HCN channels, TRIP8b also binds directly to the cyclic nucleotide-binding domain (CNBD). A small central portion of TRIP8b, termed TRIP8bcore, is involved in this interaction. Binding of TRIP8bcore to the CNBD dramatically reduces the effects of cAMP on the channel. Using spectroscopic and biochemical techniques, we sought to understand how and where TRIP8b binds to the CNBD and how it reduces the cyclic nucleotide dependence of HCN channels. To closely examine the binding of TRIP8bcore to the CNBD, we used double electron-electron resonance (DEER) at Q-band frequencies to study conformational changes in the soluble CNBD of HCN2 in the presence of TRIP8b and cAMP. We show that the overall structure of the TRIP8b bound conformation of the CNBD closely resembles the apo state. In addition, we use DEER between the CNBD and TRIP8bcore and nuclear magnetic resonance (NMR) to localize the binding site for TRIP8b on the CNBD. Finally, to understand the mechanism of TRIP8b inhibition of cAMP regulation of HCN channels, we performed binding studies of cAMP and TRIP8b on the CNBD and developed a multi-state mathematical model to explore 1) whether cAMP and TRIP8b can bind simultaneously to HCN channels, 2) whether TRIP8b reduces the affinity for cAMP binding, and 3) whether TRIP8b reduces the effect of cAMP on HCN channels by preventing the structural rearrangements associated with channel opening.