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PKA holoenzyme is functionally coupled to CFTR by AKAPs
P Huang1, K Trotter, R C Boucher
1Departments of Medicine and CF/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA. Pingbo_Huang@med.unc.edu
American Journal of Physiology. Cell Physiology
|February 9, 2000
Summary
A kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) to the cystic fibrosis transmembrane regulator (CFTR). This AKAP-PKA-CFTR complex is crucial for CFTR regulation by physiological stimuli.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- The cystic fibrosis transmembrane regulator (CFTR) is a crucial ion channel.
- Membrane-bound protein kinase A (PKAII) is thought to regulate CFTR.
- A kinase anchoring proteins (AKAPs) are known to localize PKA to specific cellular sites.
Purpose of the Study:
- To investigate the physical and functional association between PKA and CFTR.
- To determine the role of AKAPs in PKA-CFTR complex formation and CFTR regulation.
Main Methods:
- Utilized inside-out membrane patches from Calu-3 cells.
- Applied 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and PKI to assess PKA activity.
- Used HT-31 peptide to disrupt AKAP-PKA interactions.
- Conducted whole-cell voltage-clamp studies with intracellular dialysis.
Main Results:
- Isolated Calu-3 membrane patches contained functional PKA holoenzyme coupled to CFTR.
- CPT-cAMP significantly increased CFTR activity, an effect blocked by PKI.
- HT-31 disrupted PKA-CFTR coupling and prevented CPT-cAMP-mediated CFTR activation in patches.
- HT-31 blocked CFTR activation by extracellular adenosine in whole-cell studies.
Conclusions:
- AKAPs mediate the compartmentalization of PKA holoenzyme with CFTR in Calu-3 cells.
- AKAP-dependent PKA localization is essential for the physiological regulation of CFTR.