Protein diversity is a fundamental biological process that enhances the functional complexity of cellular signaling pathways. This diversity arises through multiple molecular mechanisms such as gene duplication, alternative splicing, and alternative translation initiation, which together expand the proteome landscape. Calcium signaling showcases this diversity, with several channels, pumps, and regulatory proteins expressed as multiple isoforms and variants. Within the store-operated calcium entry pathway, protein diversity is evident in the existence of distinct paralogs of ORAI channels and STIM proteins. The additional presence of numerous isoforms and variants of ORAI and STIM shapes the store-operated calcium entry pathway, providing flexibility to cellular calcium regulation in various contexts. Deciphering how protein diversity modulates store-operated calcium entry function is essential for advancing our understanding of calcium signaling in both health and disease.

Angiopoietin2 (Ang2), a regulator of angiogenesis, is stored with other pro-inflammatory and pro-thrombotic mediators, in endothelial-specific vesicles called Weibel-Palade bodies (WPBs). Acute stimulation of endothelial cells with histamine, delays Ang2 secretion by activating Rab46-specific trafficking of Ang2-containing WPBs to the microtubule organising centre (MTOC), where they persist until Ca²⁺ binds to the EF-hand of Rab46, enabling detachment. Here, using Ca²⁺ imaging and high-resolution light microscopy, we pharmacologically investigated the contribution of endolysosomal two-pore channel proteins (TPC) to the Ca²⁺ signal necessary for WPB detachment and Ang2 secretion. We show an increase in the histamine-evoked clustering of Rab46 (and thus WPBs) at the MTOC in the presence of TPC inhibitors Ned-19 and tetrandrine, and a decrease in the presence of a TPC2 agonist, TPC2-A1-N. Histamine-evoked secretion of Ang2 was decreased by pharmacological inhibition of TPC channels but potentiated in the presence of TPC2-A1-N. These data suggest that histamine-mediated Ca²⁺ release via TPC2 channels is necessary for the Rab46-dependent detachment of Ang2-positive WPBs from the MTOC and thus Ang2 secretion. SUMMARY: Ca²⁺ binding to the EF-hand of Rab46 in endothelial cells has previously been reported but the molecular mechanisms and functional relevance are unclear. Here, the authors show that Ca²⁺ released from TPC channels regulates the detachment of Rab46-positive WPBs from the MTOC, which thereby promotes secretion of Ang2.

Dietary calcium intake modifies the action of active vitamin D [1,25(OH)2D3], which promotes the expression of transient receptor potential vanilloid (TRPV) 6, an epithelial calcium channel, to initiate intestinal calcium absorption in response to biological requirements. However, it is unclear whether the change caused by dietary intake results from endocrine regulation or the direct responses to luminal contents. In this study, to reveal the underlying mechanisms of intestinal calcium transport in response to dietary intake, we assessed the early postprandial responses in mice. Although mice lacking intestinal vitamin D receptor function (Int Vdr-) exhibited severe calcium deficiency, a high-calcium diet (1 % calcium) containing 2-fold calcium compared to a control diet reversed impaired calcium absorption and compensated for the mechanisms of 1,25(OH)2D3-dependent transcellular calcium transport. Additionally, the calcium-sensing receptor (CaSR) was abundantly present at the basolateral site in the intestine and the signals were emphasized by a high-calcium diet. To examine the direct response of intestinal epithelium to dietary intake, wild-type (Int Vdr+) and Int Vdr- mice were fed a control or high-calcium diet for 30- or 60-min after 23 h fasting. Serum glucose levels increased 30 min post-feeding in either genotype. TRPV6 expression increased 30 min post-feeding, whereas serum calcium levels were unaltered, suggesting that dietary intake stimulates TRPV6 expression. These data suggest that the regulation of calcium absorption activated immediately after feeding differs from the mechanism involving endocrine responses. Factors altered in the early phase of feeding, such as glucose, may contribute to the regulation of calcium absorption.

The pleiotropic cytokine macrophage migration inhibitory factor (MIF) elevates sarcoplasmic reticulum (SR) Ca2+ content and enhances Ca2+ transient in cardiac muscle. Our imaging and immunoblot data indicated that the MIF-evoked effect is caused mainly by the phosphorylation of the SR Ca2+-pump regulator phospholamban (PLN). Gene expression data suggested that the cluster of differentiation 74 (CD74) and the C-X-C motif chemokine receptor 7 (CXCR7) form a major MIF receptor complex in cardiomyocytes, but CXCR7 activation alone seemed sufficient to exert the MIF-evoked effect. Our pharmacological assessments suggested that phosphoinositide 3-kinase (PI3K), AKT kinase and endothelial nitric oxide synthase (eNOS) were continuously stimulated in the downstream of CXCR7 activation. Furthermore, NO thus generated likely reacted to activate Ca2+/calmodulin-dependent protein kinase II (CaMKII), leading to PLN phosphorylation and subsequent SR Ca2+-pump activation. Therefore, the CXCR7-PI3K-AKT-eNOS-CaMKII-PLN axis is proposed as a central pathway for MIF-evoked potentiation of cardiac Ca2+ signaling.

Paclitaxel (PTX) is one of the most widely used antineoplastic drugs for the treatment of solid cancers. Its mechanism of action involves binding to β-tubulin subunits, leading to the stabilization of microtubule polymers and subsequent cell cycle arrest. Despite its efficacy, PTX is associated with significant adverse effects, most notably peripheral neurotoxicity and neuropathic pain, which represent the primary dose-limiting side effects. In sensory neurons, PTX affects multiple molecular pathways, with early alterations in excitability and calcium signaling following acute drug exposure. To investigate these mechanisms, we employed a combination of calcium imaging, electrophysiological techniques, and pharmacological approaches to explore the role of ORAI channels in the excitability and calcium dynamics of mouse dorsal root ganglion neurons. Our findings reveal that acute exposure to low doses of PTX triggers IP3-dependent calcium release and activates a store-operated calcium entry through STIM-ORAI dependent ICRAC. Moreover, acute PTX application induced the activation of a sustained calcium inward current, Vm depolarization and triggered action potential firing that was strongly attenuated by ICRAC inhibition. Molecular analyses further revealed a significant upregulation of Orai1, Orai2, and Stim2 mRNA levels, accompanied by elevated ORAI1 protein expression, in a rat model of paclitaxel-induced peripheral neuropathy. These results suggest that ORAI and STIM proteins represent promising molecular targets for developing therapies aimed at mitigating the side effects of PTX.