A synthetic peptide that prevents cAMP regulation in mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.

Andrea Saponaro,Chiara Donadoni,Annalisa Bucchi,Bina Santoro,Alessandro Porro,Lucia Banci,Bianca Introini,Gerhard Thiel,Pietro Mesirca,Francesca Cantini,Dario Difrancesco,Matteo E Mangoni,Anna Moroni,Vincenzo Maione

doi:10.7554/elife.35753

Abstract

Binding of TRIP8b to the cyclic nucleotide binding domain (CNBD) of mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels prevents their regulation by cAMP. Since TRIP8b is expressed exclusively in the brain, we envisage that it can be used for orthogonal control of HCN channels beyond the central nervous system. To this end, we have identified by rational design a 40-aa long peptide (TRIP8bnano) that recapitulates affinity and gating effects of TRIP8b in HCN isoforms (hHCN1, mHCN2, rbHCN4) and in the cardiac current If in rabbit and mouse sinoatrial node cardiomyocytes. Guided by an NMR-derived structural model that identifies the key molecular interactions between TRIP8bnano and the HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8bnano) which successfully prevented β-adrenergic activation of mouse If leaving the stimulation of the L-type calcium current (ICaL) unaffected. TRIP8bnano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers.

Highlights

Hyperpolarization-activated cyclic nucleotide-gated (HCN1-4) channels are the molecular correlate of the If/Ih current, which plays a key role in controlling several higher order physiological functions, including dendritic integration and intrinsic rhythmicity both in cardiac and neuronal cells (Robinson and Siegelbaum, 2003)
We have previously shown that TRIP8bcore interacts with two elements of the isolated cyclic nucleotide binding domain (CNBD) protein fragment from hyperpolarization-activated cyclic nucleotide-gated (HCN) channels: the C-helix and the N-bundle loop, a sequence connecting helix E’ of the C-linker with helix A of the CNBD (Saponaro et al, 2014)
We have identified the minimal binding peptide that reproduces the effects of TRIP8b on HCN channel gating

Summary

Introduction

Hyperpolarization-activated cyclic nucleotide-gated (HCN1-4) channels are the molecular correlate of the If/Ih current, which plays a key role in controlling several higher order physiological functions, including dendritic integration and intrinsic rhythmicity both in cardiac and neuronal cells (Robinson and Siegelbaum, 2003). CAMP-dependent modulation of HCN channels underlies distinct roles of cAMP in heart rate regulation (DiFrancesco, 1993) and development of peripheral neuropathic pain (Emery et al, 2012; Herrmann et al, 2017), which can be dissected by using a TRIP8b-based tool In this regard, peptide-based drugs (2–50 aa long) are emerging as a fascinating application area as they open new therapeutic possibilities with an advantage over small molecules in terms of specificity and affinity for the target (Fosgerau and Hoffmann, 2015; Henninot et al, 2018). Our study opens the possibility of selective in vivo control of the cAMP-dependent facilitation of HCN channel opening, by local supply of TATTRIP8bnano peptide

Results

Discussion

Materials and methods

Ethics statement

Funding Funder