Biogenesis and topology of the transient receptor potential Ca2+ channel TRPC1.
The TRPC ion channels are candidates for the store-operated Ca(2+) entry pathway activated in response to depletion of intracellular Ca(2+) stores. Hydropathy analyses indicate that these proteins contain eight hydrophobic regions (HRs) that could potentially form alpha-helical membrane-spanning segments. Based on limited sequence similarities to other ion channels, it has been proposed that only six of the eight HRs actually span the membrane and that the last two membrane-spanning segments (HRs 6 and 8) border the ion-conducting pore of which HR 7 forms a part. Here we study the biogenesis and transmembrane topology of human TRPC1 to test this model. We have employed a truncation mutant approach combined with insertions of glycosylation sites into full-length TRPC1. In our truncation mutants, portions of the TRPC1 sequence containing one or more HRs were fused between the enhanced green fluorescent protein and a C-terminal glycosylation tag. These chimeras were transiently expressed in the human embryonic cell line HEK-293T. Glycosylation of the tag was used to monitor its location relative to the lumen of the endoplasmic reticulum and thereby HR orientation. Our data indicate that HRs 1, 4, and 6 cross the membrane from cytosol to the ER lumen, that HRs 2, 5, and 8 have the opposite orientation, and that HR 3 is left out of the membrane on the cytosolic side. Our results also show that the sequence downstream of HR 8 plays an important role in anchoring its C-terminal end on the cytosolic side of the membrane. This effect appears to prevent HR 7 from spanning the bilayer and to result in its forming a pore-like structure of the type previously envisioned for the TRPC channels. We speculate that a similar mechanism may be responsible for the formation of other ion channel pores.
Dohke
Yoko
Y
Membrane Biology Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health/DHHS, Building 10, Room 1A01, 10 Center Drive, Bethesda, MD 20892, USA.
Oh
Young S
YS
Ambudkar
Indu S
IS
Turner
R James
RJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000737:chemistry*; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D006801:Humans; D007477:Ions; D008164:Luminescent Proteins / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D050052:TRPC Cation Channels; D014162:Transfection
D000596:Amino Acids; D015220:Calcium Channels; D007477:Ions; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D049452:Green Fluorescent Proteins; D004247:DNA; D002118:Calcium