Anti-ORAI1 Monoclonal antibody, clone 4G7-I6

Store-operated calcium entry (SOCE) is one of regulatory mechanisms which regulates Ca2+ cycling in the heart. SOCE alterations in pathological conditions contribute to progression of heart failure and cardiac hypertrophy by multiple signaling pathways such as Cn/NFAT and CaMKII/MEF2. Several components mediating SOCE have been identified, such as STIM and Orai. Different isoforms of both Orai and STIM have been detected in animal studies, exhibiting distinct functional properties. This study is focused on the analysis of STIM and Orai isoforms expression in the end-stage human failing myocardium. Left ventricle samples isolated from 43 explanted hearts from patients undergoing heart transplant and from 5 healthy donor hearts were used to determine the mRNA levels of Orai1, Orai2 and Orai3, STIM1, STIM2 and STIM2.1 by qRT-PCR. The expression was further analyzed for connection with gender, related co-morbidities, pathoetiology, clinical data and biochemical parameters. We show that Orai1 expression is decreased by 30 % in failing myocardium, even though we detected no significant changes in expression of Orai2 or Orai3. Interestingly, this decrease in Orai1 was gender-specific and was present only in men, with no change in women. The ratio Orai1/Orai3 was significantly lower in males as well. The novel STIM2.1 isoform was detected both in healthy and failing human myocardium. In the end-stage heart failure, the expression of STIM2.1 was significantly decreased. The lower ratio of STIM2.1/STIM2 in failing hearts indicates a switch from SOCE-inhibiting STIM2.1 isoform to stimulatory STIM2.2. STIM1 mRNA levels were not significantly changed. These observed alterations in Orai and STIM expression were independent of functional heart parameters, clinical or biochemical patient characteristics. These results provide detailed insight into the alterations of SOCE regulation in human failing myocardium. Gender-specific change in Orai1 expression might represent a possible mechanism of cardioprotective effects of estrogens. The switch from STIM2.1 to STIM2.2 indicates an amplification of SOCE and could contribute to the hypertrophy development in the filing heart.

Simple Summary Store-operated calcium channels constituted from the proteins Orai and STIM are important targets for development of new drugs, especially for the treatment of auto-immune diseases. Also, interference with channel function is linked to reduced cancer cell progression, making these channels potential targets for anti-cancer drug development. Therefore, inhibitors need to be evaluated for both their binding selectivity and their potential to interfere with cancer progression. Here, we investigated the inhibitor Synta66 and determined its site of binding via both patch clamp recordings and computational approaches and evaluated its potency as anti-cancer agent in glioblastoma multiforme cells. Our findings show that Synta66 is a highly selective ligand to the Orai1 pore and efficiently blocks store operated calcium entry in glioblastoma cells. Still, in the tested cell lines, Synta66 did not reduce cell viability. We therefore suggest Synta66 as a precise tool to observe interference of store-operated Orai1 channel function in vitro and of resulting downstream effects. The Ca2+ sensor STIM1 and the Ca2+ channel Orai1 that form the store-operated Ca2+ (SOC) channel complex are key targets for drug development. Selective SOC inhibitors are currently undergoing clinical evaluation for the treatment of auto-immune and inflammatory responses and are also deemed promising anti-neoplastic agents since SOC channels are linked with enhanced cancer cell progression. Here, we describe an investigation of the site of binding of the selective inhibitor Synta66 to the SOC channel Orai1 using docking and molecular dynamics simulations, and live cell recordings. Synta66 binding was localized to the extracellular site close to the transmembrane (TM)1 and TM3 helices and the extracellular loop segments, which, importantly, are adjacent to the Orai1-selectivity filter. Synta66-sensitivity of the Orai1 pore was, in fact, diminished by both Orai1 mutations affecting Ca2+ selectivity and permeation of Na+ in the absence of Ca2+. Synta66 also efficiently blocked SOC in three glioblastoma cell lines but failed to interfere with cell viability, division and migration. These experiments provide new structural and functional insights into selective drug inhibition of the Orai1 Ca2+ channel by a high-affinity pore blocker.