University thesis:
Dissertation, Friedrich-Schiller-Universität Jena, 2021
Footnote:
Tag der Verteidigung: 18.05.2021
Zusammenfassungen in deutscher und englischer Sprache
Description:
HCN channels are activated by hyperpolarizing-voltages and modulated by the binding of cAMP. They are tetrameric channels and the four subunits are arranged as homotetramers or heterotetramers. Each subunit of the channel consists of four structural domains: the HCN domain, the voltage-sensing domain (VSD), the C-linker (CL) domain and the cyclic nucleotide-binding domain (CNBD). The cryo-EM structure of human HCN1 (hHCN1) suggests that a small portion of the CL is in a proximate distance to the residues of the respective opposite subunits. Previous studies have shown the functional importance of diverse interactions of adjacent subunits. This work focuses on characterizing the opposite subunit interactions. The results herein show that the residue K464 of the CL contacts the residue M155 of the HCN domain of the respective opposite subunit. These contacts are essential in close state stabilization of the channel by mediating the opposite subunit interactions. The experimental mutant K464E shows a destabilization of closed state and mimics the phenotype of a cAMP-bound wild-type channel. Such a phenotype shows a relief of auto-inhibition posed by the tetrameric CL-CNBD, as also observed in wild-type cAMP bound phenotype. Furthermore, the loss of auto-inhibition increases the apparent affinity of cAMP binding. The results from this work are in excellent agreement with already published crystal structures and the MD simulations performed by the collaborators. In conclusion, the data here show that the interactions between opposite subunits are essential in HCN channel gating. These interactions play a significant role in maintaining the auto-inhibitory property of the channel. Both binding of cAMP and mutagenesis, disrupt these opposite subunit interactions and cause a weakening of auto-inhibition, leading to enhanced activation of the channel.