title: Dominant-negative suppression of HCN channels markedly reduces the native pacemaker current I(f) and undermines spontaneous beating of neonatal cardiomyocytes.
abstract: BACKGROUND
  The pacemaker current I(f) contributes to spontaneous diastolic depolarization of cardiac autonomic cells. In heterologous expression, HCN channels exhibit a hyperpolarization-activated inward current similar to I(f). However, the links between HCN genes and native I(f) are largely inferential, and it remains unknown whether I(f) is essential for cardiac pacing.
  
  
  RESULTS
  To clarify this situation, we generated a GYG(402-404)AYA pore mutation of HCN2, which rendered the channel nonfunctional and suppressed wild-type HCN2 in a dominant-negative manner in Chinese hamster ovary cells. In addition, HCN2-AYA suppressed I(HCN4) in a dominant-negative manner when coexpressed with wild-type HCN4, indicating that the 2 isoforms HCN2 and HCN4 are able to coassemble to form heteromultimeric complexes. Given that HCN2 and HCN4 are the dominant HCN mRNA transcripts in neonatal rat ventricle, we expressed HCN2-AYA in neonatal cardiocytes using adenoviral gene transfer to test the effect of HCN suppression on native I(f). I(f) density was indeed reduced markedly, from 7.8+/-1.6 pA/pF (n=13) in control cells to 0.3+/-0.2 pA/pF (n=11) in HCN2-AYA-infected cells when measured at -130 mV (P<0.001). To probe the effect of HCN on cardiac pacing, we infected spontaneously beating neonatal monolayers with adenoviral vectors expressing wild-type and mutant HCN channels. Infection with HCN2 and HCN4 accelerated the beating rate significantly, to 230.5+/-8.6 bpm (n=12) and 223.5+/-12.3 bpm (n=10), respectively, compared with control cultures (83.4+/-4.5 bpm, n=13, P<0.001). Conversely, HCN2-AYA completely undermined spontaneous pacing of neonatal cardiocytes.
  
  
  CONCLUSIONS
  HCN channels are the major molecular component of native I(f) and are critical for spontaneous beating of neonatal cardiomyocytes.
authors.affiliation: Department of Medicine III, University of Cologne, Germany.
authors.forename: Fikret
authors.initials: F
authors.lastname: Er
authors.forename: Robert
authors.initials: R
authors.lastname: Larbig
authors.forename: Andreas
authors.initials: A
authors.lastname: Ludwig
authors.forename: Martin
authors.initials: M
authors.lastname: Biel
authors.forename: Franz
authors.initials: F
authors.lastname: Hofmann
authors.forename: Dirk J
authors.initials: DJ
authors.lastname: Beuckelmann
authors.forename: Uta C
authors.initials: UC
authors.lastname: Hoppe
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D001683:Biological Clocks; D016466:CHO Cells; D006224:Cricetinae; D054815:Cyclic Nucleotide-Gated Cation Channels; D004553:Electric Conductivity; D005822:Genetic Vectors; D006321:Heart / Q000502:physiology*; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000502:physiology; D009124:Muscle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000502:physiology; D009154:Mutation; D009200:Myocardial Contraction; D018408:Patch-Clamp Techniques; D015221:Potassium Channels; D051381:Rats
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; C578710:HCN2 protein, human; C119587:HCN4 protein, human; C578712:Hcn2 protein, rat; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D009124:Muscle Proteins; D015221:Potassium Channels