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. 2023 May 5;146(8):3444–3454. doi: 10.1093/brain/awad146

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© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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GPRC5B variants alter regulatory volume decrease, and GPRC5B modulates VRAC. (A) GPRC5B schematic, highlighting duplicated amino acids in the fourth transmembrane segment (red). (B) RVD measurements in lymphoblasts exposed to a hypotonic shock. Averaged cell surface dynamics for five control lymphoblast lines and three GPRC5B patient lines. (C) RVD percentage in lymphoblasts. Points indicate individual cells, bars represent average ± SEM per cell line, dotted lines indicate mean RVD for all control and all GPRC5B patient lines. Patient cells show reduced RVD (controls: 60.1 ± 3.4%, n = 103 cells from five subjects versus patients 44.4 ± 3.4%, n = 100 cells from three subjects, P = 0.001). (D) Western blot for GPRC5B, MLC1 and TRPV4 (β-actin as loading control) on three control and three GPRC5B patient lymphoblast cell lines. Molecular weight markers on the left. See Supplementary Fig. 4 for full-length blots. (E) Densitometric analysis of protein bands normalized to β-actin (2–4 replicate experiments). Patient cells show increased GPRC5B levels (controls: 10.0 ± 2.8, patients: 23.0 ± 1.4, P = 0.003), but unaltered MLC1 levels (controls: 16.7 ± 0.5, patients: 16.7 ± 0.8, P = 0.95). TRPV4 levels were reduced (controls: 25.8 ± 4.5, patients: 9.2 ± 1.4, P = 0.005). (F) Representative patch-clamp current traces under isotonic and hypotonic conditions in a non-transfected (empty; left) and a wild-type GPRC5B (isoform 1) transfected U251 cell (right). Traces in response to voltage steps (−100 mV to +100 mV). Scale bars = 200 ms (x), 1 NA (y). (G) Averaged I/V relationship of hypotonically activated VRAC current density in empty, wild-type GPRC5B, Ala177dup GPRC5B, and Ile176dup GPRC5B expressing U251 cells. (H) Averaged VRAC current density at +100 mV in U251 cells. Overexpression of all GPRC5B variants of both isoforms significantly increased VRAC current (empty: 14.9 ± 4.3 pA/pF, n = 15, isoform 1 wild-type: 60.0 ± 10.3 pA/pF, n = 12, P = 0.01; isoform 1 Ala177dup: 52.0 ± 9.2 pA/pF, n = 17, P = 0.02; isoform 1 Ile176dup: 67.9 ± 11.5 pA/pF, n = 16, P = 0.001; isoform 2 wild-type: 89.5 ± 17.6, n = 12, P < 0.001; isoform 2 Ala177dup: 57.4 ± 13.3, n = 15, P = 0.03; isoform 2 Ile176dup: 55.9 ± 12.4, n = 15, P = 0.04). No significant differences between Ala177dup, Ile176dup and wild-type GPRC5B were found (P-values >0.99). (I) VRAC current density in MLC1-overexpressing U251 cells. GPRC5B overexpression (isoform 1) has no additional effect on MLC1 overexpression-augmented VRAC activation (empty: 53.0 ± 17.5 pA/pF, n = 8, wild-type: 49.7 ± 16.5 pA/pF, n = 6, P > 0.99; Ala177dup: 60.6 ± 19.2 pA/pF, n = 5, P > 0.99; Ile176dup: 54.0 ± 19.6 pA/pF, n = 4, P > 0.99). Also, no significant differences between Ala177dup and Ile176dup GPRC5B variants and wild-type GPRC5B were found (P > 0.99). All data presented as mean ± SEM.