Developing Synthetic Peptides to Regulate Native HCN Channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are the molecular correlate of the Ih (or If) current, which plays a key role in controlling rhythmic activity in cardiac pacemaker cells and spontaneously firing neurons. HCN channels are activated by voltage and modulated by the direct binding of cAMP to their cytoplasmic C-terminal region named CNBD (cyclic nucleotide binding domain). HCN channels are further regulated by TRIP8b, a brain-specific auxiliary subunit which controls the channels’ trafficking and gating. In particular, TRIP8b interacts with the HCN channel CNBD and antagonizes the facilitatory effect of cAMP on channel opening. Recently, we have identified by rational design a 40 aa long peptide (TRIP8bnano) that recapitulates affinity and gating effects of full length TRIP8b in all HCN isoforms (HCN1, HCN2 and HCN4) and in the cardiac current If. Guided by a NMR-derived structural model that identifies the key molecular interactions between TRIP8bnano and HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8bnano) which successfully prevented β-adrenergic activation of If leaving the stimulation of calcium current unaffected. Moreover, we fused TRIP8bnano to an engineered LOV (Light Oxygen Voltage) domain of the plant blue-light receptor phototropin. Our preliminary results show that the synthetic LOV-TRIP8bnano protein is able to inhibit the cAMP regulation of HCN channels in a blue-light dependent manner. TRIP8bnano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers.