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. KyteDoolittle 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 ␣1 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.

The mouse C-type transient receptor potential 2 (TRPC2) channel: Alternative splicing and calmodulin binding to its N terminus

“…1 shows typical Kyte and Doolittle plots of TRPC2 and TRPC3, highlighting eight hydrophobic domains. Of these, the first was shown to be intracellular by glycosylation scanning mutagenesis and epitope insertion mapping (17). One of the remaining seven comprises a region that forms the selectivity pore of the channel.…”

Functional characterization and physiological relevance of the TRPC3/6/7 subfamily of cation channels

“…A structural analysis of heterologously expressed TRPC3 by glycosylation scanning mutagenesis revealed a naturally occurring glycosylation site in the second extracellular loop of the cation channel (Vannier et al 1998). Although its functional significance remains poorly understood, the introduction of a second glycosylation site in the second extracellular loop in order to imitate the situation in TRPC6 reduced the high TRPC3 basal activity (Dietrich et al 2003b).…”

N-Glycosylation of TRPM8 Ion Channels Modulates Temperature Sensitivity of Cold Thermoreceptor Neurons

“…Besides TRPM8, several other TRP channels are also known to undergo glycosylation, with important functional consequences (reviewed in Refs. [22][23][24]. In TRPV5, a channel critical for Ca 2ϩ reabsorption in the distal convoluted tubule of the kidney, cleavage of a single N-linked oligosaccharide by Klotho, a glucuronidase enzyme, stabilizes the channel at the apical plasma membrane (25).…”