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 implicat ed in the calcium entry observed after stimulation of the phospholipas e 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 vo ltage-gated calcium channel alpha 1 subunits lead to the prediction of six transmembrane (TM) segments flanked by intracellular N and C term ini and a putative pore region between TM5 and TM6, However, experimen tal evidence supporting this model is missing, Using human Trp 3 to te st Trp topology, we now confirm the intracellular nature of the termin i by immunocytochemistry, We also demonstrate presence of a unique gly cosylation site in position 418, which defines one extracellular loop between H2 and H3, After removal of this site and insertion of ten sep arate glycosylation sites, we defined two additional extracellular loo ps between H4 and H5, and H6 and H7, This demonstrated the existence o f six transmembrane segments formed of H2-H7, Thus, the first hydropho bic region of Trp rather than being a transmembrane segment is intrace llular and available for protein-protein interactions, A site placed i n the center of the putative pore region was glycosylated, suggesting that this region may have been luminal and was reinserted into the mem brane at a late stage of channel assembly.