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Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice
Published on: October 23, 2016
Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node
1Department of Physiology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.
Insights
Researchers identified HAC4, a gene encoding a hyperpolarization-activated cation channel (If), crucial for heart rhythm. This finding advances understanding of cardiac pacemaking in the sinoatrial node.
Area of Science:
- Cardiology
- Molecular Biology
- Biophysics
Background:
- The sinoatrial (SA) node generates the heart's electrical impulse.
- Hyperpolarization-activated cation channels (If) play a critical role in cardiac pacemaking.
- Previous cloning efforts identified HAC1-3, but the specific If channel in the SA node remained elusive.
Purpose of the Study:
- To clone and characterize the hyperpolarization-activated cation channel (If) responsible for pacemaking in the rabbit SA node.
- To investigate the molecular and electrophysiological properties of the newly identified channel, termed HAC4.
Main Methods:
- Screening of a rabbit SA node cDNA library using a rat brain If cDNA fragment.
- Sequence analysis of the cloned cDNA (HAC4) to determine its structural features.
- Northern blotting to assess HAC4 mRNA expression in rabbit cardiac tissues.
- Whole-cell patch-clamp electrophysiology in COS-7 cells transfected with HAC4 cDNA.
Main Results:
- Cloning of HAC4 cDNA, encoding a 1150-amino acid protein with distinct N- and C-terminal regions.
- HAC4 transmembrane domain showed high homology to mouse If BCNG-3 (96%).
- HAC4 mRNA was predominantly expressed in the SA node.
- Electrophysiological studies revealed that HAC4 forms slowly activating inward currents upon hyperpolarization, modulated by cAMP.
Conclusions:
- HAC4 is a novel hyperpolarization-activated cation channel identified in the rabbit SA node.
- HAC4 possesses electrophysiological properties consistent with the If current essential for cardiac pacemaking.
- This discovery provides a molecular basis for understanding the generation of the heart's electrical rhythm.
Abstract:
We cloned a cDNA (HAC4) that encodes the hyperpolarization-activated cation channel (If or Ih) by screening a rabbit sinoatrial (SA) node cDNA library using a fragment of rat brain If cDNA. HAC4 is composed of 1150 amino acid residues, and its cytoplasmic N- and C-terminal regions are longer than those of HAC1-3. The transmembrane region of HAC4 was most homologous to partially cloned mouse If BCNG-3 (96%), whereas the C-terminal region of HAC4 showed low homology to all HAC family members so far cloned. Northern blotting revealed that HAC4 mRNA was the most highly expressed in the SA node among the rabbit cardiac tissues examined. The electrophysiological properties of HAC4 were examined using the whole cell patch-clamp technique. In COS-7 cells transfected with HAC4 cDNA, hyperpolarizing voltage steps activated slowly developing inward currents. The half-maximal activation was obtained at -87.2 +/- 2.8 mV under control conditions and at -64.4 +/- 2.6 mV in the presence of intracellular 0.3 mM cAMP. The reversal potential was -34.2 +/- 0.9 mV in 140 mM Na+o and 5 mM K+o versus 10 mM Na+i and 145 mM K+i. These results indicate that HAC4 forms If in rabbit heart SA node.
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