A calmodulin/inositol 1,4,5-trisphosphate (IP3) receptor-binding region targets TRPC3 to the plasma membrane in a calmodulin/IP3 receptor-independent process.
Conformational coupling with the inositol 1,4,5-trisphosphate (IP3) receptor has been suggested as a possible mechanism of activation of TRPC3 channels and a region in the C terminus of TRPC3 has been shown to interact with the IP3 receptor as well as calmodulin (calmodulin/IP3 receptor-binding (CIRB) region). Here we show that internal deletion of 20 amino acids corresponding to the highly conserved CIRB region results in the loss of diacylglycerol and agonist-mediated channel activation in HEK293 cells. By using confocal microscopy to examine the cellular localization of Topaz fluorescent protein fusion constructs, we demonstrate that this loss in activity is caused by faulty targeting of CIRB-deleted mutants to intracellular compartments. Wild type TRPC3 and mutants lacking a C-terminal predicted coiled coil region downstream of CIRB were targeted to the plasma membrane correctly in HEK293 cells and exhibited TRPC3-mediated calcium entry in response to agonist activation. Mutation of conserved YQ and MKR motifs to alanine within the CIRB region in TRPC3-Topaz, which would be expected to interfere with IP3 receptor and/or calmodulin binding, had no effect on channel function or targeting. Additionally, TRPC3 targets to the plasma membrane of DT40 cells lacking all three IP3 receptors and forms functional ion channels. These findings indicate that the previously identified CIRB region of TRPC3 is involved in its targeting to the plasma membrane by a mechanism that does not involve interaction with IP3 receptors.
Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA.
Barbara J
BJ
Wedel
Guillermo
G
Vazquez
Richard R
RR
McKay
Gary
G
St  J  Bird
James W
JW
Putney
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002645:Chickens; D018679:Cholinergic Agonists / Q000494:pharmacology; D017124:Conserved Sequence; D004075:Diglycerides / Q000378:metabolism; D005434:Flow Cytometry; D017353:Gene Deletion; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D050052:TRPC Cation Channels; D013997:Time Factors; D014162:Transfection
D015220:Calcium Channels; D002147:Calmodulin; D018679:Cholinergic Agonists; D004075:Diglycerides; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007473:Ion Channels; D018160:Receptors, Cytoplasmic and Nuclear; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D002217:Carbachol; D002118:Calcium