Loss-of-function mutations in stromal interaction molecule 1 (STIM1) impair the activation of Ca2+ release-activated Ca2+ (CRAC) channels and store-operated Ca2+ entry (SOCE), resulting in a disease syndrome called CRAC channelopathy that is characterized by severe dental enamel defects. The cause of these enamel defects has remained unclear given a lack of animal models. We generated Stim1/2K14cre mice to delete STIM1 and its homolog STIM2 in enamel cells. These mice showed impaired SOCE in enamel cells. Enamel in Stim1/2K14cre mice was hypomineralized with decreased Ca content, mechanically weak, and thinner. The morphology of SOCE-deficient ameloblasts was altered, showing loss of the typical ruffled border, resulting in mislocalized mitochondria. Global gene expression analysis of SOCE-deficient ameloblasts revealed strong dysregulation of several pathways. ER stress genes associated with the unfolded protein response were increased in Stim1/2-deficient cells, whereas the expression of components of the glutathione system were decreased. Consistent with increased oxidative stress, we found increased ROS production, decreased mitochondrial function, and abnormal mitochondrial morphology in ameloblasts of Stim1/2K14cre mice. Collectively, these data show that loss of SOCE in enamel cells has substantial detrimental effects on gene expression, cell function, and the mineralization of dental enamel.
Background and Purpose Transient receptor potential vanilloid subtype 3 ( TRPV3 ) is implicated in nociception and certain skin conditions. As such, it is an attractive target for pharmaceutical research. Understanding of endogenous TRPV3 function and pharmacology remains elusive as selective compounds and native preparations utilizing higher throughput methodologies are lacking. In this study, we developed medium‐throughput recombinant and native cellular assays to assess the detailed pharmacological profile of human, rat and mouse TRPV3 channels. Experimental Approach Medium‐throughput cellular assays were developed using a Ca 2+ ‐sensitive dye and a fluorescent imaging plate reader. Human and rat TRPV3 pharmacology was examined in recombinant cell lines, while the mouse 308 keratinocyte cell line was used to assess endogenous TRPV3 activity. Key Results A recombinant rat TRPV3 cellular assay was successfully developed after solving a discrepancy in the published rat TRPV3 protein sequence. A medium‐throughput, native, mouse TRPV3 keratinocyte assay was also developed and confirmed using genetic approaches. Whereas the recombinant human and rat TRPV3 assays exhibited similar agonist and antagonist profiles, the native mouse assay showed important differences, namely, TRPV3 activity was detected only in the presence of potentiator or during agonist synergy. Furthermore, the native assay was more sensitive to block by some antagonists. Conclusions and Implications Our findings demonstrate similarities but also notable differences in TRPV3 pharmacology between recombinant and native systems. These findings offer insights into TRPV3 function and these assays should aid further research towards developing TRPV3 therapies. Linked Articles This article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue‐10
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