title: 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.
authors.affiliation: Victor Chang Cardiac Research Institute, Lowy Packer Building, NSW 2010.|St Vincent's Clinical School, University of New South Wales, Sydney, NSW 2010, Australia.
authors.forename: Maryrose
authors.initials: M
authors.lastname: Constantine
authors.affiliation: Victor Chang Cardiac Research Institute, Lowy Packer Building, NSW 2010.
authors.forename: Chu Kong
authors.initials: CK
authors.lastname: Liew
authors.affiliation: School of Medical Sciences, The Bosch Institute, The University of Sydney, NSW 2006.
authors.forename: Victor
authors.initials: V
authors.lastname: Lo
authors.affiliation: Biomedical Imaging Facility, Lowy Cancer Research Centre, The University of New South Wales, Kensington, NSW 2052, Australia.
authors.forename: Alex
authors.initials: A
authors.lastname: Macmillan
authors.affiliation: School of Life Sciences, University of Technology Sydney.
authors.forename: Charles G
authors.initials: CG
authors.lastname: Cranfield
authors.affiliation: School of Medical Sciences, The Bosch Institute, The University of Sydney, NSW 2006.
authors.forename: Margaret
authors.initials: M
authors.lastname: Sunde
authors.affiliation: Biomedical Imaging Facility, Lowy Cancer Research Centre, The University of New South Wales, Kensington, NSW 2052, Australia.
authors.forename: Renee
authors.initials: R
authors.lastname: Whan
authors.affiliation: Victor Chang Cardiac Research Institute, Lowy Packer Building, NSW 2010.|St Vincent's Clinical School, University of New South Wales, Sydney, NSW 2010, Australia.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Graham
authors.affiliation: Victor Chang Cardiac Research Institute, Lowy Packer Building, NSW 2010.|St Vincent's Clinical School, University of New South Wales, Sydney, NSW 2010, Australia.
authors.forename: Boris
authors.initials: B
authors.lastname: Martinac
mesh_terms: D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D008081:Liposomes / Q000737:chemistry; D057054:Molecular Imaging; D055503:Protein Multimerization; D021381:Protein Transport; D059748:Proteolysis; D011993:Recombinant Fusion Proteins; D050053:TRPM Cation Channels / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D008081:Liposomes; D011993:Recombinant Fusion Proteins; D050053:TRPM Cation Channels; C437888:TRPM4 protein, human; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins