Les lysosomes sont importants pour la survie et la fonction des cellules du système nerveux central et en particulier des neurones. Le mécanisme de la reformation des lysosomes est crucial pour maintenir une quantité adéquate de lysosomes fonctionnels dans les cellules. La spatacsine, qui joue un rôle dans le ce mécanisme est impliquée dans la paraplégie spastique de type SPG11 ; une maladie caractérisée par des troubles moteurs et cognitifs sévères. L’utilisation de modèles cellulaires de cette pathologie permet d’étudier les mécanismes physiopathologiques à l’origine d’altérations de la reformation des lysosomes. J’ai montré que la perte de fonction de la spatacsine est responsable de l’accumulation de lipides dans les lysosomes. Ces accumulations sont constituées de gangliosides et de cholestérol et sont présentes dans les autolysosomes perturbant leur recyclage en lysosomes, notamment en empêchant le recrutement de protéines impliquées dans le mécanisme. Les accumulations de gangliosides rendent les neurones à l’exposition au glutamate ce qui suggère que ces altérations pourraient avoir un rôle dans la neurodégénérescence. J’ai aussi montré que l’absence de spatacsine provoque une dérégulation de l’import de Ca2+ extracellulaire par le « store-operated calcium entry » ce qui conduit à altération de l’homéostasie calcique. L’inhibition de l’import de calcium par le SOCE permet de réduire les accumulations de lipides et de rétablir partiellement le recyclage des lysosomes. Ainsi, l’absence de spatacsine induit une altération de l’homéostasie calcique qui participe à l’accumulation de lipides dans le système lysosomal ce qui est délétère pour la survie des neurones. / Lysosomal dysfunctions are involved in a large number of neurodegenerative diseases highlightingthe crucial function of lysosomes in neuron survival and function. The mechanism of lysosomereformation from autolysosomes allow cells to maintain the ool of functional lysosomes.Disruptions of this rocess are involved in athologies affecting the central nervous system. Inparticular, spatacsin that lays a role in lysosome recycling is implicated in hereditary spasticparaplegia type SPG11, a severe disease characterized by motors and cognitive alterations. Thispathology is caused by loss of function mutations in SPG11, encoding spatacsin. The study ofSPG11 cellular models gives the opportunity to decipher the hysiopathological mechanismsunderlying lysosomal reformation disruptions.During my thesis, I showed that loss of spatacsin function induces lipid accumulation in lysosomesand articularly in autolysosomes both in fibroblasts and neurons from Spg11-/- mice. Gangliosidesand cholesterol are among lipids that accumulate in autolysosomes impairing lysosomal membranerecycling by disrupting the recruitment of keys roteins. Neurons with ganglioside accumulationsare more sensitive to glutamate induced neuronal death, suggesting that these accumulations areinvolved in neurodegeneration. These results could be of great importance since accumulations ofgangliosides in lysosomes arise in many diseases.I also showed that loss of spatacsin disrupts extracellular calcium import by the store-operatedcalcium entry (SOCE) leading to an increase in cytosolic calcium levels. Lysosomal calcium contentis also increased in Spg11-/- cells and calcium release from lysosome by TRPML1 is reduced.Inhibiting SOCE and stimulating lysosomal calcium release by TRPML1 reduced lipidsaccumulations in lysosomes and artially restored lysosome reformation.Our data suggest that absence of spatacsin is responsible for a disruption of calcium homeostasisthat contributes to lipid accumulation in autolysosomes, disturbing reformation of lysosomes fromautolysosomes. Inhibiting gangliosides synthesis could be used as a therapeutic strategy. However,understanding how loss of function of spatacsin alters these cellular athways will allow thedevelopment of targeted therapeutic approaches.

No description available.

Résumé : La variation de la [Ca2+] intracellulaire participe à nombreux de processus biologiques. Les cellules eucaryotes expriment à la membrane plasmique une variété de canaux par lesquelles le calcium peut entrer. Dans les cellules non excitables, deux mécanismes principaux permettent l'entrée calcique; l'entrée capacitative de Ca2+ via Orai1 (SOCE) et l'entrée calcique activé par un récepteur (ROCE). Plusieurs protéines clés sont impliquées dans la régulation de ces voies d'entrée calcique, ainsi que dans l'homéostasie calcique. TRPC6 est un canal calcique impliquée dans l'entrée calcique dans les cellules à la suite d’une stimulation d’un récepteur hormonal. TRPC6 transloque à la membrane cellulaire et il y demeure jusqu'à ce que le stimulus soit retiré. Les mécanismes qui régulent le trafic et l'activation de TRPC6 sont cependant encore peu connus. Des découvertes récentes ont démontré qu'il y a un rôle potentiel de Rho kinase dans l'activité de TRPC6. Rho kinase est activée par la petite protéine G RhoA qui peut être activée par les protéines G hétérotrimériques Gα12 et Gα13. En plus de Gα12 et Gα13, les protéines de désensibilisation des GPCR β -arrestin 1 et / ou β-arrestin 2 peuvent aussi activer RhoA. Le but de notre étude est d'examiner la participation des protéines Gα12/13 et β-arrestin 1/ β-arrestin 2 dans l'activation de TRPC6 et de la protéine Orai1. Nous avons utilisé des ARN interférant (siRNA) spécifiques pour induire une réduction de l'expression de Gα12/13 ou β-arrestin 1/β-arrestin 2. La conséquence sur l’entrée de Ca2+ dans les cellules a été ensuite déterminée par imagerie calcique en temps réel suite à une stimulation par la vasopressine (AVP), thapsigargin ou carbachol. Nous avons donc identifié que dans des cellules A7r5, une lignée cellulaire de musculaires lisses vasculaires où le canal TRPC6 exprimé de manière endogène, la diminution de l’expression des protéines Gα12 ou Gα13 ne semble pas modifier l’entrée Ca2+ induit par l’AVP par rapport aux cellules témoins. D'autre part, la diminution de l’expression β-arrestin 1 ou β-arrestin 2 dans des cellules HEK 293 ainsi que des cellules HEK 293 exprimant de façon stable TRPC6 (cellules T6.11) ont augmenté l’entrée de Ca2+ induite par thapsigargin, un activateur pharmacologique de SOCE. Des études de co-immunoprécipitation démontrent une interaction entre la β-arrestin 1 et STIM1, alors qu'aucune interaction n'a été observée entre les β-arrestin 1 et Orai1. Nous avons de plus montré à l'aide d'analyse en microscopie confocale que la diminution de l’expression β-arrestin 1 ou β-arrestin 2 n’influence pas la quantité d’Orai1 à la périphérie cellulaire. Cependant, des résultats préliminaires indiquent que la diminution de l’expression β-arrestin 1 ou β-arrestin 2 augmente la quantité de STIM1-YFP dans l'espace intracellulaire et diminue sa quantité à la périphérie cellulaire. En conclusion, nous avons montré que les β-arrestin 1 ou β-arrestin 2 sont impliquées dans l'entrée capacitative de Ca2+ (SOCE) et contrôlent la quantité de STIM1 dans le réticulum endoplasmique. / Abstract : In an organism, intracellular [Ca2+] takes part in many biological processes. Eukaryotic cells express a variety of channels in the plasma membrane through which calcium can enter. In non-excitable cells, two main mechanisms allow calcium entry; the store-operated calcium entry via Orai1 (SOCE) and receptor-operated calcium entry (ROCE). Several key proteins are involved in the regulation of these calcium entry pathways as well as in calcium homeostasis. TRPC6 is a calcium channel implied in calcium entrance into the cells following hormonal stimulation and translocates to the plasma membrane. TRPC6 channel appear to the plasma membrane until the stimulus is present. Although, the mechanisms that regulate the trafficking and activation of TRPC6 are still little known. Recent findings have demonstrated that there is a potential role of Rho kinase in activity of TRPC6. Rho kinase is activated by the small G protein RhoA that itself can be activated by the heterotrimeric G proteins Gα12 and Gα13. In addition to Gα12 and Gα13 proteins, cytosolic GPCR desensitizing proteins β-arrestin 1 and/or β-arrestin 2 could also activate RhoA. The purpose of our study is to investigate the involvement of the proteins Gα12/13 and β-arrestin 1/β-arrestin 2 in the activation of TRPC6 and Orai1 protein. We used siRNA specific to Gα12/13 or β-arrestin 1/β-arrestin 2 to knockdown their endogenous expression. Then, calcium imaging in real time was performed in order to see the quantity of calcium entered into the cell following stimulation by vasopressin (AVP), thapsigargin, or carbachol. We hence identified that in A7r5 cell, vascular smooth muscle cell where TRPC6 channel expressed endogenously; reduced expression of Gα12 or Gα13 proteins does not seem to modify the AVP-induced Ca2+ entry compared to control cells. On the other hand, calcium imaging experiment in knocked down β-arrestin 1 or β-arrestin 2 in HEK 293 cells as well as HEK 293 cells stably transfected with TRPC6 (T6.11 cells) resulted in an increased thapsigargin-induced calcium entry. The co-immunoprecipitation studies demonstrate an interaction between β-arrestin 1 and STIM1, a calcium sensor in SOCE influx, while no interaction was observed between β-arrestin 1 and Orai1.We moreover showed by confocal microscopy that reduced expression of β-arrestin 1/ β-arrestin 2 does not influence the quantity of Orai1 at the cell periphery. Preliminary results showed that reduced expression of β-arrestin 1 or β-arrestin 2 increases the quantity of STIM1-YFP in the intracellular space and less it’s in peri-membrane space. In conclusion, we showed that β-arrestin 1 or β-arrestin 2 are involved in the store-operated calcium entry (SOCE) and control the quantity of STIM1 in the endoplasmic reticulum.

BEZERRA DE LIMA, BJS. Efeito da nifedipina por via sistêmica na analgesia pós-operatória induzida pelo sufentanil intratecal em pacientes submetidas à lipoabdominoplastia em caráter ambulatorial. 2011. 72 f. Tese (Doutorado) Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo Canais de cálcio dependentes da voltagem têm um importante papel na transmissão de impulsos nociceptivos e alterações das concentrações extracelulares de íons cálcio podem modificar a analgesia opioide. No entanto, a utilização de bloqueadores de canais de cálcio produziu resultados conflitantes no tocante à potencialização ou prolongamento da analgesia opioide. O sufentanil, pela alta lipossolubilidade e consequente baixo potencial para depressão respiratória tardia, pode ser utilizado pela via subaracnoidea em pacientes ambulatoriais. Para ser considerado o opioide ideal para uso subaracnoideo falta-lhe a capacidade de produzir analgesia prolongada. O objetivo deste trabalho foi testar se a nifedipina oral pode aumentar a sua duração e potência. Trinte e seis pacientes do sexo feminino, ASA I ou II e idade entre 20 e 60 anos, submetidas à lipoabdominoplastia sob regime ambulatorial foram casualisada em 3 grupos, de acordo com a quantidade de nifedipina recebida no pré-operatório. Foram confeccionadas 13 cápsulas semelhantes que continham amido ou 10 mg de nifedipina e entregues para cada uma das pacientes durante a avaliação pré-anestésica que aconteceu com a antecedência mínima de cinco dias do procedimento cirúrgico. As pacientes foram orientadas a ingerir 3 (três) cápsulas por dia, com intervalo de 8 horas entre cada ingestão, durante 4 (quatro) dias, totalizando 12 cápsulas. A 13ª cápsula foi ingerida 60 minutos antes do procedimento anestésico. As pacientes do Grupo A ingeriram 13 cápsulas de amido, as do Grupo B ingeriram 12 cápsulas contendo amido e a 13ª contendo 10 mg de nifedipina e as do Grupo C ingeriram 13 cápsulas contendo 10 mg de nifedipina. A PA e a FC foram aferidas em seis momentos: imediatamente antes do procedimento anestésico (M1), 5 (cinco) minutos após a realização da punção subaracnoidea (M2), 60 (sessenta) minutos após a realização da punção subaracnóidea (M3), ao término do procedimento cirúrgico (M4), no momento da primeira queixa dolorosa (M5) e no momento da alta hospitalar (M6).A técnica anestésica foi semelhante em todos os grupos e constou de raquianestesia e administração de solução contendo 20 mg de bupivacaína pesada e 5 microgramas de sufentanil. Após a punção e administração das medicações, o nível do bloqueio pretendido (T4) foi controlado pelo posicionamento da mesa cirúrgica. As pacientes foram orientadas a informar o momento em que sentissem dor pela primeira vez. Considerou-se como tempo de analgesia, o tempo (minutos) decorrido entre a punção subaracnoidea e esta primeira queixa. Neste momento, foi realizada a mensuração da intensidade da dor por meio da escala analógica visual. A intensidade da dor foi novamente avaliada no momento da alta hospitalar, da mesma forma que no momento da primeira queixa. Não houve diferença no comportamento hemodinâmico entre os grupos. O grupo C apresentou maior tempo de analgesia e menor escore de dor no momento da primeira queixa. Não houve diferença entre os grupos quanto ao escore de dor no momento da alta hospitalar. A nifedipina administrada durante 4 dias no pré-operatório prolongou, de forma tênue, o tempo e a intensidade da analgesia induzida pelo fentanil. / BEZERRA DE LIMA, BJS. Effect of nifedipine in a systemic way in postoperative analgesia induced by intrathecal sufentanil in patients undergoing ambulatory lipoabdominoplasty. 2011. 72 f. Tese (Doutorado) Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo Calcium channel voltage-gated play an important role in transmitting nociceptive impulses and changes in extracellular concentrations of calcium ions may reverse opioid analgesia. However, the use of calcium channel blockers produced conflicting results regarding the extension or enhancement of opioid analgesia. Sufentanil, high lipid solubility and consequent low potential for late respiratory depression, can be used by subarachnoid in outpatients. To be considered the ideal opioid for use subarachnoid lacks the ability to produce prolonged analgesia. The aim of this study was to test whether oral nifedipine may increase the duration and potency of sufentanil. Thirty-six female patients, ASA I or II and aged between 20 and 60 years undergoing ambulatory lipoabdominoplasty were randomized into three groups according to the amount received nifedipine preoperatively. 13 capsules were prepared containing similar starch or 10 mg of nifedipine and delivered to each of the patients during the preanesthetic evaluation that took place with at least five days after surgery. The patients were instructed to ingest three (3) capsules per day with 8 hours interval between each ingestion, during 4 (four) days, totaling 12 capsules. The 13th capsule was ingested 60 minutes before anesthesia. The Group A patients ingested 13 capsules of starch, the Group B ingested 12 capsules containing starch and 13th with 10 mg of nifedipine and those in Group C ingested 13 capsules containing 10 mg of nifedipine. BP and HR were measured on six occasions: just before anesthesia (M1), 5 (five) minutes after subarachnoid puncture (M2), 60 (sixty) minutes after spinal puncture (M3), the after surgery (T4), when the first complaint of pain (M5) and at discharge (M6). Anesthetic technique was similar in all groups and consisted of spinal anesthesia and administration of a solution containing 20 mg of heavy bupivacaine and 5 micrograms of sufentanil. After puncture and administration of medications, the desired level of the block (T4) was controlled by positioning the operating table. The patients were instructed to inform when they felt pain the first time. It was considered as a time of analgesia, time (minutes) elapsed between the subarachnoid puncture and this first complaint. At this point, we performed the measurement of pain intensity by visual analog scale. Pain intensity was evaluated again at discharge from hospital, the same way as when the first complaint. There was no difference in hemodynamic performance between the groups. The C group showed longer duration of analgesia and lower pain scores at the first complaint. There was no difference between the groups in pain scores at discharge from hospital. Nifedipine administered for 4 days preoperatively prolonged, weakly, the time and intensity of analgesia induced by fentanyl.

Store-operated calcium entry (SOCE) is the major Ca2+ entry pathway in most non-excitable cells maintaining prolonged elevation of cytosolic Ca2+ levels required for gene transcription. SOCE is activated by the loss of endoplasmic reticulum (ER) Ca2+ through stromal interaction molecules (STIM), ER-membrane associated Ca2+ sensors. In humans, STIM1 and STIM2 share 65% sequence similarity but differentially regulate SOCE. Biophysical studies on the luminal Ca2+-binding region suggests that STIM2 EF-SAM is more stable than STIM1. The NMR structure of Ca2+-loaded STIM2 EF-SAM determined in this work suggests a more stable SAM and a tighter EF-hand and SAM interaction in STIM2 may be account for its higher stability. Chimeric swapping of the EF-hand and SAM domains generates an unstable ES211. Introducing ES211 into cherryFP-STIM1 shows constitutive puncta which activate SOCE independent of ER depletion. The current work demonstrates that the instability of the EF-SAM plays an important role in regulating SOCE initiation.

Store-operated calcium entry (SOCE) is the major Ca2+ entry pathway in most non-excitable cells maintaining prolonged elevation of cytosolic Ca2+ levels required for gene transcription. SOCE is activated by the loss of endoplasmic reticulum (ER) Ca2+ through stromal interaction molecules (STIM), ER-membrane associated Ca2+ sensors. In humans, STIM1 and STIM2 share 65% sequence similarity but differentially regulate SOCE. Biophysical studies on the luminal Ca2+-binding region suggests that STIM2 EF-SAM is more stable than STIM1. The NMR structure of Ca2+-loaded STIM2 EF-SAM determined in this work suggests a more stable SAM and a tighter EF-hand and SAM interaction in STIM2 may be account for its higher stability. Chimeric swapping of the EF-hand and SAM domains generates an unstable ES211. Introducing ES211 into cherryFP-STIM1 shows constitutive puncta which activate SOCE independent of ER depletion. The current work demonstrates that the instability of the EF-SAM plays an important role in regulating SOCE initiation.

BEZERRA DE LIMA, BJS. Efeito da nifedipina por via sistêmica na analgesia pós-operatória induzida pelo sufentanil intratecal em pacientes submetidas à lipoabdominoplastia em caráter ambulatorial. 2011. 72 f. Tese (Doutorado) Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo Canais de cálcio dependentes da voltagem têm um importante papel na transmissão de impulsos nociceptivos e alterações das concentrações extracelulares de íons cálcio podem modificar a analgesia opioide. No entanto, a utilização de bloqueadores de canais de cálcio produziu resultados conflitantes no tocante à potencialização ou prolongamento da analgesia opioide. O sufentanil, pela alta lipossolubilidade e consequente baixo potencial para depressão respiratória tardia, pode ser utilizado pela via subaracnoidea em pacientes ambulatoriais. Para ser considerado o opioide ideal para uso subaracnoideo falta-lhe a capacidade de produzir analgesia prolongada. O objetivo deste trabalho foi testar se a nifedipina oral pode aumentar a sua duração e potência. Trinte e seis pacientes do sexo feminino, ASA I ou II e idade entre 20 e 60 anos, submetidas à lipoabdominoplastia sob regime ambulatorial foram casualisada em 3 grupos, de acordo com a quantidade de nifedipina recebida no pré-operatório. Foram confeccionadas 13 cápsulas semelhantes que continham amido ou 10 mg de nifedipina e entregues para cada uma das pacientes durante a avaliação pré-anestésica que aconteceu com a antecedência mínima de cinco dias do procedimento cirúrgico. As pacientes foram orientadas a ingerir 3 (três) cápsulas por dia, com intervalo de 8 horas entre cada ingestão, durante 4 (quatro) dias, totalizando 12 cápsulas. A 13ª cápsula foi ingerida 60 minutos antes do procedimento anestésico. As pacientes do Grupo A ingeriram 13 cápsulas de amido, as do Grupo B ingeriram 12 cápsulas contendo amido e a 13ª contendo 10 mg de nifedipina e as do Grupo C ingeriram 13 cápsulas contendo 10 mg de nifedipina. A PA e a FC foram aferidas em seis momentos: imediatamente antes do procedimento anestésico (M1), 5 (cinco) minutos após a realização da punção subaracnoidea (M2), 60 (sessenta) minutos após a realização da punção subaracnóidea (M3), ao término do procedimento cirúrgico (M4), no momento da primeira queixa dolorosa (M5) e no momento da alta hospitalar (M6).A técnica anestésica foi semelhante em todos os grupos e constou de raquianestesia e administração de solução contendo 20 mg de bupivacaína pesada e 5 microgramas de sufentanil. Após a punção e administração das medicações, o nível do bloqueio pretendido (T4) foi controlado pelo posicionamento da mesa cirúrgica. As pacientes foram orientadas a informar o momento em que sentissem dor pela primeira vez. Considerou-se como tempo de analgesia, o tempo (minutos) decorrido entre a punção subaracnoidea e esta primeira queixa. Neste momento, foi realizada a mensuração da intensidade da dor por meio da escala analógica visual. A intensidade da dor foi novamente avaliada no momento da alta hospitalar, da mesma forma que no momento da primeira queixa. Não houve diferença no comportamento hemodinâmico entre os grupos. O grupo C apresentou maior tempo de analgesia e menor escore de dor no momento da primeira queixa. Não houve diferença entre os grupos quanto ao escore de dor no momento da alta hospitalar. A nifedipina administrada durante 4 dias no pré-operatório prolongou, de forma tênue, o tempo e a intensidade da analgesia induzida pelo fentanil. / BEZERRA DE LIMA, BJS. Effect of nifedipine in a systemic way in postoperative analgesia induced by intrathecal sufentanil in patients undergoing ambulatory lipoabdominoplasty. 2011. 72 f. Tese (Doutorado) Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo Calcium channel voltage-gated play an important role in transmitting nociceptive impulses and changes in extracellular concentrations of calcium ions may reverse opioid analgesia. However, the use of calcium channel blockers produced conflicting results regarding the extension or enhancement of opioid analgesia. Sufentanil, high lipid solubility and consequent low potential for late respiratory depression, can be used by subarachnoid in outpatients. To be considered the ideal opioid for use subarachnoid lacks the ability to produce prolonged analgesia. The aim of this study was to test whether oral nifedipine may increase the duration and potency of sufentanil. Thirty-six female patients, ASA I or II and aged between 20 and 60 years undergoing ambulatory lipoabdominoplasty were randomized into three groups according to the amount received nifedipine preoperatively. 13 capsules were prepared containing similar starch or 10 mg of nifedipine and delivered to each of the patients during the preanesthetic evaluation that took place with at least five days after surgery. The patients were instructed to ingest three (3) capsules per day with 8 hours interval between each ingestion, during 4 (four) days, totaling 12 capsules. The 13th capsule was ingested 60 minutes before anesthesia. The Group A patients ingested 13 capsules of starch, the Group B ingested 12 capsules containing starch and 13th with 10 mg of nifedipine and those in Group C ingested 13 capsules containing 10 mg of nifedipine. BP and HR were measured on six occasions: just before anesthesia (M1), 5 (five) minutes after subarachnoid puncture (M2), 60 (sixty) minutes after spinal puncture (M3), the after surgery (T4), when the first complaint of pain (M5) and at discharge (M6). Anesthetic technique was similar in all groups and consisted of spinal anesthesia and administration of a solution containing 20 mg of heavy bupivacaine and 5 micrograms of sufentanil. After puncture and administration of medications, the desired level of the block (T4) was controlled by positioning the operating table. The patients were instructed to inform when they felt pain the first time. It was considered as a time of analgesia, time (minutes) elapsed between the subarachnoid puncture and this first complaint. At this point, we performed the measurement of pain intensity by visual analog scale. Pain intensity was evaluated again at discharge from hospital, the same way as when the first complaint. There was no difference in hemodynamic performance between the groups. The C group showed longer duration of analgesia and lower pain scores at the first complaint. There was no difference between the groups in pain scores at discharge from hospital. Nifedipine administered for 4 days preoperatively prolonged, weakly, the time and intensity of analgesia induced by fentanyl.

No description available.

No description available.

Store-operated Ca2+ entry (SOCE) triggered by the depletion of endoplasmic reticulum (ER) luminal Ca2+ stores is a major Ca2+ entry pathway in non-excitable cells and is essential in physiological Ca2+ signaling and homeostasis. STIM proteins are sensors of ER luminal Ca2+, which translocate to ER-plasma membrane (PM) junctional regions to activate the family of Orai channels mediating Ca2+ entry. This study is focused on dissecting the mechanism of STIM interacting with Orai. A powerful modifier of SOCE, 2-aminoethoxydiphenyl borate (2-APB) is utilized. First, the action of 2-APB on the mammalian Orai homologues are characterized using the DT40 STIM knockout cells. 50 ìM 2-APB directly activates Orai3 but not Orai1 or Orai2. Second, while it stimulates the STIM2-mediated constitutive Ca2+ entry through Orai, 2-APB also induces the cytosolic STIM C-terminus fragments to translocate to the PM and activate Orai1. These data reveal 50 ìM 2-APB enhances STIM-Orai coupling. Further, this enhanced binding of STIM and Orai leads to a conformational change within the STIM-Orai complex, which is possibly the underlying mechanism for the 50 ìM 2-APB inhibitory effect on SOCE. Finally, six residues (344-349) at the N-terminus of the STIM-Orai activation region (SOAR) prove to be critical for this inhibitory action. These same six amino acid region also constitutes an ancillary Orai binding site within SOAR, in addition to the main polybasic region. The deletion of this ancillary site abolishes the ability of SOAR to bind to and activate Orai1, but can be compensated for by the STIM-Orai binding enhancing effect of 50 ìM 2-APB. The majority of STIM1 is located on the ER membrane, while a small proportion of STIM1 is on the PM. Using an extracellularly applied STIM1 antibody, the PM STIM1 can be aggregated to exert an influence on the ER STIM1. Although the PM STIM1 is not obligatory for STIM1-mediated Orai activation, it nevertheless may have a functional presence in the PM. Lastly, a regulatory link between voltage-gated Ca2+ channels (Cav channels) and the STIM proteins is established. After activation by store depletion, STIM strongly suppresses the Cav1.2 channels. There is a biochemical interaction between STIM1 and the Cav1.2 pore subunit á1C. This inhibitory effect is independent of Orai1 activation. Hence, STIM1 interacts with and reciprocally controls two major Ca2+ channels. / Biochemistry