title: Oligomeric state of purified transient receptor potential melastatin-1 (TRPM1), a protein essential for dim light vision.
abstract: Transient receptor potential melastatin-1 (TRPM1) is essential for the light-induced depolarization of retinal ON bipolar cells. TRPM1 likely forms a multimeric channel complex, although almost nothing is known about the structure or subunit composition of channels formed by TRPM1 or any of its close relatives. Recombinant TRPM1 was robustly expressed in insect cells, but only a small fraction was localized to the plasma membrane. Similar intracellular localization was observed when TRPM1 was heterologously expressed in mammalian cells. TRPM1 was affinity-purified from Sf9 cells and complexed with amphipol, followed by detergent removal. In blue native gels and size exclusion chromatography, TRPM1 migrated with a mobility consistent with detergent- or amphipol-bound dimers. Cross-linking experiments were also consistent with a dimeric subunit stoichiometry, and cryoelectron microscopy and single particle analysis without symmetry imposition yielded a model with approximate 2-fold symmetrical features. Finally, electron microscopy of TRPM1-antibody complexes revealed a large particle that can accommodate TRPM1 and two antibody molecules. Taken together, these data indicate that purified TRPM1 is mostly dimeric. The three-dimensional structure of TRPM1 dimers is characterized by a small putative transmembrane domain and a larger domain with a hollow cavity. Blue native gels of solubilized mouse retina indicate that TRPM1 is present in two distinct complexes: one similar in size to the recombinant protein and one much larger. Because dimers are likely not functional ion channels, these results suggest that additional partner subunits participate in forming the transduction channel required for dim light vision and the ON pathway.
authors.affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: Melina A
authors.initials: MA
authors.lastname: Agosto
authors.affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: Zhixian
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: Feng
authors.initials: F
authors.lastname: He
authors.affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: Ivan A
authors.initials: IA
authors.lastname: Anastassov
authors.affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: Sara J
authors.initials: SJ
authors.lastname: Wright
authors.affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: Jennifer
authors.initials: J
authors.lastname: McGehee
authors.affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030. Electronic address: twensel@bcm.edu.
authors.forename: Theodore G
authors.initials: TG
authors.lastname: Wensel
mesh_terms: D000818:Animals; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D055503:Protein Multimerization / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D050053:TRPM Cation Channels / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014785:Vision, Ocular / Q000502:physiology*
chemical_list: D005136:Eye Proteins; D011994:Recombinant Proteins; D050053:TRPM Cation Channels; C116297:Trpm1 protein, mouse
keywords: Cryoelectron Microscopy; G Protein Signaling; Ion Channel; Membrane Protein; Transient Receptor Potential Channels (TRP Channels); Vision
references.citation: J Biol Chem. 2011 Nov 4;286(44):38168-38176
references.pmid: 21908607
references.citation: Am J Hum Genet. 2009 Nov;85(5):720-9
references.pmid: 19896113
references.citation: Prog Retin Eye Res. 2008 Jul;27(4):450-63
references.pmid: 18524666
references.citation: J Biol Chem. 2006 Dec 15;281(50):38748-56
references.pmid: 17060318
references.citation: Annu Rev Biophys. 2011;40:379-408
references.pmid: 21545287
references.citation: J Biol Chem. 2003 Oct 3;278(40):39014-9
references.pmid: 12857742
references.citation: Science. 1981 Jan 9;211(4478):182-5
references.pmid: 6255566
references.citation: Bioinformatics. 2005 Apr 1;21(7):951-60
references.pmid: 15531603
references.citation: J Neurosci. 2000 Dec 15;20(24):9053-8
references.pmid: 11124982
references.citation: Cell. 1995 Mar 10;80(5):757-65
references.pmid: 7889569
references.citation: Proc Natl Acad Sci U S A. 2010 Jan 5;107(1):332-7
references.pmid: 19966281
references.citation: Neuron. 2002 May 30;34(5):759-72
references.pmid: 12062022
references.citation: J Mol Biol. 2002 Apr 5;317(4):591-600
references.pmid: 11955011
references.citation: Nature. 1981 Dec 10;294(5841):592-4
references.pmid: 6273752
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: J Biochem. 2009 Jun;145(6):811-8
references.pmid: 19270057
references.citation: Hum Mol Genet. 2005 Jul 1;14(13):1877-87
references.pmid: 15905181
references.citation: Am J Hum Genet. 2012 Feb 10;90(2):321-30
references.pmid: 22325361
references.citation: J Struct Biol. 1999 Dec 1;128(1):82-97
references.pmid: 10600563
references.citation: Am J Hum Genet. 2009 Nov;85(5):730-6
references.pmid: 19896109
references.citation: Proc Natl Acad Sci U S A. 2012 May 29;109(22):8752-7
references.pmid: 22586107
references.citation: Proc Natl Acad Sci U S A. 2009 Nov 10;106(45):19174-8
references.pmid: 19861548
references.citation: Proc Natl Acad Sci U S A. 2008 May 27;105(21):7451-5
references.pmid: 18490661
references.citation: Am J Hum Genet. 2013 Jan 10;92(1):67-75
references.pmid: 23246293
references.citation: Vision Res. 2011 Dec 8;51(23-24):2440-52
references.pmid: 22037305
references.citation: Invest Ophthalmol Vis Sci. 2010 Feb;51(2):1121-9
references.pmid: 19797214
references.citation: J Mol Biol. 1998 Feb 6;275(5):861-72
references.pmid: 9480774
references.citation: J Neurosci. 2010 Jan 20;30(3):1064-72
references.pmid: 20089915
references.citation: Invest Ophthalmol Vis Sci. 2013 Oct 29;54(10):7153-61
references.pmid: 24114537
references.citation: J Comp Neurol. 1998 May 25;395(1):43-52
references.pmid: 9590545
references.citation: J Neurophysiol. 2007 Nov;98(5):3023-33
references.pmid: 17881478
references.citation: Anal Biochem. 1991 Dec;199(2):223-31
references.pmid: 1812789
references.citation: J Neurophysiol. 2012 Nov;108(9):2442-51
references.pmid: 22896717
references.citation: Proc Natl Acad Sci U S A. 2002 May 28;99(11):7461-6
references.pmid: 12032305
references.citation: Am J Hum Genet. 2009 Nov;85(5):711-9
references.pmid: 19878917
references.citation: Nat Genet. 2000 Nov;26(3):319-23
references.pmid: 11062471
references.citation: PLoS One. 2014 Mar 05;9(3):e90342
references.pmid: 24598786
references.citation: EMBO J. 2003 Feb 17;22(4):776-85
references.pmid: 12574114
references.citation: Neuron. 2001 Mar;29(3):645-55
references.pmid: 11301024
references.citation: Mol Cell Proteomics. 2010 Oct;9(10):2149-61
references.pmid: 20173216
references.citation: J Neurosci. 2009 Jun 17;29(24):7753-65
references.pmid: 19535587
references.citation: Biophys J. 2008 May 1;94(9):3523-37
references.pmid: 18192360
references.citation: Neuron. 2002 Feb 28;33(5):715-29
references.pmid: 11879649
references.citation: J Biol Chem. 2011 Apr 8;286(14):12221-33
references.pmid: 21278253
references.citation: Proc Natl Acad Sci U S A. 2009 Jul 14;106(28):11558-63
references.pmid: 19556541
references.citation: Neuron. 2006 Jul 20;51(2):201-12
references.pmid: 16846855
references.citation: Cell. 1994 May 6;77(3):361-9
references.pmid: 8181056
references.citation: FASEB J. 2011 Mar;25(3):822-9
references.pmid: 21115852
references.citation: J Neurosci. 1999 Apr 15;19(8):2938-44
references.pmid: 10191311
references.citation: PLoS One. 2012;7(4):e33137
references.pmid: 22485138
references.citation: J Gen Physiol. 2007 Mar;129(3):191-207
references.pmid: 17325193
references.citation: Neuron. 2003 Nov 13;40(4):763-74
references.pmid: 14622580
references.citation: Proc Natl Acad Sci U S A. 2012 May 15;109(20):7905-10
references.pmid: 22547806
references.citation: Biochemistry. 1984 Dec 18;23(26):6544-9
references.pmid: 6529569
references.citation: J Neurosci. 2011 Jul 6;31(27):10060-6
references.pmid: 21734298
references.citation: Mol Vis. 2010 Mar 12;16:425-37
references.pmid: 20300565
references.citation: Nature. 1990 Jul 19;346(6281):269-71
references.pmid: 1695713
references.citation: J Neurosci. 2009 May 13;29(19):6088-93
references.pmid: 19439586
references.citation: J Biol Chem. 2010 Apr 9;285(15):11210-8
references.pmid: 20044482
references.citation: Biochemistry. 1983 Feb 1;22(3):653-60
references.pmid: 6188482
references.citation: J Mol Biol. 2001 Jan 19;305(3):567-80
references.pmid: 11152613
references.citation: Nature. 2013 Dec 5;504(7478):107-12
references.pmid: 24305160
references.citation: Sci Signal. 2009 May 12;2(70):ra21
references.pmid: 19436059
references.citation: Anal Biochem. 1995 Jan 1;224(1):28-31
references.pmid: 7710083
references.citation: J Neurosci. 2011 Aug 10;31(32):11521-6
references.pmid: 21832182
references.citation: Am J Hum Genet. 2012 Feb 10;90(2):331-9
references.pmid: 22325362
references.citation: J Neurosci. 2014 Apr 30;34(18):6334-43
references.pmid: 24790204
references.citation: J Neurosci. 2009 Jul 22;29(29):9301-13
references.pmid: 19625520
references.citation: Anal Biochem. 1986 May 1;154(2):525-35
references.pmid: 3728966
references.citation: J Neurosci. 2011 Mar 16;31(11):3962-7
references.pmid: 21411639
references.citation: Structure. 2014 Feb 4;22(2):260-8
references.pmid: 24373766
references.citation: Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2894-9
references.pmid: 14976260
references.citation: Prog Brain Res. 2005;147:205-18
references.pmid: 15581708
references.citation: Curr Protoc Bioinformatics. 2006 Oct;Chapter 5:Unit-5.6
references.pmid: 18428767
references.citation: Nat Genet. 2000 Nov;26(3):324-7
references.pmid: 11062472
references.citation: Invest Ophthalmol Vis Sci. 2003 Jan;44(1):378-84
references.pmid: 12506099
references.citation: J Mol Biol. 2008 Nov 21;383(4):854-70
references.pmid: 18782578
references.citation: J Biol Chem. 1993 Jun 5;268(16):11868-73
references.pmid: 8389366