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Role of TRPC3 channels in ATP-induced Ca2+ signaling in principal cells of the inner medullary collecting duct.

ABSTRACT: The transient receptor potential channel TRPC3 is exclusively expressed in the apical membrane of principal cells of the collecting duct (CD) both in vivo and in the mouse CD cell line IMCD-3. Previous studies revealed that ATP-induced apical-to-basolateral transepithelial Ca(2+) flux across IMCD-3 monolayers is increased by overexpression of TRPC3 and attenuated by a dominant negative TRPC3 construct, suggesting that Ca(2+) entry across the apical membrane occurs via TRPC3 channels. To test this hypothesis, we selectively measured the Ca(2+) permeability of the apical membrane of fura-2-loaded IMCD-3 cells using the Mn(2+) quench technique. Mn(2+) influx across the apical membrane was increased 12- to 16-fold by apical ATP and was blocked by the pyrazole derivative BTP2, a known inhibitor of TRPC3 channels, with an IC(50) value <100 nM. In contrast, Mn(2+) influx was only increased approximately 2-fold by basolateral ATP. Mn(2+) influx was also activated by apical, but not basolateral, 1-stearoyl-2-acetyl-sn-glycerol (SAG), a known activator of TRPC3 channels. Apical ATP- and SAG-induced Mn(2+) influx was increased by overexpression of TRPC3 and completely blocked by expression of the dominant negative TRPC3 construct. Mn(2+) influx was also stimulated approximately 2-fold by thapsigargin applied to either the apical or basolateral side. Thapsigargin-induced flux was blocked by BTP2 but was unaffected by overexpression of TRPC3 or by dominant negative TRPC3. Apical ATP, but not basolateral ATP, increased transepithelial (45)Ca(2+) flux. These results demonstrate that the apical membrane of IMCD-3 cells has two distinct Ca(2+) influx pathways: 1) a store-operated channel activated by thapsigargin and basolateral ATP and 2) TRPC3 channels activated by apical ATP. Only activation of TRPC3 leads to net transepithelial apical-to-basolateral Ca(2+) flux. Furthermore, these results demonstrate that native TRPC3 is not a store-operated channel in IMCD-3 cells.

SUBMITTER: Goel M

PROVIDER: S-EPMC2904168 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Role of TRPC3 channels in ATP-induced Ca2+ signaling in principal cells of the inner medullary collecting duct.

Goel Monu M Schilling William P WP

American journal of physiology. Renal physiology 20100421 1

The transient receptor potential channel TRPC3 is exclusively expressed in the apical membrane of principal cells of the collecting duct (CD) both in vivo and in the mouse CD cell line IMCD-3. Previous studies revealed that ATP-induced apical-to-basolateral transepithelial Ca(2+) flux across IMCD-3 monolayers is increased by overexpression of TRPC3 and attenuated by a dominant negative TRPC3 construct, suggesting that Ca(2+) entry across the apical membrane occurs via TRPC3 channels. To test thi ...[more]

PMID: 20410214

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Project description:Transient receptor potential channels TRPC3 and TRPC6 are expressed in principal cells of the collecting duct (CD) along with the water channel aquaporin-2 (AQP2) both in vivo and in the cultured mouse CD cell line IMCD-3. The channels are primarily localized to intracellular vesicles, but upon stimulation with the antidiuretic hormone arginine vasopressin (AVP), TRPC3 and AQP2 translocate to the apical membrane. In the present study, the effect of various activators and inhibitors of the adenylyl cyclase (AC)/cAMP/PKA signaling cascade on channel trafficking was examined using immunohistochemical techniques and by biotinylation of surface membrane proteins. Both in vivo in rat kidney and in IMCD-3 cells, translocation of AQP2 and TRPC3 (but not TRPC6) was stimulated by [deamino-Cys(1), d-Arg(8)]-vasopressin (dDAVP), a specific V2-receptor agonist, and blocked by [adamantaneacetyl(1), O-Et-d-Tyr(2), Val(4), aminobutyryl(6), Arg(8,9)]-vasopressin (AEAVP), a specific V2-receptor antagonist. In IMCD-3 cells, translocation of TRPC3 and AQP2 was activated by forskolin, a direct activator of AC, or by dibutyryl-cAMP, a membrane-permeable cAMP analog. AVP-, dDAVP-, and forskolin-induced translocation in IMCD-3 cells was blocked by SQ22536 and H89, specific inhibitors of AC and PKA, respectively. Translocation stimulated by dibutyryl-cAMP was unaffected by AEAVP but could be blocked by H89. AVP- and forskolin-induced translocation of TRPC3 in IMCD-3 cells was also blocked by two additional inhibitors of PKA, specifically Rp-cAMPS and the myristoylated inhibitor of PKA (m-PKI). Quantification of TRPC3 membrane insertion in IMCD-3 cells under each assay condition using a surface membrane biotinylation assay, confirmed the translocation results observed by immunofluorescence. Importantly, AVP-induced translocation of TRPC3 as estimated by biotinylation was blocked on average 95.2 +/- 1.0% by H89, Rp-cAMPS, or m-PKI. Taken together, these results demonstrate that AVP stimulation of V2 receptors in principal cells of the CD causes translocation of TRPC3 to the apical membrane via stimulation of the AC/cAMP/PKA signaling cascade.

Dataset Information

Role of TRPC3 channels in ATP-induced Ca2+ signaling in principal cells of the inner medullary collecting duct.

Publications

Role of TRPC3 channels in ATP-induced Ca2+ signaling in principal cells of the inner medullary collecting duct.

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