Abstract
Hyperpolarization-activated cation-nonselective (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. Cyclic AMP potentiation has been explained by an autoinhibition model which views the unliganded CNB fold as an inhibitory module whose influence is disrupted by cAMP binding. However, the HCN2 subtype uses two other CNB fold-mediated mechanisms called open-state trapping and Quick-Activation to respectively slow the deactivation kinetics and speed the activation kinetics, against predictions of an autoinhibition model. To test how these multiple mechanisms are influenced by the TM region, we replaced the TM region of HCN2 with that of HCN4. This HCN4 TM-replacement preserved cAMP potentiation but augmented the magnitude of autoinhibition by the unliganded CNB fold; it moreover disrupted open-state trapping and Quick-Activation so that autoinhibition became the dominant mechanism contributed by the C-terminal region to determine kinetics. Truncation within the CNB fold partially relieved this augmented autoinhibition. This argues against the C-terminal region acting like a portable module with consistent effects on TM regions of different subtypes. Our findings provide evidence that functional interactions between the HCN2 TM region and C-terminal region govern multiple CNB fold-mediated mechanisms, implying that the molecular mechanisms of autoinhibition, open-state trapping, and Quick-Activation include participation of TM region structures.
Highlights
Hyperpolarization-activated cation-nonselective (HCN) channels produce the Ih or If "pacemaker" currents that regulate rhythmic firing in the brain and heart
Autoinhibition of HCN channels is mediated by the cytoplasmic cyclic nucleotide-binding (CNB) fold, but this study shows for the first time that the effects of this autoinhibition are not solely determined by the sequence of the C-terminal region and can be strongly altered by substitution of the TM region
This augmentation relied on structures in the C-terminal region since it was abolished by truncation within the CNB fold after the beta-roll
Summary
Hyperpolarization-activated cation-nonselective (HCN) channels produce the Ih or If "pacemaker" currents that regulate rhythmic firing in the brain and heart (reviewed in Wahl-Schott and Biel 2009). The autoinhibition model envisions that a ΔCNB channel has maximally favoured hyperpolarization-activation energetics, and applying a Leffler-type transition state model (Leffler 1953) would predict that CNB fold deletion should enable maximally fast hyperpolarization-dependent activation kinetics and maximally slow depolarization-dependent deactivation. These autoinhibition-based predictions are notably contradicted for kinetics of at least one HCN subtype, mouse HCN2 (Magee et al 2015). A full understanding of HCN channel gating requires elucidation of multiple, co-existing mechanisms that all depend on the CNB fold, and yet have different structural determinants and target rate-limiting reaction steps of distinct pathways
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