N-linked protein glycosylation is a major determinant for basal TRPC3 and TRPC6 channel activity.
The TRPC family of receptor-activated cation channels (TRPC channels) can be subdivided into four subfamilies based on sequence homology as well as functional similarities. Members of the TRPC3/6/7 subfamily share common biophysical characteristics and are activated by diacylglycerol in a membrane-delimited manner. At present, it is only poorly understood whether members of the TRPC3/6/7 subfamily are functionally redundant or whether they serve distinct cellular roles. By electrophysiological and fluorescence imaging strategies we show that TRPC3 displays considerable constitutive activity, while TRPC6 is a tightly regulated channel. To identify potential molecular correlates accounting for the functional difference, we analyzed the glycosylation pattern of TRPC6 compared with TRPC3. Two NX(S/T) motifs in TRPC6 were mutated (Asn to Gln) by in vitro mutagenesis to delete one or both extracellular N-linked glycosylation sites. Immunoblotting analysis of HEK 293 cell lysates expressing TRPC6 wild type and mutants favors a model of TRPC6 that is dually glycosylated within the first (e1) and second extracellular loop (e2) as opposed to the monoglycosylated TRPC3 channel (Vannier, B., Zhu, X., Brown, D., and Birnbaumer, L. (1998) J. Biol. Chem. 273, 8675-8679). Elimination of the e2 glycosylation site, missing in the monoglycosylated TRPC3, was sufficient to convert the tightly receptor-regulated TRPC6 into a constitutively active channel, displaying functional characteristics of TRPC3. Reciprocally, engineering of an additional second glycosylated site in TRPC3 to mimic the glycosylation status in TRPC6 markedly reduced TRPC3 basal activity. We conclude that the glycosylation pattern plays a pivotal role for the tight regulation of TRPC6 through phospholipase C-activating receptors.
Dietrich
Alexander
A
Institut für Pharmakologie und Toxikologie, Philipps-Universität-Marburg, 35033 Marburg, Germany.
Mederos y Schnitzler
Michael
M
Emmel
Jens
J
Kalwa
Hermann
H
Hofmann
Thomas
T
Gudermann
Thomas
T
D020816:Amino Acid Motifs; D015220:Calcium Channels / Q000737:chemistry*; D002412:Cations; D002460:Cell Line; D004075:Diglycerides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D007473:Ion Channels / Q000737:chemistry*; D008345:Manganese / Q000378:metabolism; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; D050053:TRPM Cation Channels; D013997:Time Factors; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000737:chemistry
D015220:Calcium Channels; D002412:Cations; D004075:Diglycerides; D007473:Ion Channels; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D014415:Tunicamycin; D008345:Manganese; D010738:Type C Phospholipases