The natural compound TMYX reduces SAN cells rate by antagonizing the cAMP modulation of f-channels

Tongmai Yangxin (TMYX), is a complex compound of a Traditional Chinese Medicine (TCM) used to treat several cardiac rhythm disorders; however, no information regarding its mechanism of action is available. In this study we provide a detailed characterization of the effects of TMYX on the electrical activity of pacemaker cells and unravel its mechanism of action. Single-cell electrophysiology revealed that TMYX elicits a reversible and dose-dependent (2/6 mg/ml) slowing of spontaneous action potentials rate (-20.8/-50.2%) by a selective reduction of the diastolic phase (-50.1/-76.0%). This action is mediated by a negative shift of the If activation curve (-6.7/-11.9 mV) and is caused by a reduction of the cAMP-induced stimulation of pacemaker channels. We provide evidence that TMYX acts by directly antagonizes the cAMP-induced allosteric modulation of the pacemaker channels. Noticeably, this mechanism functionally resembles the pharmacological actions of muscarinic stimulation or β-blockers, but it does not require generalized changes in cytoplasmic cAMP levels thus ensuring a selective action on rate. In agreement with a competitive inhibition mechanism, TMYX exerts its maximal antagonistic action at submaximal cAMP concentrations and then progressively becomes less effective thus ensuring a full contribution of If to pacemaker rate during high metabolic demand and sympathetic stimulation.

Identification of novel pulmonary vein nodes as generators of ectopic arrhythmic foci for atrial fibrillation: An immuno-histochemical proof

Introduction: The atrial muscle sleeve (AMS) of the pulmonary vein is the most common source of the arrhythmogenic triggers in atrial fibrillation (AF). Anatomical substrate generating these ectopic currents is still elusive. The present study was designed to study the AMS of pulmonary veins with an emphasis on the structural basis which might govern AF initiation and perpetuation. Materials and Methods: The study was conducted on longitudinal tissue section of pulmonary vein, taken from 15 human cadaveric non-diseased hearts. Tissue was studied histologically using H&E and Gomori trichrome stain. The pacemaker channels were identified by immunohistochemistry using monoclonal HCN4 and HCN1 antibodies. Results: The AMS was identified in each pulmonary vein, located between the tunica adventitia and tunica media. A node like arrangement of myocytes was seen within the AMS in 30% of veins. It had a compact zone limited by a fibrous capsule and contained much smaller, paler and interconnected myocytes. Outside the capsule there was a zone of dispersed, singly placed myocytes separating the compact zone from the working myocytes of the AMS. HCN4 and HCN1 antibodies were expressed on the cell membrane of nodal myocytes, while the working myocytes demonstrated none to minimal staining. Conclusion: Pulmonary veins nodes are similar to the specialized cardiac conductive tissue in, histological arrangement of compact and transitional zones, cellular characteristics, and the presence of pacemaker channels. They might be the anatomical basis of ectopic arrhythmogenic foci. To our knowledge these nodes are being described for the first time in human.

Prolyl isomerization controls activation kinetics of a cyclic nucleotide-gated ion channel

SthK, a cyclic nucleotide-modulated ion channel from Spirochaeta thermophila, activates slowly upon cAMP increase. This is reminiscent of the slow, cAMP-induced activation reported for the hyperpolarization-activated and cyclic nucleotide-gated channel HCN2 in the family of so-called pacemaker channels. Here, we investigate slow cAMP-induced activation in purified SthK channels using stopped-flow assays, mutagenesis, enzymatic catalysis and inhibition assays revealing that the cis/trans conformation of a conserved proline in the cyclic nucleotide-binding domain determines the activation kinetics of SthK. We propose that SthK exists in two forms: trans Pro300 SthK with high ligand binding affinity and fast activation, and cis Pro300 SthK with low affinity and slow activation. Following channel activation, the cis/trans equilibrium, catalyzed by prolyl isomerases, is shifted towards trans, while steady-state channel activity is unaffected. Our results reveal prolyl isomerization as a regulatory mechanism for SthK, and potentially eukaryotic HCN channels. This mechanism could contribute to electrical rhythmicity in cells.

A family of hyperpolarization-activated channels selective for protons

Proton (H+) channels are special: They select protons against other ions that are up to a millionfold more abundant. Only a few proton channels have been identified so far. Here, we identify a family of voltage-gated “pacemaker” channels, HCNL1, that are exquisitely selective for protons. HCNL1 activates during hyperpolarization and conducts protons into the cytosol. Surprisingly, protons permeate through the channel’s voltage-sensing domain, whereas the pore domain is nonfunctional. Key to proton permeation is a methionine residue that interrupts the series of regularly spaced arginine residues in the S4 voltage sensor. HCNL1 forms a tetramer and thus contains four proton pores. Unlike classic HCN channels, HCNL1 is not gated by cyclic nucleotides. The channel is present in zebrafish sperm and carries a proton inward current that acidifies the cytosol. Our results suggest that protons rather than cyclic nucleotides serve as cellular messengers in zebrafish sperm. Through small modifications in two key functional domains, HCNL1 evolutionarily adapted to a low-Na+freshwater environment to conserve sperm’s ability to depolarize.