RESUMEN
Ca2+ release-activated Ca2+ (CRAC) channels constitute the major Ca2+ entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca2+-selective Orai channel and the Ca2+-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating. Although the extended transmembrane Orai N-terminal region (Orai1 amino acids 73-91; Orai3 amino acids 48-65) is fully conserved in the Orai1 and Orai3 isoforms, Orai3 tolerates larger N-terminal truncations than Orai1 in retaining store-operated activation. In an attempt to uncover the reason for these isoform-specific structural requirements, we analyzed a series of Orai mutants and chimeras. We discovered that it was not the N termini, but the loop2 regions connecting TM2 and TM3 of Orai1 and Orai3 that featured distinct properties, which explained the different, isoform-specific behavior of Orai N-truncation mutants. Atomic force microscopy studies and MD simulations suggested that the remaining N-terminal portion in the non-functional Orai1 N-truncation mutants formed new, inhibitory interactions with the Orai1-loop2 regions, but not with Orai3-loop2. Such a loop2 swap restored activation of the N-truncation Orai1 mutants. To mimic interactions between the N terminus and loop2 in full-length Orai1 channels, we induced close proximity of the N terminus and loop2 via cysteine cross-linking, which actually caused significant inhibition of STIM1-mediated Orai currents. In aggregate, maintenance of Orai activation required not only the conserved N-terminal region but also permissive communication of the Orai N terminus and loop2 in an isoform-specific manner.
Asunto(s)
Canales de Calcio/química , Proteína ORAI1/química , Canales de Calcio/genética , Canales de Calcio/metabolismo , Células HEK293 , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , Molécula de Interacción Estromal 1/química , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismoRESUMEN
STIM1 (stromal interaction molecule 1) and Orai proteins are the essential components of Ca(2+) release-activated Ca(2+) (CRAC) channels. We focused on the role of cholesterol in the regulation of STIM1-mediated Orai1 currents. Chemically induced cholesterol depletion enhanced store-operated Ca(2+) entry (SOCE) and Orai1 currents. Furthermore, cholesterol depletion in mucosal-type mast cells augmented endogenous CRAC currents, which were associated with increased degranulation, a process that requires calcium influx. Single point mutations in the Orai1 amino terminus that would be expected to abolish cholesterol binding enhanced SOCE to a similar extent as did cholesterol depletion. The increase in Orai1 activity in cells expressing these cholesterol-binding-deficient mutants occurred without affecting the amount in the plasma membrane or the coupling of STIM1 to Orai1. We detected cholesterol binding to an Orai1 amino-terminal fragment in vitro and to full-length Orai1 in cells. Thus, our data showed that Orai1 senses the amount of cholesterol in the plasma membrane and that the interaction of Orai1 with cholesterol inhibits its activity, thereby limiting SOCE.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Colesterol/metabolismo , Biotinilación , Línea Celular , Membrana Celular/metabolismo , Colesterol Oxidasa/metabolismo , Dicroismo Circular , Fenómenos Electrofisiológicos , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Histamina/metabolismo , Humanos , Mastocitos/metabolismo , Mutación , Proteína ORAI1 , Péptidos/metabolismo , Mutación Puntual , Estructura Terciaria de Proteína , Transducción de Señal , Espectrometría de FluorescenciaRESUMEN
The developmental potency of pre-implant parthenogentic goat embryos were compared under two chemical activation protocols in three different culture media groups. The in vitro matured oocytes were chemically activated by two protocols viz. P1 (CB-CHX-6DMAP) and P2 (Ca-CHX-6DMAP). The activated oocytes under both the protocols were developed in three culture media, viz. modified synthetic oviductal fluid (mSOF), research vitro cleave medium (RVCL), and RVCL-Blast. While comparing the developmental potential of activated oocytes, it was observed that the oocytes activated under P2 protocol pooled over three culture media group producing significantly higher mean cleavage rate (43.2±0.9 vs 40.6±1.5), blastocyst development (16.4±1.1 vs 12.6±0.8), and blastomere count (120.7±4.7 vs 113.2±4.1) as compared to P1 protocol. The comparison of effect of culture media pooled over protocol groups revealed that the mean cleavage rate observed under RVCL-Blast (44.8±1.3) and RVCL (45.3±0.5) were significantly higher (P≤0.01) than mSOF (35.8±1.2). However, the mean blastocyst development observed under RVCL-Blast group (18.8±3.2) was significantly higher than RVCL (14.0±0.8) and mSOF (10.8±0.4). Similarly, the mean blastomere count under RVCL-Blast group (136.0±3.7) was significantly higher (P≤0.01) than RVCL (114.7±1.0) and mSOF (100.2±0.5) groups. The semiquantitative RT PCR analysis showed the expression of pro-apoptotic caspase 3 gene in P1 and anti-apoptotic Mcl-1 gene in P2. This study concludes that the activation protocol P2 and embryo cultured under RVCL-Blast group were optimum for chemical activation and culture medium, respectively.