title: Regulation of gating and rundown of HCN hyperpolarization-activated channels by exogenous and endogenous PIP2.
abstract: The voltage dependence of activation of the HCN hyperpolarization-activated cation channels is shifted in inside-out patches by -40 to -60 mV relative to activation in intact cells, a phenomenon referred to as rundown. Less than 20 mV of this hyperpolarizing shift can be due to the influence of the canonical modulator of HCN channels, cAMP. Here we study the role of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in HCN channel rundown, as hydrolysis of PI(4,5)P(2) by lipid phosphatases is thought to underlie rundown of several other channels. We find that bath application of exogenous PI(4,5)P(2) reverses the effect of rundown, producing a large depolarizing shift in HCN2 activation. A synthetic short chain analogue of PI(4,5)P(2), dioctanoyl phosphatidylinositol 4,5-bisphosphate, shifts the HCN2 activation curve to more positive potentials in a dose-dependent manner. Other dioctanoyl phosphatidylinositides with one or more phosphates on the lipid headgroup also shift activation, although phosphatidylinositol (PI) is ineffective. Several lines of evidence suggest that HCN2 is also regulated by endogenous PI(4,5)P(2): (a) blockade of phosphatases slows the hyperpolarizing shift upon patch excision; (b) application of an antibody that binds and depletes membrane PIP(2) causes a further hyperpolarizing shift in activation; (c) the shift in activation upon patch excision can be partially reversed by MgATP; and (d) the effect of MgATP is blocked by wortmannin, an inhibitor of PI kinases. Finally, recordings from rabbit sinoatrial cells demonstrate that diC(8) PI(4,5)P(2) delays the rundown of native HCN currents. Thus, both native and recombinant HCN channels are regulated by PI(4,5)P(2).
authors.affiliation: Center for Neurobiology and Behavior, Columbia University Medical Center, New York, NY 10032, USA.
authors.forename: Phillip
authors.initials: P
authors.lastname: Pian
authors.forename: Annalisa
authors.initials: A
authors.lastname: Bucchi
authors.forename: Richard B
authors.initials: RB
authors.lastname: Robinson
authors.forename: Steven A
authors.initials: SA
authors.lastname: Siegelbaum
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000242:Cyclic AMP / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005260:Female; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology*; D007473:Ion Channels / Q000235:genetics / Q000502:physiology*; D051379:Mice; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology* / Q000502:physiology*; D015221:Potassium Channels; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011817:Rabbits; D012849:Sinoatrial Node / Q000166:cytology / Q000502:physiology; D000077191:Wortmannin; D014982:Xenopus laevis
chemical_list: D000730:Androstadienes; C578711:Hcn2 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D019269:Phosphatidylinositol 4,5-Diphosphate; D015221:Potassium Channels; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D000077191:Wortmannin