PTPN6 regulates the cell-surface expression of TRPM4 channels in HEK293 cells

Dong Kun Lee, Jung Yeon Park, Jae Cheal Yoo, Eun Hye Byun, Yeon Ju Bae, Young Sun Lee, Nammi Park, Dawon Kang, Jaehee Han, Jae-Yong Park, Eunmi Hwang, Seong Geun Hong

Research output: Contribution to journalArticle

Abstract

Transient receptor-potential, cation channel, subfamily M, member 4 (TRPM4) channels regulate a variety of physiological and pathological processes; however, their roles as functional channels under diverse conditions remain unclear. In this study, cytosolic protein tyrosine phosphatase non-receptor type 6 (PTPN6) interacted with TRPM4 channels. We confirmed their interaction by performing co-immunoprecipitation (Co-IP) assays following heterologous PTPN6 and TRPM4 channel expression in HEK293 cells. Furthermore, biomolecular fluorescence complementation (BiFC) image analysis confirmed TRPM4–PTPN6 binding. In addition, immunoblotting and Co-IP analyses revealed that TRPM4 expression significantly decreased in the membrane fraction of cells after PTPN6 was silenced with a specific short-hairpin RNA (shRNA-PTPN6). In agreement, TRPM4-induced changes in whole-cell currents were not detected in PTPN6-silenced HEK cells, in contrast to cells transfected with a scrambled RNA (scRNA) or in naïve HEK cells. These data suggest that PTPN6 inhibits TRPM4 channel activity by disrupting TRPM4 expression. Furthermore, TRPM4 channels were expressed in the membrane of naïve cells and scRNA transfectants, but not in those of PTPN6-silenced cells. These results indicated that PTPN6 is critically associated with TRPM4 trafficking. This role of PTPN6 in TRPM4 membrane localization was also demonstrated in HeLa cells. TRPM4 overexpression significantly enhanced cell proliferation in untreated HeLa cells, but not in HeLa cells with silenced PTPN6 expression. These findings indicate that PTPN6-dependent TRPM4 expression and trafficking to the plasma membrane is critical for cell proliferation in both HEK293 and HeLa cells. Therefore, PTPN6 is a novel therapeutic target for treating pathologic diseases involving TRPM4.

Original languageEnglish
Pages (from-to)1449-1458
Number of pages10
JournalPflugers Archiv European Journal of Physiology
Volume470
Issue number10
DOIs
Publication statusPublished - 2018 Oct 1

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Non-Receptor Type 6 Protein Tyrosine Phosphatase
HEK293 Cells
HeLa Cells
Cell Membrane
Cell proliferation
Membranes
Immunoprecipitation
Small Interfering RNA
Cell Proliferation
RNA
Physiological Phenomena
Transient Receptor Potential Channels
Pathologic Processes
Cell membranes
Immunoblotting
Image analysis

Keywords

  • Membrane trafficking
  • PTPN6
  • RTK
  • TRPM4
  • Tyrosine phosphorylation

ASJC Scopus subject areas

  • Physiology
  • Clinical Biochemistry
  • Physiology (medical)

Cite this

Lee, D. K., Park, J. Y., Yoo, J. C., Byun, E. H., Bae, Y. J., Lee, Y. S., ... Hong, S. G. (2018). PTPN6 regulates the cell-surface expression of TRPM4 channels in HEK293 cells. Pflugers Archiv European Journal of Physiology, 470(10), 1449-1458. https://doi.org/10.1007/s00424-018-2161-9

PTPN6 regulates the cell-surface expression of TRPM4 channels in HEK293 cells. / Lee, Dong Kun; Park, Jung Yeon; Yoo, Jae Cheal; Byun, Eun Hye; Bae, Yeon Ju; Lee, Young Sun; Park, Nammi; Kang, Dawon; Han, Jaehee; Park, Jae-Yong; Hwang, Eunmi; Hong, Seong Geun.

In: Pflugers Archiv European Journal of Physiology, Vol. 470, No. 10, 01.10.2018, p. 1449-1458.

Research output: Contribution to journalArticle

Lee, DK, Park, JY, Yoo, JC, Byun, EH, Bae, YJ, Lee, YS, Park, N, Kang, D, Han, J, Park, J-Y, Hwang, E & Hong, SG 2018, 'PTPN6 regulates the cell-surface expression of TRPM4 channels in HEK293 cells', Pflugers Archiv European Journal of Physiology, vol. 470, no. 10, pp. 1449-1458. https://doi.org/10.1007/s00424-018-2161-9
Lee, Dong Kun ; Park, Jung Yeon ; Yoo, Jae Cheal ; Byun, Eun Hye ; Bae, Yeon Ju ; Lee, Young Sun ; Park, Nammi ; Kang, Dawon ; Han, Jaehee ; Park, Jae-Yong ; Hwang, Eunmi ; Hong, Seong Geun. / PTPN6 regulates the cell-surface expression of TRPM4 channels in HEK293 cells. In: Pflugers Archiv European Journal of Physiology. 2018 ; Vol. 470, No. 10. pp. 1449-1458.
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abstract = "Transient receptor-potential, cation channel, subfamily M, member 4 (TRPM4) channels regulate a variety of physiological and pathological processes; however, their roles as functional channels under diverse conditions remain unclear. In this study, cytosolic protein tyrosine phosphatase non-receptor type 6 (PTPN6) interacted with TRPM4 channels. We confirmed their interaction by performing co-immunoprecipitation (Co-IP) assays following heterologous PTPN6 and TRPM4 channel expression in HEK293 cells. Furthermore, biomolecular fluorescence complementation (BiFC) image analysis confirmed TRPM4–PTPN6 binding. In addition, immunoblotting and Co-IP analyses revealed that TRPM4 expression significantly decreased in the membrane fraction of cells after PTPN6 was silenced with a specific short-hairpin RNA (shRNA-PTPN6). In agreement, TRPM4-induced changes in whole-cell currents were not detected in PTPN6-silenced HEK cells, in contrast to cells transfected with a scrambled RNA (scRNA) or in na{\"i}ve HEK cells. These data suggest that PTPN6 inhibits TRPM4 channel activity by disrupting TRPM4 expression. Furthermore, TRPM4 channels were expressed in the membrane of na{\"i}ve cells and scRNA transfectants, but not in those of PTPN6-silenced cells. These results indicated that PTPN6 is critically associated with TRPM4 trafficking. This role of PTPN6 in TRPM4 membrane localization was also demonstrated in HeLa cells. TRPM4 overexpression significantly enhanced cell proliferation in untreated HeLa cells, but not in HeLa cells with silenced PTPN6 expression. These findings indicate that PTPN6-dependent TRPM4 expression and trafficking to the plasma membrane is critical for cell proliferation in both HEK293 and HeLa cells. Therefore, PTPN6 is a novel therapeutic target for treating pathologic diseases involving TRPM4.",
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AU - Byun, Eun Hye

AU - Bae, Yeon Ju

AU - Lee, Young Sun

AU - Park, Nammi

AU - Kang, Dawon

AU - Han, Jaehee

AU - Park, Jae-Yong

AU - Hwang, Eunmi

AU - Hong, Seong Geun

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