A single residue exerts two contrasting effects on autoinhibition of HCN channels

Abstract

Hyperpolarization‐activated cation (HCN) channels regulate electrical activity in the brain and heart in a cAMP‐dependent manner. The voltage‐gating of these channels is mediated by a transmembrane (TM) region but is additionally regulated by direct binding of cAMP to a cyclic nucleotide‐binding (CNB) fold in the cytoplasmic C‐terminal region. In the unliganded form, the CNB fold mediates an autoinhibitory mechanism that kinetically and thermodynamically stabilizes the closed state, and cAMP binding relieves this inhibition. This autoinhibition is an important regulatory mechanism, relief of which provides a molecular mechanism for how cAMP potentiation occurs. The magnitude of this autoinhibition can be calculated by comparing thermodynamic or kinetic parameters of a channel to the parameters from a channel that is autoinhibition‐free due to truncation of the CNB fold. Recently, I have found that the magnitude of autoinhibition is regulated by interdomain interactions between the C‐terminal region and TM region structures. Specifically, the magnitude of both kinetic and thermodynamic autoinhibition was augmented by replacing the TM region of the HCN2 channel with the TM region of HCN4. I hypothesized that interdomain interactions between the HCN4 TM region and charged residues in the C‐linker, which connects the CNB fold to the TM region, can modify the magnitude of autoinhibition. I used site directed mutagenesis to identify that the E457 residue formed an electrostatic interaction with the HCN4 TM region to stabilize the open state. This residue also has an important role in both the kinetic and thermodynamic components of the autoinhibition mechanism. A charge reversal mutation (E457R) limited the thermodynamic effect of autoinhibition while augmenting the kinetic effect of autoinhibition. Overall, this work suggests that the molecular mechanisms of autoinhibition must include participation of TM region structures and provides functional evidence for interdomain interactions that facilitate HCN channel regulation by CNB fold‐mediated mechanisms.Support or Funding InformationNSERC, Simon Fraser University