The membrane topology of human transient receptor potential 3 as inferred from glycosylation-scanning mutagenesis and epitope immunocytochemistry.
Transient receptor potential (Trp) proteins form ion channels implicated in the calcium entry observed after stimulation of the phospholipase C pathway. Kyte-Doolittle analysis of the amino acid sequence of Trp proteins identifies seven hydrophobic regions (H1-H7) with potential of forming transmembrane segments. A limited sequence similarity to voltage-gated calcium channel alpha1 subunits lead to the prediction of six transmembrane (TM) segments flanked by intracellular N and C termini and a putative pore region between TM5 and TM6. However, experimental evidence supporting this model is missing. Using human Trp 3 to test Trp topology, we now confirm the intracellular nature of the termini by immunocytochemistry. We also demonstrate presence of a unique glycosylation site in position 418, which defines one extracellular loop between H2 and H3. After removal of this site and insertion of ten separate glycosylation sites, we defined two additional extracellular loops between H4 and H5, and H6 and H7. This demonstrated the existence of six transmembrane segments formed of H2-H7. Thus, the first hydrophobic region of Trp rather than being a transmembrane segment is intracellular and available for protein-protein interactions. A site placed in the center of the putative pore region was glycosylated, suggesting that this region may have been luminal and was reinserted into the membrane at a late stage of channel assembly.
Vannier
B
B
Department of Anesthesiology, University of California, Los Angeles, California, USA.
Zhu
X
X
Brown
D
D
Birnbaumer
L
L
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D015220:Calcium Channels / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D017931:DNA Primers; D000939:Epitopes / Q000032:analysis / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D007473:Ion Channels / Q000096:biosynthesis* / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D050052:TRPC Cation Channels; D014162:Transfection
D015220:Calcium Channels; D017931:DNA Primers; D000939:Epitopes; D007473:Ion Channels; D011994:Recombinant Proteins; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel