Heterologously-expressed and Liposome-reconstituted Human Transient Receptor Potential Melastatin 4 Channel (TRPM4) is a Functional Tetramer

Abstract

Mutation, irregular expression and sustained activation of the Transient Receptor Potential Channel, type Melastatin 4 (TRPM4), have been linked to various cardiovascular diseases. However, much remains unknown about the structure of this important ion channel. Here, we have purified a heterologously expressed TRPM4-eGFP fusion protein and investigated the oligomeric state of TRPM4-eGFP in detergent micelles using crosslinking, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data indicate that TRPM4 is tetrameric, like other TRP channels studied to date. Furthermore, the functionality of liposome reconstituted TRPM4-eGFP was examined using electrophysiology. Single-channel recordings from TRPM4-eGFP proteoliposomes showed inhibition of the channel using Flufenamic acid, a well-established inhibitor of TRPM4, suggesting that the channels are functional upon reconstitution. Our characterisation of the oligomeric structure of TRPM4 and the ability to reconstitute functional channels in liposomes should facilitate future studies into the structure, function and pharmacology of this therapeutically relevant channel.

Mammalian TRP ion channels are insensitive to membrane stretch

“…Notably, inherent mechanosensitivitywhich would suggest TRP channels to be primary mechanosensorswas never provided for any of these channels. In agreement with our findings, some studies do not support the inherent mechanosensitivity of TRPC6 (Gottlieb et al, 2008), TRPV4 (Servin-Vences et al, 2017) and TRPM4 (Constantine et al, 2016). Given that cells are highly heterogeneous systems, with diverse protein and lipid composition, the cellular environment can be expected to play an essential role in the function of mechanosensitive, i.e.…”

TRPM4 expression is associated with activated B cell subtype and poor survival in diffuse large B cell lymphoma

“…In conclusion, we have identified TRPM4 as a novel protein that confers an inferior clinical outcome in DLBCL patients treated with R‐CHOP and its association with the poor prognostic ABC‐DLBCL subtype. Our results suggest an oncogenic role of TRPM4 in DLBCL and hence the functional relevance of TRPM4 in the disease, as well as TRPM4 inhibition by known inhibitors (glibenclamide, flufenamic acid or 9‐phenanthrol) and its subsequent effects in DLBCL cell survival and migration, warrant future investigation.…”

“…In GEP studies, TRPM4 is among the top up‐regulated genes in CD5‐positive DLBCL, which belong commonly to the ABC‐DLBCL subtype, and TRPM4 mRNA shows significantly higher expression levels in ABC‐DLBCL compared to the GCB‐DLBCL subtype . Moreover, the opportunity to target TRPM4 is represented by the discovery and characterization of small molecules that inhibit TRPM4 activities, including the Food and Drug Administration (FDA)‐approved anti‐diabetic drug glibenclamide, the non‐steroidal anti‐inflammatory compound flufenamic acid and the phenanthrene derivative 9‐phenanthrol …”

“…18,19 In GEP studies, TRPM4 is among the top up-regulated genes in CD5-positive DLBCL, 20 which belong commonly to the ABC-DLBCL subtype, 21 and TRPM4 mRNA shows significantly higher expression levels in ABC-DLBCL compared to the GCB-DLBCL subtype. 22,23 Moreover, the opportunity to target TRPM4 is represented by the discovery and characterization of small molecules that inhibit TRPM4 activities, including the Food and Drug Administration (FDA)-approved anti-diabetic drug glibenclamide, 24 the non-steroidal anti-inflammatory compound flufenamic acid 25 and the phenanthrene derivative 9-phenanthrol. 26 Therefore, in this study we set out to investigate whether TRPM4 could be a rational therapeutic target in DLBCL by profiling TRPM4 protein expression in various non-malignant human tissues (n = 16) and primary DLBCL cases (n = 189) treated with the R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) regimen, and to correlate TRPM4 protein expression with patients' clinicodemographic data and treatment outcomes.…”

Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched

“…The dn/dc values were according to previous reports (Constantine et al, 2016), namely, 0.185 mL/g for proteins, 0.1424 mL/g for A8-35. The UV extinction coefficient for A8-35 is 0.0386 (Constantine et al, 2016). Data were analyzed with Astra6 software.…”