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Calmodulin-binding autoinhibitory domain controls "pH-sensing" in the Na+/H+ exchanger NHE1 through sequence-specific
S Wakabayashi1, T Ikeda, T Iwamoto
1Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565, Japan. wak@ri.ncvc.go.jp
Biochemistry
|October 23, 1997
Summary
The calmodulin-binding domain of Na+/H+ exchanger NHE1 inhibits its function. Replacing this domain with segments from NHE2 or NHE4 revealed specific sequence requirements for autoinhibition and Ca2+-dependent activation in the NHE1 protein.
Area of Science:
- Molecular Biology
- Cell Physiology
- Biochemistry
Background:
- The Na+/H+ exchanger 1 (NHE1) plays a crucial role in regulating intracellular pH.
- Its calmodulin (CaM)-binding domain exerts autoinhibition, reducing affinity for intracellular H+ in quiescent cells.
Purpose of the Study:
- To investigate the role of the autoinhibitory domain in NHE1 function.
- To determine the sequence specificity and interaction requirements for CaM-mediated regulation of NHE1.
Main Methods:
- Chimeric Na+/H+ exchanger constructs were created by replacing the NHE1 CaM-binding domain with homologous segments from NHE2 and NHE4, or other CaM-binding regions.
- These constructs were expressed in exchanger-deficient PS120 cells.
- Intracellular pH (pHi) dependence of 22Na+ uptake was measured following stimulation with Ca2+-mobilizing agents; CaM binding affinities (Kds) were determined; crosslinking assays were performed.
Main Results:
- NHE1 incorporating the NHE2 segment exhibited Ca2+-dependent activation, similar to native NHE2.
- NHE1 with NHE4 or other CaM-binding segments showed constitutive activation and lost Ca2+-dependent regulation.
- Specific residues (Leu639, Lys651, Tyr652) conserved in NHE1 and NHE2 segments were identified as critical for autoinhibition.
Conclusions:
- The autoinhibitory function of the NHE1 CaM-binding domain is highly sequence-specific.
- The NHE4 segment and other tested CaM-binding regions cannot functionally replace the autoinhibitory domain in NHE1.
- The inhibitory domain likely interacts with specific sites on NHE1 to regulate H+ affinity.