Korean J Physiol Pharmacol.  2023 Jul;27(4):417-426. 10.4196/kjpp.2023.27.4.417.

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

• Hwang EM
• Lee BH
• Byun EH
• Lee S
• Kang D
• Lee DK
• Song MS
• Hong SG

• KMID: 2544143
• DOI: http://doi.org/10.4196/kjpp.2023.27.4.417

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 SHP-1) 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.

Keyword

Hemagglutinin; Protein tyrosine phosphatase, non-receptor type 6 (PTPN6); Trafficking; TRPM4; 9-phenanthrol

Figure

• 

  Fig. 1 Expression of HA-tagged TRPM4 in the intact membrane. (A) Schematic illustration of TRPM4 with a HA epitope (nine amino acids) inserted in the extracellular loop between the fifth (S5) and sixth (S6) transmembrane domains. (B) Western blot of the expression of the HA-tagged TRPM4 channel, TRPM4-HA, in the cell membrane and determination of its PTPN6 dependency. The membrane expression of both TRPM4 and TRPM4-HA were disrupted by silencing PTPN6 (PTPN6 shRNA). The data were obtained from three replicate experiments. (C) Fluorescence confocal microscopy image of cells labeled with anti-HA antibody and Alexa 594. GFP images (left) of cells expressing IRES-GFP vector containing TRPM4-HA, captured at a wavelength of 488 nm. The red fluorescent images (right) were also captured at a wavelength of 488 nm. The top and middle images are of permeabilized cells and the lower images are of non-treated cells with intact membranes. Asterisks indicate significant differences from the control group: *p < 0.05, ***p < 0.001; scale bar represents 10 μm, and all data are shown as means ± SEM. HA, hemagglutinin; TRPM4, transient receptor potential melastatin 4; PTPN6, protein tyrosine phosphatase, non-receptor type 6; WT, wild-type.

• 

  Fig. 2 The channel properties of TRPM4-HA were consistent with those of TRPM4 (WT). (A) A representative sequence from the electrophysiological experiments. Currents were elicited using ramp pulses every 5 sec in the presence of 100 μM Ca2+. A bathing solution containing 9-phenantrol or NMDG was applied following stable basal currents observed for at least 1 min post-desensitization. The figure shows representative tracings obtained in the excised inside-out configuration. (B) The I-V relationships affected by 9-phenanthrol are presented as bar charts. Representative I-V current relationships from cells transfected with GFP only as a control (left, white), WT (middle, gray), and TRPM4-HA (right, dark gray) are shown. The rates of inhibition by 9-phenanthrol are summarized, with error bars on the right. (C) Similar to (B), the I-V relationship and data summaries from the excised inside-out configuration are shown on the right. The recording configurations are represented as cartoons on the left side of each panel. The black and red current traces were recorded immediately before (control) and 3 min after applying 9-phenanthrol, respectively. Asterisks indicate significant differences between the data before and after applying 9-phenanthrol: **p < 0.01, ***p < 0.001. All data are presented as means ± SEM. HA, hemagglutinin; TRPM4, transient receptor potential melastatin 4; NMDG, N-Methyl-D-glucamine; WT, wild-type; NS, not significant.

• 

  Fig. 3 The TRPM4-HA was found to regulate cell proliferation and migration, and its activity was dependent on PTPN6. (A) Representative images of the wound-healing assay (n = 6) with cells overexpressing TRPM4 (WT) and TRPM4-HA, and a summary bar chart is presented in (B). The rate of wound closure 24 h post-scratching was assessed in six replicate experiments. Solid lines indicate images and data from cells with silencing of PTPN6 expression. Statistical analysis revealed significant differences compared with the control, with **p < 0.01, ****p < 0.0001. All data are presented as means ± SEM. HA, hemagglutinin; TRPM4, transient receptor potential melastatin 4; PTPN6, protein tyrosine phosphatase, non-receptor type 6; WT, wild-type.

• 

  Fig. 4 The surface expression of the YF mutants is not independent of PTPN6. (A) Western blot analysis clearly showing that both TRPM4-HA as a control and TRPM4-HA-YF mutants were expressed at similar levels (0.90 ± 0.03–1.16 ± 0.04, n = 3) on the cell surface, compared to the levels in the cytosolic fraction (0.13 ± 0.06–0.20 ± 0.11, n = 3). (B) In repeated analysis under PTPN6 knockdown conditions to interfere with channel trafficking, silencing PTPN6 greatly increased the levels of all TRPM4-HA-YF mutants in the cytosolic fraction (0.35 ± 0.11–0.48 ± 0.10, n = 3). The Y256F (∇) mutant exhibited a degree of resistance to trafficking, although the difference was not statistically significant. The Western blot images are representative of three replicate experiments. Statistical analysis revealed significant differences compared with the control, with *p < 0.05, **p < 0.01. All data are presented as means ± SEM. HA, hemagglutinin; TRPM4, transient receptor potential melastatin 4; PTPN6, protein tyrosine phosphatase, non-receptor type 6.

• 

  Fig. 5 Channel currents in TRPM4-HA-YF mutants were found to be resistant to 9-phenanthrol. (A) The I-V relationships for whole-cell currents of the TRPM4-HA-YF mutants were lower than that of TRPM4-HA, although not statistically significant, except for Y256F (p = 0.003, n = 12). (B) The I-V relationships of the excised inside-out currents in the TRPM4-HA-YF mutants were also evaluated. In isolated membrane patches without cytosol, the currents in cells with the TRPM4-HA-YF mutants were found to be more resistant to inhibition by 9-phenanthrol, with inhibition rates ranging from approximately 25% to half the level of inhibition observed in TRPM4-HA (57.3 ± 2.5%, n = 6). The black and red current traces were recorded immediately before (control) and 3 min after applying 9-phenanthrol, respectively. Bar charts were obtained from currents measured at +100 mV. Asterisks indicate significant differences between data before and after the application of 9-phenanthrol, **p < 0.01, ***p < 0.001. ****p < 0.0001. All data are shown as means ± SEM. HA, hemagglutinin; TRPM4, transient receptor potential melastatin 4.

• 

  Fig. 6 Effects of TRPM4-HA-YF mutation on cell proliferation and migration and the dependence on PTPN6. (A) Representative images of cell proliferation and migration assessed by wound-healing assay (WHA) with naive (N/C), TRPM4-HA, and TRPM4-HA-YF mutant-overexpressing cells. TRPM4-HA-YF mutations resulted in a significant reduction in cell proliferation and migration by approximately 30% or more, compared with the inhibition rate of 10% observed for TRPM4-HA. (B) In replicate experiments, cells transfected with TRPM4-HA with scrambled RNA or with TRPM4-HA-YF mutants and PTPN6 shRNA were subjected to WHA. Under PTPN6 knockdown conditions, wound closure was delayed by approximately 50% in the cases indicated by a solid bar. Wound closure was assessed 24 h post-scratching in replicate experiments (n = 8). Solid lines indicate images and data from PTPN6 knockdown cells. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control. All data are presented as means ± SEM. HA, hemagglutinin; TRPM4, transient receptor potential melastatin 4; PTPN6, protein tyrosine phosphatase, non-receptor type 6; N/C, negative control.

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