Abstract 2067 Tuning HCN channels by interaction of the cytoplasmic C-linker with the transmembrane region

Hyperpolarization-activated cation (HCN) channels regulate oscillatory electrical activity in the brain and heart. Voltage-gating mediated by their transmembrane (TM) region is inhibited by a cytoplasmic cyclic nucleotide-binding (CNB) fold, and direct cAMP binding to this fold relieves the autoinhibition. We investigated HCN homomers expressed in intact Xenopus oocytes to explore mechanistic interactions between TM and CNB regions. Results: (Part 1) Slow gating kinetics of HCN channels complicates conventional gating assessments relying on activation midpoint-voltage, VmACT. Time courses for hyperpolarization-dependent activation of HCN2 failed to reach endpoint even after 8 s. This meant that the equilibria between multiple open and closed states were not efficiently sampled in VmACT determination. Therefore we measured the deactivation midpoint-voltage, VmDEACT, using an activating prepulse to populate a broad mixture of states before testing variable voltages; the subsequent deactivation time courses efficiently reached endpoint, enabling more accurate assessment of open vs. closed state energetics. (Part 2) We tested the effect of substituting the TM region in HCN2 with the HCN4 TM region, in a chimeric channel Ch4-2. Compared to HCN2, activation kinetics for Ch4-2 were significantly slower but strikingly the VmDEACT for Ch4-2 was more positive. That is, the HCN4 TM substitution destabilized the activation barriers (transition states) yet stabilized the open ground state. We then repeated the HCN4 TM substitution in the background of HCN2 with a point mutation (E457R). This mutation lies in the C-linker region that links the TM and CNB regions, and by itself has no observable effect on HCN2 - no significant change in VmDEACT and gating kinetics, as tested in intact oocytes and excised inside-out membrane patches. Remarkably, the presence of E457R in HCN2 eliminates the VmDEACT-shift phenotype of the HCN4 TM substitution; that is, the TM substitution effect critically depends on the cytoplasmic residue E457. (Part 3) Does this interdependence of E457R and HCN4 TM substitution depend on cAMP binding in the CNB fold? We repeated our substitution tests on HCN2 whose cAMP-binding was abolished by a CNB fold point mutation (HCN2 R591E). Compared to HCN2 wild-type (WT), the HCN2 R591E channels have more negative VmDEACT as expected due to lack of cAMP potentiation. For the E457R mutation alone, HCN2 R591E channels exhibited the same lack of phenotype as HCN2 WT, which indicates E457R preserved the CNB fold's autoinhibition. For the HCN4 TM substitution alone, an HCN2 R591E background exhibited the same positive VmDEACT-shift phenotype as an HCN2 WT background. This indicates that the open-state stabilization by HCN4 TM substitution works by a different mechanism than relief of CNB-mediated autoinhibition. But when HCN4 TM substitution was performed in HCN2 E457R R591E, the result was an even greater positive VmDEACT-shift, combined with a marked slowing of deactivation kinetics. Our findings uncover a key modulatory interaction between the C-linker and TM regions. When cAMP is bound, the potentiating mechanism involved in HCN4 TM substitution is vulnerable to disruption by introduction of Arg at in C-linker position 457. With no ligand, the C-linker is re-oriented so that Arg 457 now amplifies the potentiating mechanism involved in HCN4 TM substitution. Research supported by Discovery Grant from Natural Sciences & Engineering Research Council (NSERC) of Canada.