A pathogenic C terminus-truncated polycystin-2 mutant enhances receptor-activated Ca2+ entry via association with TRPC3 and TRPC7.
Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca(2+) release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca(2+) influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca(2+) influx that may lead to dysregulated cell growth in ADPKD.
Miyagi
Kyoko
K
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan.
Kiyonaka
Shigeki
S
Yamada
Kazunori
K
Miki
Takafumi
T
Mori
Emiko
E
Kato
Kenta
K
Numata
Tomohiro
T
Sawaguchi
Yuichi
Y
Numaga
Takuro
T
Kimura
Toru
T
Kanai
Yoshikatsu
Y
Kawano
Mitsuhiro
M
Wakamori
Minoru
M
Nomura
Hideki
H
Koni
Ichiro
I
Yamagishi
Masakazu
M
Mori
Yasuo
Y
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004594:Electrophysiology / Q000379:methods; D005091:Exons; D016368:Frameshift Mutation; D006801:Humans; D007668:Kidney / Q000378:metabolism; D018374:LLC-PK1 Cells; D009154:Mutation; D017434:Protein Structure, Tertiary; D011976:Receptors, Muscarinic / Q000378:metabolism; D013552:Swine; D050052:TRPC Cation Channels / Q000737:chemistry* / Q000378:metabolism; D050396:TRPP Cation Channels / Q000737:chemistry* / Q000235:genetics*
D002118:Calcium; D011976:Receptors, Muscarinic; D050052:TRPC Cation Channels; D050396:TRPP Cation Channels; C104603:TRPC3 cation channel; C498501:TRPC7 protein, human