Gα11 mutation in mice causes hypocalcemia rectifiable by calcilytic therapy
Caroline M. Gorvin, Fadil M. Hannan, Sarah A. Howles, Valerie N. Babinsky, Sian E. Piret, Angela Rogers, Andrew J. Freidin, Michelle Stewart, Anju Paudyal, Tertius A. Hough, M. Andrew Nesbit, Sara Wells, Tonia L. Vincent, Stephen D.M. Brown, Roger D. Cox, Rajesh V. Thakker
Caroline M. Gorvin, Fadil M. Hannan, Sarah A. Howles, Valerie N. Babinsky, Sian E. Piret, Angela Rogers, Andrew J. Freidin, Michelle Stewart, Anju Paudyal, Tertius A. Hough, M. Andrew Nesbit, Sara Wells, Tonia L. Vincent, Stephen D.M. Brown, Roger D. Cox, Rajesh V. Thakker
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Research Article Endocrinology Metabolism

Gα11 mutation in mice causes hypocalcemia rectifiable by calcilytic therapy

Abstract

Heterozygous germline gain-of-function mutations of G-protein subunit α11 (Gα11), a signaling partner for the calcium-sensing receptor (CaSR), result in autosomal dominant hypocalcemia type 2 (ADH2). ADH2 may cause symptomatic hypocalcemia with low circulating parathyroid hormone (PTH) concentrations. Effective therapies for ADH2 are currently not available, and a mouse model for ADH2 would help in assessment of potential therapies. We hypothesized that a previously reported dark skin mouse mutant (Dsk7) — which has a germline hypermorphic Gα11 mutation, Ile62Val — may be a model for ADH2 and allow evaluation of calcilytics, which are CaSR negative allosteric modulators, as a targeted therapy for this disorder. Mutant Dsk7/+ and Dsk7/Dsk7 mice were shown to have hypocalcemia and reduced plasma PTH concentrations, similar to ADH2 patients. In vitro studies showed the mutant Val62 Gα11 to upregulate CaSR-mediated intracellular calcium and MAPK signaling, consistent with a gain of function. Treatment with NPS-2143, a calcilytic compound, normalized these signaling responses. In vivo, NPS-2143 induced a rapid and marked rise in plasma PTH and calcium concentrations in Dsk7/Dsk7 and Dsk7/+ mice, which became normocalcemic. Thus, these studies have established Dsk7 mice, which harbor a germline gain-of-function Gα11 mutation, as a model for ADH2 and have demonstrated calcilytics as a potential targeted therapy.

Authors

Caroline M. Gorvin, Fadil M. Hannan, Sarah A. Howles, Valerie N. Babinsky, Sian E. Piret, Angela Rogers, Andrew J. Freidin, Michelle Stewart, Anju Paudyal, Tertius A. Hough, M. Andrew Nesbit, Sara Wells, Tonia L. Vincent, Stephen D.M. Brown, Roger D. Cox, Rajesh V. Thakker

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Figure 3

Intracellular calcium responses of the Val62 Gα11 mutant and effect of NPS-2143 treatment.

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Intracellular calcium responses of the Val62 Gα11 mutant and effect of N...
(A) Fluorescence microscopy of HEK293 cells stably expressing CaSR (HEK-CaSR) and transiently transfected with WT Ile62 or mutant (m) Val62 pBI-CMV2-GNA11 constructs. GFP expression in these cells indicates successful transfection and expression by these constructs. Scale bars: 10 μm. (B) Western blot analysis of lysates from HEK-CaSR cells used for flow cytometry experiments. Transient transfection with WT or mutant Val62 expression constructs resulted in overexpression of Gα11 and GFP. Calnexin, a housekeeping protein, and untransfected cells (Supplemental Figure 2) were used as controls. (C) Ca2+i response to changes in [Ca2+]o of HEK-CaSR cells transfected with WT or Val62 Gα11 mutant. The Ca2+i responses to changes in [Ca2+]o are expressed as a percentage of the maximum normalized responses and shown as the mean ± SEM of 5–8 assays from 2 independent transfections. The Val62 Gα11 mutant led to a leftward shift in the concentration-response curve (red line). The addition of 20 nM NPS-2143 rectified the leftward shift of the Val62 Gα11 mutant (red dashed line), whereas 40 nM NPS-2143 led to a rightward shift of the mutant concentration-response curve (pink dashed line) compared with WT (black line). (D–F) Histograms showing mean ± SEM EC50 values, % maximal signaling responses, and Hill coefficients, respectively, for cells expressing WT (open bar) or Val62 Gα11 mutant (black bar) proteins and for mutant-expressing cells treated with NPS-2143 (patterned bars). **P < 0.01, ***P <0.001. F-test for C–D. Mann-Whitney U test for E–F.