Combined single channel and single molecule detection identifies subunit composition of STIM1-activated transient receptor potential canonical (TRPC) channels
Por:
Asanov A., Sampieri A., Moreno C., Pacheco J., Salgado A., Sherry R., Vaca L.
Publicada:
1 ene 2015
Resumen:
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. © 2014 Elsevier Ltd.
Filiaciones:
Asanov A.:
TIRF Labs, 106 Grendon Place, Cary, NC 27519, United States
Sampieri A.:
Univ Nacl Autonoma Mexico, Inst Fisiol Celular, Mexico City 04510, DF, Mexico
Moreno C.:
Deparment of Physiology and Biophysics, University of Washington, 1705 NE Pacific St, Seattle, WA 98195, United States
Pacheco J.:
Univ Nacl Autonoma Mexico, Inst Fisiol Celular, Mexico City 04510, DF, Mexico
Salgado A.:
Instituto Nacional de Enfermedades Respiratorias (INER), México, DF, 04510, Mexico
Sherry R.:
BitLevel Controls, 416 Market Street, Suite 205, Lewisburg, PA 17837, United States
Vaca L.:
Univ Nacl Autonoma Mexico, Inst Fisiol Celular, Mexico City 04510, DF, Mexico
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