Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Lentiviral Transduction
2.3. Immunoblot and Antibodies
2.4. Membrane Preparations and CLCA2 Cleavage Experiments
2.5. Co-Transfections and Co-Immunoprecipitation Analysis
2.6. DNA Sequencing
2.7. CLCA2 E165Q Mutation and Δ35 Mutation
2.8. SOCE Measurements
2.9. Statistics
3. Results
3.1. CLCA2 Cleavage Is Zinc- and pH-Dependent
3.2. A point Mutation in the CLCA-N Domain Abolishes CLCA2 Precursor Cleavage
3.3. Concentration Dependence Suggests Trans Cleavage
3.4. Cleavage of E165Q Mutant in Trans by Wildtype CLCA2
3.5. CLCA2 Precursors Interact to Form a Stable Complex
3.6. E165Q Mutation Does Not Affect the Ability to Form a Stable Complex
3.7. Autocleavage by Immunopurified CLCA2 Requires Denaturation/Renaturation
3.8. CLCA2 Ectodomain Undergoes Conformational Shift upon Cleavage
3.9. E165Q Abolishes SOCE Stimulation by CLCA2
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Ramena, G.T.; Sharma, A.; Chang, Y.; Pan, Z.; Elble, R.C. Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry. Biomedicines 2023, 11, 2915. https://doi.org/10.3390/biomedicines11112915
Ramena GT, Sharma A, Chang Y, Pan Z, Elble RC. Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry. Biomedicines. 2023; 11(11):2915. https://doi.org/10.3390/biomedicines11112915
Chicago/Turabian StyleRamena, Grace T., Aarushi Sharma, Yan Chang, Zui Pan, and Randolph C. Elble. 2023. "Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry" Biomedicines 11, no. 11: 2915. https://doi.org/10.3390/biomedicines11112915