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

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.

TRPM4 in Cancer—A New Potential Drug Target

“…Several interaction partners of TRPM4 have been reported, such as sulfonylurea receptor (SUR1) [ 29 , 95 , 96 ], 14-3-3 [ 97 ], potassium channel tetramerization domain-containing protein 5 (KCTD5) [ 39 ], small ubiquitin-related modifier (SUMO) [ 16 , 17 ], TRPC3 [ 98 ], protein tyrosine phosphatase non-receptor type 6 (PTPN6) [ 99 ], and subunits Glu2A and Glu2B of the N-methyl-D-aspartate receptor (NMDA) receptor [ 100 ]. KCTD5 has been shown to correlate with particular classes of breast cancer [ 39 ].…”

Machine Learning for Prediction of Drug Targets in Microbe Associated Cardiovascular Diseases by Incorporating Host‐pathogen Interaction Network Parameters

“…PTPN6 has been identified to play a critical role in a network study for carotid atheroma. It also regulates the cell‐surface expression of Transient receptor‐potential, cation channel, subfamily M, member 4 (TRPM4) channels [61] which are involved in the development of cardiac ischemia‐reperfusion injury causing myocardial infarction [62] . NOTCH1 in previous studies has been found to play a critical role in cardiac cell differentiation [63] and it has been suggested that the maintenance or reactivation of Notch signaling in cardiac cells can be a therapeutic target to protect against myocardial damage [64] .…”

“…In this study, SMURF2 is an important predicted host drug target, targeting which has already been proposed as a novel therapeutic approach for increasing the antitumor efficacy of MEK inhibitors, in a previous study. [80] Similarly PTPN6 (predicted host drug target) has also been proposed as novel therapeutic target for treating pathologic diseases involving TRPM4 [81] eg. multiple sclerosis.…”

Monitoring trafficking and expression of hemagglutinin-tagged transient receptor potential melastatin 4 channel in mammalian cells

“…The cytosolic PTPN6, interacts with and dephosphorylates TRPM4, thereby increasing the activity of TRPM4 [ 23 ]. To confirm whether PTPN6 is required for activation of TRPM4, pSicoR-PTPN6-shRNA was generated using oligonucleotide-directed mutagenesis (Enzynomics).…”

“…Epitope tagging of TRPM4 is a useful strategy for studying its channel trafficking and expression. While previous studies have inserted an epitope at either the amino (N) or carboxy (C) terminus of TRPM4, this study suggests that the N-terminus is a more promising site for epitope insertion, as attempts to tag the C-terminus have been unsuccessful for other proteins like protein tyrosine phosphatase, non-receptor type 6 (PTPN6) [ 23 ]. To address this issue, we designed a mutant by inserting an epitope motif between the fifth (S5) and sixth (S6) transmembrane domains, allowing it to protrude from the cell membrane [ 24 , 25 ].…”