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Kv4.2 phosphorylation by cyclic AMP-dependent protein kinase.
A E Anderson1, J P Adams, Y Qian
1Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030, USA. aander@cns.neusc.bcm.tmc.edu
The Journal of Biological Chemistry
|February 22, 2000
Summary
This study identifies specific sites on Kv4.2 potassium channels phosphorylated by cAMP-dependent protein kinase (PKA). These findings reveal key regulatory mechanisms for Kv4.2 channel function in neurons and cardiac cells.
Area of Science:
- Neuroscience
- Molecular Biology
- Cardiology
Background:
- Kv4.2 alpha-subunits form transient potassium channels in hippocampal CA1 neurons and ventricular myocytes.
- The cAMP second messenger cascade modulates this transient current, suggesting a role for protein kinase A (PKA).
Purpose of the Study:
- To investigate whether Kv4.2 alpha-subunits are directly phosphorylated by PKA.
- To identify specific phosphorylation sites on Kv4.2 regulated by PKA.
Main Methods:
- Expression of Kv4.2 intracellular domains as glutathione S-transferase fusion proteins.
- In vitro kinase assays with PKA, phosphopeptide mapping, and amino acid sequencing.
- Generation of phospho-specific antibodies and in situ phosphorylation studies in COS-7 cells and hippocampal CA1.
Main Results:
- Both amino- and carboxyl-terminal Kv4.2 fusion proteins were identified as PKA substrates in vitro.
- Specific PKA phosphorylation sites were mapped to Thr(38) and Ser(552) within the Kv4.2 sequence.
- PKA stimulation increased phosphorylation at Thr(38) and Ser(552) in intact cells and hippocampal CA1.
Conclusions:
- Kv4.2 alpha-subunits are directly phosphorylated by PKA at Thr(38) and Ser(552).
- These sites represent key regulatory points for Kv4.2 channel function in neuronal and cardiac tissues.
- Provides molecular insights into PKA-mediated modulation of Kv4.2 potassium channels.