Abstract 13045: Reduced Tyrosine Phosphorylation Inhibits HCN4-573x Channel Independent of cAMP Signaling
Recently, a mutated cardiac pacemaker channel, HCN4-573x, was identified in a patient with sinus bradycardia (41 beats per minute) and intermittent atrial fibrillation. Lacking the cyclic nucleotide binding domain (CNBD), HCN4-573x is irresponsive to cAMP and acts in a dominant negative fashion in transiently transfected cells in vitro and in a knock-in mice model. We tested a new strategy to modulate HCN4-573x channel activity by altering its tyrosine phosphorylation state independent of cAMP signaling pathway. We found that reduced tyrosine phosphorylation of HCN4 and HCN4-573x by a type IV receptor protein tyrosine phosphatase (RPTP) epsilon is directly associated with the decreased whole cell current density (HCN4: 105±27pA/pF, n=8, HCN4+RPTP epsilon: 12±3pA/pF, n=21, p<0.0001; HCN4-573x: 82±17pA/pF, n=9, HCN4-573x+RPTP epsilon: 2±1pA/pF, n=18, p<0.0001). To mimic the heterogeneous condition of the patient, we studied effects of PP2 (a selective inhibitor of Src kinases) on a dual-expression plasmid containing both the wild-type HCN4 and the 573x mutant, HCN4/HCN4-573x. On average of six cells, PP2 at 10μM for 5-8 minutes of perfusion, slowed its activation kinetics by 77±8% at -125mV; prolonged perfusion time (15 minutes) slowed activation kinetics by over 3-fold. PP2 also significantly inhibited the current density by 23.6±5.5% at -125mV, shifted its threshold activation to a more negative potential by 20mV, and shifted the midpoint of activation curve to a more negative potential by 11.6±1.8 mV. The effects of PP2 on these properties were reversible and similar to those on wild-type HCN4, indicating that CNBD is not required for tyrosine phosphorylation modulation of the HCN4 channel. In conclusion, our data has provided a molecular strategy that increased tyrosine phosphorylation of HCN4/HCN4-573x, the cAMP-insensitive heterogeneous mutant, can potentially enhance the channel activity, thereby increasing the amount of time-dependent inward current available at the early diastolic depolarization of cardiac action potential, leading to the increased heart rate.
- © 2011 by American Heart Association, Inc.