title: Inhibition of cardiac pacemaker channel hHCN2 depends on intercalation of lipopolysaccharide into channel-containing membrane microdomains.
abstract: Depressed heart rate variability in severe inflammatory diseases can be partially explained by the lipopolysaccharide (LPS)-dependent modulation of cardiac pacemaker channels. Recently, we showed that LPS inhibits pacemaker current in sinoatrial node cells and in HEK293 cells expressing cloned pacemaker channels, respectively. The present study was designed to verify whether this inhibition involves LPS-dependent intracellular signalling and to identify structures of LPS responsible for pacemaker current modulation. We examined the effect of LPS on the activity of human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) stably expressed in HEK293 cells. In whole-cell recordings, bath application of LPS decreased pacemaker current (IhHCN2) amplitude. The same protocol had no effect on channel activity in cell-attached patch recordings, in which channels are protected from the LPS-containing bath solution. This demonstrates that LPS must interact directly with or close to the channel protein. After cleavage of LPS into lipid A and the polysaccharide chain, neither of them alone impaired IhHCN2, which suggests that modulation of channel activity critically depends on the integrity of the entire LPS molecule. We furthermore showed that β-cyclodextrin interfered with LPS-dependent channel modulation predominantly via scavenging of lipid A, thereby abrogating the capability of LPS to intercalate into target cell membranes. We conclude that LPS impairs IhHCN2 by a local mechanism that is restricted to the vicinity of the channels. Furthermore, intercalation of lipid A into target cell membranes is a prerequisite for the inhibition that is suggested to depend on the direct interaction of the LPS polysaccharide chain with cardiac pacemaker channels.
authors.affiliation: Julius Bernstein Institut für Physiologie, Martin Luther Universität Halle-Wittenberg, Magdeburger Strasse 6, 06112 Halle (Saale), Germany. udo.kloeckner@medizin.uni-halle.de.
authors.forename: Udo
authors.initials: U
authors.lastname: Klöckner
authors.forename: Uwe
authors.initials: U
authors.lastname: Rueckschloss
authors.forename: Claudia
authors.initials: C
authors.lastname: Grossmann
authors.forename: Saskia
authors.initials: S
authors.lastname: Matzat
authors.forename: Katja
authors.initials: K
authors.lastname: Schumann
authors.forename: Henning
authors.initials: H
authors.lastname: Ebelt
authors.forename: Ursula
authors.initials: U
authors.lastname: Müller-Werdan
authors.forename: Harald
authors.initials: H
authors.lastname: Loppnow
authors.forename: Karl
authors.initials: K
authors.lastname: Werdan
authors.forename: Michael
authors.initials: M
authors.lastname: Gekle
mesh_terms: D002784:Cholesterol / Q000378:metabolism; D055724:Electrophysiological Phenomena; D006031:Glycosylation; D057809:HEK293 Cells; D006339:Heart Rate / Q000502:physiology; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D007364:Intercalating Agents / Q000737:chemistry / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D021962:Membrane Microdomains / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D009102:Multiple Organ Failure / Q000503:physiopathology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015290:Second Messenger Systems; D018805:Sepsis / Q000503:physiopathology; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: C578710:HCN2 protein, human; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007364:Intercalating Agents; D008070:Lipopolysaccharides; D015221:Potassium Channels; D011994:Recombinant Proteins; D047392:beta-Cyclodextrins; D002784:Cholesterol; C031215:betadex