RESUMO
Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Potenciais de Ação/efeitos dos fármacos , Cálcio/metabolismo , Calmodulina/farmacologia , Fenômenos Eletrofisiológicos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imunoprecipitação , Técnicas de Patch-Clamp , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Molécula 1 de Interação Estromal , Canais de Potencial de Receptor Transitório/genéticaRESUMO
The assembly of STIM1 protein puncta near endoplasmic reticulum-plasma membrane (ER-PM) junctions is required for optimal activation of store-operated channels (SOC). The mechanisms controlling the translocation of STIM1 puncta to ER-PM junctions remain largely unknown. In the present study, we have explored the role of the microtubule binding protein adenomatous polyposis coli (APC), on STIM1 puncta and store-operated calcium entry (SOCE). APC-depleted cells showed reduced STIM1 puncta near ER-PM junctions, instead puncta is found at the ER surrounding the cell nucleus. Reduced STIM1 puncta near ER-PM junctions in APC-depleted cells correlates with a strong inhibition of SOCE and diminished Orai whole-cell currents. Immunoprecipitation and confocal microscopy co-localization studies indicate that, upon depletion of the ER, STIM1 dissociates from EB1 and associates to APC. Deletion analysis identified an APC-binding domain in the carboxyl terminus of STIM1 (STIM1 650-685). These results together position APC as an important element in facilitating the translocation of STIM1 puncta near ER-PM junctions, which in turn is required for efficient SOCE and Orai activation upon depletion of the ER.
