title: HCN Channel C-Terminal Region Speeds Activation Rates Independently of Autoinhibition.
abstract: Hyperpolarization- and cyclic nucleotide-activated (HCN) channels contribute to rhythmic oscillations in excitable cells. They possess an intrinsic autoinhibition with a hyperpolarized V 1/2, which can be relieved by cAMP binding to the cyclic nucleotide binding (CNB) fold in the C-terminal region or by deletion of the CNB fold. We questioned whether V 1/2 shifts caused by altering the autoinhibitory CNB fold would be accompanied by parallel changes in activation rates. We used two-electrode voltage clamp on Xenopus oocytes to compare wildtype (WT) HCN2, a constitutively autoinhibited point mutant incapable of cAMP binding (HCN2 R591E), and derivatives with various C-terminal truncations. Activation V 1/2 and deactivation t 1/2 measurements confirmed that a truncated channel lacking the helix αC of the CNB fold (ΔαC) had autoinhibition comparable to HCN2 R591E; however, ΔαC activated approximately two-fold slower than HCN2 R591E over a 60-mV range of hyperpolarizations. A channel with a more drastic truncation deleting the entire CNB fold (ΔCNB) had similar V 1/2 values to HCN2 WT with endogenous cAMP bound, confirming autoinhibition relief, yet it surprisingly activated slower than the autoinhibited HCN2 R591E. Whereas CNB fold truncation slowed down voltage-dependent reaction steps, the voltage-independent closed-open equilibrium subject to autoinhibition in HCN2 was not rate-limiting. Chemically inhibiting formation of the endogenous lipid PIP2 hyperpolarized the V 1/2 of HCN2 WT but did not slow down activation to match ΔCNB rates. Our findings suggest a "quickening conformation" mechanism, requiring a full-length CNB that ensures fast rates for voltage-dependent steps during activation regardless of potentiation by cAMP or PIP2.
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, V5A 1S6, Canada.
authors.forename: Kaylee E A
authors.initials: KE
authors.lastname: Magee
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, V5A 1S6, Canada.
authors.forename: Zarina
authors.initials: Z
authors.lastname: Madden
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, V5A 1S6, Canada. youngec@sfu.ca.
authors.forename: Edgar C
authors.initials: EC
authors.lastname: Young
mesh_terms: D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D051379:Mice; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D014982:Xenopus laevis
chemical_list: D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D008024:Ligands; D000242:Cyclic AMP
keywords: Autoinhibition; HCN channels; Kinetics; PIP2; Two-electrode voltage clamp; Voltage-gating