Constitutively active Src tyrosine kinase changes gating of HCN4 channels through direct binding to the channel...

Area of Science:

• Cardiology
• Molecular Biology
• Biophysics

Background:

• Cardiac pacemaker current (If) is crucial for heart rhythm.
• Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate If.
• Tyrosine phosphorylation is known to modulate HCN channel properties.

Purpose of the Study:

• To investigate the role of intracellular tyrosine kinase Src in modulating If via tyrosine phosphorylation.
• To determine if Src interacts with and alters HCN4 channel function.

Main Methods:

• HEK293 cells were cotransfected with HCN4 and constitutively active or dominant-negative Src proteins.
• Voltage-dependent activation and activation kinetics of HCN4 channels were measured.
• Coimmunoprecipitation assays were used to detect Src-HCN4 complex formation.

Main Results:

• Constitutively active Src (Src529) positively shifted HCN4 activation (V1/2 from -93 mV to -80 mV) and accelerated kinetics.
• Dominant-negative Src (Src296) slightly and insignificantly shifted HCN4 activation more negative and slowed kinetics.
• Src529 and Src296 did not alter HCN4 current density.
• Coimmunoprecipitation confirmed Src forms a complex with HCN4 in cells and myocytes.

Conclusions:

• Src tyrosine kinase regulates HCN4 channel function and cardiac pacemaker current (If).
• Src modulates HCN4 channel gating properties through tyrosine phosphorylation.
• This study reveals a novel mechanism for If regulation involving Src-mediated phosphorylation of HCN channels.