Differential regulation of Ca ²⁺ influx by ORAI channels mediates enamel mineralization

Abstract

Store-operated Ca2+ entry (SOCE) channels are highly selective Ca2+ channels activated by the endoplasmic reticulum (ER) sensors STIM1 and STIM2. Their direct interaction with the pore-forming plasma membrane ORAI proteins (ORAI1, ORAI2, and ORAI3) leads to sustained Ca2+ fluxes that are critical for many cellular functions. Mutations in the human ORAI1 gene result in immunodeficiency, anhidrotic ectodermal dysplasia, and enamel defects. In our investigation of the role of ORAI proteins in enamel, we identified enamel defects in a patient with an ORAI1 null mutation. Targeted deletion of the Orai1 gene in mice showed enamel defects and reduced SOCE in isolated enamel cells. However, Orai2−/− mice showed normal enamel despite having increased SOCE in the enamel cells. Knockdown experiments in the enamel cell line LS8 suggested that ORAI2 and ORAI3 modulated ORAI1 function, with ORAI1 and ORAI2 being the main contributors to SOCE. ORAI1-deficient LS8 cells showed altered mitochondrial respiration with increased oxygen consumption rate and ATP, which was associated with altered redox status and enhanced ER Ca2+ uptake, likely due to S-glutathionylation of SERCA pumps. Our findings demonstrate an important role of ORAI1 in Ca2+ influx in enamel cells and establish a link between SOCE, mitochondrial function, and redox homeostasis.

The Number and Position of Orai3 Units within Heteromeric Store-Operated Ca2+ Channels Alter the Pharmacology of ICRAC

“…When store depletion was prevented or only endogenous STIM1 was present at the channel, no I CRAC was observed. In line with previous studies [15,61], the expression of one or more Orai3 subunit(s) within these channels result in a significant reduction of I CRAC that was also observed in endogenous systems [15][16][17][18]23,24]. In concatenated channels, expression of two or more Orai3 subunits resulted in 2-APB-induced outward currents in STIM1-dependent and store-independent 2-APB-activated channels.…”

“…Endogenous heteromeric SOCE channels have been reported in different cell types [15][16][17][18][19][20][21][22]. Orai3 may be a negative modulator as, upon knockdown of Orai3, SOCE can be increased [15][16][17][18]23,24]. In contrast, in other studies, SOCE is decreased upon knockout of Orai3, indicating a role as a positive modulator of SOCE [25][26][27][28].…”

“…In addition to forming homomeric Orai1 SOCE channels, Orai1 proteins multimerize with Orai family members Orai2 and Orai3 when overexpressed [14]. Endogenous heteromeric SOCE channels have been reported in different cell types [15][16][17][18][19][20][21][22]. Orai3 may be a negative modulator as, upon knockdown of Orai3, SOCE can be increased [15][16][17][18]23,24].…”

Type 3 Inositol 1,4,5-Trisphosphate Receptor is a Crucial Regulator of Calcium Dynamics Mediated by Endoplasmic Reticulum in HEK Cells

“…Since ER Ca 2+ levels are determined by the relative balance between Ca 2+ leak by leak channels and refilling by SERCA, un-altered resting ER Ca 2+ content together with a weaker ER Ca 2+ leakage would imply a less SERCA pumping activity. We thus directly measured refilling of ER Ca 2+ store with R-CEPIA1er and a protocol adapted from one previous report [35]. In permeabilized, ER Ca 2+ store depleted RIPK cells, addition of 1 mM ATP and 100 nM Ca 2+ would enable the refilling of Ca 2+ into ER by SERCA, as indicated by increases in R-CEPIA1er signals ( Figure 2C).…”

Distinct and Overlapping Expression Patterns of the Homer Family of Scaffolding Proteins and Their Encoding Genes in Developing Murine Cephalic Tissues

“…In developing rodent teeth, it has been shown that PMCA pumps and their encoding genes [42] as well as Orai and Stim proteins and their encoding genes [43][44][45][46][47] are expressed in ameloblasts, the enamel-forming cells, and genetic studies in humans and mice have revealed a critical role for Orai and Stim proteins for proper enamel formation [43,[46][47][48][49].…”