title: Orai1 determines calcium selectivity of an endogenous TRPC heterotetramer channel.
abstract: BACKGROUND
  Canonical transient receptor potential 4 (TRPC4) contributes to the molecular composition of a channel encoding for a calcium selective store-operated current, I(SOC), whereas Orai1 critically comprises a channel encoding for the highly selective calcium release activated calcium current, I(CRAC). However, Orai1 may interact with TRPC proteins and influence their activation and permeation characteristics. Endothelium expresses both TRPC4 and Orai1, and it remains unclear as to whether Orai1 interacts with TRPC4 and contributes to calcium permeation through the TPRC4 channel.
  
  
  OBJECTIVE
  We tested the hypothesis that Orai1 interacts with TRPC4 and contributes to the channel's selective calcium permeation important for endothelial barrier function.
  
  
  RESULTS
  A novel method to purify the endogenous TRPC4 channel and probe for functional interactions was developed, using TRPC4 binding to protein 4.1 as bait. Isolated channel complexes were conjugated to anti-TRPC protein antibodies labeled with cy3-cy5 pairs. Förster Resonance Energy Transfer among labeled subunits revealed the endogenous protein alignment. One TRPC1 and at least 2 TRPC4 subunits constituted the endogenous channel (TRPC1/4). Orai1 interacted with TRPC4. Conditional Orai1 knockdown reduced the probability for TRPC1/4 channel activation and converted it from a calcium-selective to a nonselective channel, an effect that was rescued on Orai1 reexpression. Loss of Orai1 improved endothelial cell barrier function.
  
  
  CONCLUSIONS
  Orai1 interacts with TRPC4 in the endogenous channel complex, where it controls TRPC1/4 activation and channel permeation characteristics, including calcium selectivity, important for control of endothelial cell barrier function.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of South Alabama College of Medicine, Mobile, AL 36688, USA. dlcioffi@ usouthal.edu
authors.forename: Donna L
authors.initials: DL
authors.lastname: Cioffi
authors.forename: Songwei
authors.initials: S
authors.lastname: Wu
authors.forename: Hairu
authors.initials: H
authors.lastname: Chen
authors.forename: Mikhail
authors.initials: M
authors.lastname: Alexeyev
authors.forename: Claudette M
authors.initials: CM
authors.lastname: St Croix
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Pitt
authors.forename: Stefan
authors.initials: S
authors.lastname: Uhlig
authors.forename: Troy
authors.initials: T
authors.lastname: Stevens
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002199:Capillary Permeability; D042783:Endothelial Cells / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D015640:Ion Channel Gating; D008564:Membrane Potentials; D000071740:ORAI1 Protein; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D055503:Protein Multimerization; D034622:RNA Interference; D051381:Rats; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
chemical_list: D015220:Calcium Channels; D000071740:ORAI1 Protein; C532493:Orai1 protein, rat; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; C096351:transient receptor potential cation channel, subfamily C, member 1; D002118:Calcium