title: Intracellular Mg2+ is a voltage-dependent pore blocker of HCN channels.
abstract: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization that creates time-dependent, inward rectifying currents, gated by the movement of the intrinsic voltage sensor S4. However, inward rectification of the HCN currents is not only observed in the time-dependent HCN currents, but also in the instantaneous HCN tail currents. Inward rectification can also be seen in mutant HCN channels that have mainly time-independent currents (5). In the present study, we show that intracellular Mg(2+) functions as a voltage-dependent blocker of HCN channels, acting to reduce the outward currents. The affinity of HCN channels for Mg(2+) is in the physiological range, with Mg(2+) binding with an IC(50) of 0.53 mM in HCN2 channels and 0.82 mM in HCN1 channels at +50 mV. The effective electrical distance for the Mg(2+) binding site was found to be 0.19 for HCN1 channels, suggesting that the binding site is in the pore. Removing a cysteine in the selectivity filter of HCN1 channels reduced the affinity for Mg(2+), suggesting that this residue forms part of the binding site deep within the pore. Our results suggest that Mg(2+) acts as a voltage-dependent pore blocker and, therefore, reduces outward currents through HCN channels. The pore-blocking action of Mg(2+) may play an important physiological role, especially for the slowly gating HCN2 and HCN4 channels. Mg(2+) could potentially block outward hyperpolarizing HCN currents at the plateau of action potentials, thus preventing a premature termination of the action potential.
authors.affiliation: Neurological Sciences Institute, Oregon Health & Science Univ., 505 NW 185th Ave., Beaverton, OR 97006, USA.
authors.forename: Sriharsha
authors.initials: S
authors.lastname: Vemana
authors.forename: Shilpi
authors.initials: S
authors.lastname: Pandey
authors.forename: H Peter
authors.initials: HP
authors.lastname: Larsson
mesh_terms: D000818:Animals; D001665:Binding Sites; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D004594:Electrophysiology; D005260:Female; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D042541:Intracellular Space / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology*; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology; D013997:Time Factors; D014982:Xenopus laevis
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; C578666:Hcn1 protein, mouse; C578711:Hcn2 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D015221:Potassium Channels; D013096:Spermine; D004492:Edetic Acid; D008274:Magnesium; D013095:Spermidine