The Korean Journal of Physiology and Pharmacology

The Korean Society of Pharmacology (대한약리학회)
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Monitoring trafficking and expression of hemagglutinin-tagged transient receptor potential melastatin 4 channel in mammalian cells

  • Eun Mi Hwang (Brain Science Institute, Korea Institute of Science and Technology (KIST)) ;
  • Bo Hyun Lee (Department of Physiology, College of Medicine, Gyeongsang National University) ;
  • Eun Hye Byun (Department of Physiology, College of Medicine, Gyeongsang National University) ;
  • Soomin Lee (Brain Science Institute, Korea Institute of Science and Technology (KIST)) ;
  • Dawon Kang (Department of Physiology, College of Medicine, Gyeongsang National University) ;
  • Dong Kun Lee (Department of Physiology, College of Medicine, Gyeongsang National University) ;
  • Min Seok Song (Department of Physiology, College of Medicine, Gyeongsang National University) ;
  • Seong-Geun Hong (Department of Physiology, College of Medicine, Gyeongsang National University)
  • Received : 2023.05.22
  • Accepted : 2023.06.05
  • Published : 2023.07.01
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Abstract

The TRPM4 gene encodes a Ca2+-activated monovalent cation channel called transient receptor potential melastatin 4 (TRPM4) that is expressed in various tissues. Dysregulation or abnormal expression of TRPM4 has been linked to a range of diseases. We introduced the hemagglutinin (HA) tag into the extracellular S6 loop of TRPM4, resulting in an HA-tagged version called TRPM4-HA. This TRPM4-HA was developed to investigate the purification, localization, and function of TRPM4 in different physiological and pathological conditions. TRPM4-HA was successfully expressed in the intact cell membrane and exhibited similar electrophysiological properties, such as the current-voltage relationship, rapid desensitization, and current size, compared to the wild-type TRPM4. The presence of the TRPM4 inhibitor 9-phenanthrol did not affect these properties. Furthermore, a wound-healing assay showed that TRPM4-HA induced cell proliferation and migration, similar to the native TRPM4. Co-expression of protein tyrosine phosphatase, non-receptor type 6 (PTPN6 or SHP1) with TRPM4-HA led to the translocation of TRPM4-HA to the cytosol. To investigate the interaction between PTPN6 and tyrosine residues of TRPM4 in enhancing channel activity, we generated four mutants in which tyrosine (Y) residues were substituted with phenylalanine (F) at the N-terminus of TRPM4. The YF mutants displayed properties and functions similar to TRPM4-HA, except for the Y256F mutant, which showed resistance to 9-phenanthrol, suggesting that Y256 may be involved in the binding site for 9-phenanthrol. Overall, the creation of HA-tagged TRPM4 provides researchers with a valuable tool to study the role of TRPM4 in different conditions and its potential interactions with other proteins, such as PTPN6.

Keywords

Acknowledgement

We thank Professor Jae Yong Park (Korea University college of Health Science) for technical advices on BiFC analysis.

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