Title : The membrane topology of human transient receptor potential 3 as inferred from glycosylation-scanning mutagenesis and epitope immunocytochemistry
Abstract :
- 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