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Control of PTH secretion by the TRPC1 ion channel
Marta Onopiuk, … , Leonidas Tsiokas, Kai Lau
Marta Onopiuk, … , Leonidas Tsiokas, Kai Lau
Published March 26, 2020
Citation Information: JCI Insight. 2020;5(8):e132496. https://doi.org/10.1172/jci.insight.132496.
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Research Article Cell biology Endocrinology

Control of PTH secretion by the TRPC1 ion channel

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Abstract

Familial hypocalciuric hypercalcemia (FHH) is a genetic condition associated with hypocalciuria, hypercalcemia, and, in some cases, inappropriately high levels of circulating parathyroid hormone (PTH). FHH is associated with inactivating mutations in the gene encoding the Ca2+-sensing receptor (CaSR), a GPCR, and GNA11 encoding G protein subunit α 11 (Gα11), implicating defective GPCR signaling as the root pathophysiology for FHH. However, the downstream mechanism by which CaSR activation inhibits PTH production/secretion is incompletely understood. Here, we show that mice lacking the transient receptor potential canonical channel 1 (TRPC1) develop chronic hypercalcemia, hypocalciuria, and elevated PTH levels, mimicking human FHH. Ex vivo and in vitro studies revealed that TRPC1 serves a necessary and sufficient mediator to suppress PTH secretion from parathyroid glands (PTGs) downstream of CaSR in response to high extracellular Ca2+ concentration. Gα11 physically interacted with both the N- and C-termini of TRPC1 and enhanced CaSR-induced TRPC1 activity in transfected cells. These data identify TRPC1-mediated Ca2+ signaling as an essential component of the cellular apparatus controlling PTH secretion in the PTG downstream of CaSR.

Authors

Marta Onopiuk, Bonnie Eby, Vasyl Nesin, Peter Ngo, Megan Lerner, Caroline M. Gorvin, Victoria J. Stokes, Rajesh V. Thakker, Maria Luisa Brandi, Wenhan Chang, Mary Beth Humphrey, Leonidas Tsiokas, Kai Lau

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Figure 5

TRPC1 is required for CaSR-induced Ca2+ signaling in PTH-C1 cells.

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TRPC1 is required for CaSR-induced Ca2+ signaling in PTH-C1 cells.
(A) E...
(A) Efficiency of TRPC1 knockdown using RNAi. PTH-C1 cells were transiently transfected with a scrambled siRNA (lane 1), siTRPC1 (lane 2), expression plasmid (positive control) of a long form of TRPC1α (lane 3), or a short form of TRPC1α (lane 4). TRPC1 was immunoprecipitated and immunoblotted with a TRPC1-specific monoclonal antibody (1F1). PTH-C1 cells express predominantly the long form of TRPC1α (indicated by an asterisk). (B) Changes in intracellular Ca2+ concentration (expressed as fluorescence ratio 340/380) in PTH-C1 cells transiently transfected with a scrambled siRNA (control, black, n = 76 cells pooled from 5 independent experiments) or a Trpc1-specific siRNA (siTRPC1, red, n = 61 cells pooled from 6 independent experiments) and cultured in 1, 5, or 10 mM extracellular Ca2+. (C) Time course of spermine-induced (5 mM) intracellular Ca2+ concentration in PTH-C1 cells transiently transfected with a scrambled siRNA (control, black, n = 144 cells pooled from 4 independent experiments) or a Trpc1-specific siRNA (siTRPC1, red, n = 178 cells pooled from 8 independent experiments). (D) Time course of intracellular Ca2+ concentration in PTH-C1 cells cultured in 0 extracellular Ca2+ and activated by spermine (3 mM) in the presence (blue, n = 279 cells from 6 experiments) or absence of NPS2143 (300 nM) (black, n = 233 cells from 5 experiments). (E) Time course of R568-induced (50 μM) intracellular Ca2+ concentration in PTH-C1 cells transiently cotransfected with GCaMP3 and scrambled siRNA (control, black line, n = 216 cells pooled from 9 independent experiments) or TRPC1 siRNA (siTRPC1, red line, n = 172 cells pooled from 9 independent experiments) in the presence of 1.8 mM extracellular Ca2+ concentration. F0 was the average fluorescence for 1 minute prior to the addition of R-568. (F and G) TRPC1 is specifically coupled to CaSR. HEK293 cells were transiently cotransfected with GCaMP3, CaSR (F), or m1 muscarinic acetylcholine receptor (m1 AchR, G) in the presence or absence of TRPC1 (red or black traces). Cells were first stimulated with thapsigargin (2 μM) to deplete the internal stores and then with spermine (2 mM, F) or carbachol (10 μM, G) to activate CaSR or m1 AchR, respectively. Changes in intracellular free Ca2+ concentration are reported as F/F0. Data were pooled from 6 independent experiments totaling 263 cells transfected with CaSR (black, F) and 316 cells transfected with CaSR plus TRPC1 (red, F) or from 8 independent experiments totaling 190 cells transfected with m1 AchR (black, G) and 173 cells transfected with m1 AchR plus TRPC1 (red, G).

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