title: Combined single channel and single molecule detection identifies subunit composition of STIM1-activated transient receptor potential canonical (TRPC) channels.
abstract: 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.
authors.affiliation: TIRF Labs, 106 Grendon Place, Cary, NC 27519, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Asanov
authors.affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, México, DF 04510, Mexico.
authors.forename: Alicia
authors.initials: A
authors.lastname: Sampieri
authors.affiliation: Deparment of Physiology and Biophysics, University of Washington, 1705 NE Pacific St, Seattle, WA 98195, USA.
authors.forename: Claudia
authors.initials: C
authors.lastname: Moreno
authors.affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, México, DF 04510, Mexico.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Pacheco
authors.affiliation: Instituto Nacional de Enfermedades Respiratorias (INER), México, DF 04510, Mexico.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Salgado
authors.affiliation: BitLevel Controls, 416 Market Street, Suite 205, Lewisburg, PA 17837, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Sherry
authors.affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, México, DF 04510, Mexico. Electronic address: lvaca@ifc.unam.mx.
authors.forename: Luis
authors.initials: L
authors.lastname: Vaca
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology; D055724:Electrophysiological Phenomena; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D000071737:Stromal Interaction Molecule 1; D050051:Transient Receptor Potential Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D050051:Transient Receptor Potential Channels; D049452:Green Fluorescent Proteins; D002118:Calcium
keywords: STIM1; Single channel recording; Single molecule detection; Store-operated channels; TRPC