PMID- 26712003
OWN - NLM
STAT- MEDLINE
DCOM- 20160614
LR  - 20210604
IS  - 1091-6490 (Electronic)
IS  - 0027-8424 (Print)
IS  - 0027-8424 (Linking)
VI  - 113
IP  - 2
DP  - 2016 Jan 12
TI  - Genetically targeted single-channel optical recording reveals multiple Orai1 
      gating states and oscillations in calcium influx.
PG  - 440-5
LID - 10.1073/pnas.1523410113 [doi]
AB  - Orai1 comprises the pore-forming subunit of the Ca(2+) release-activated Ca(2+) 
      (CRAC) channel. When bound and activated by stromal interacting molecule 1 
      (STIM1), an endoplasmic reticulum (ER)-resident calcium sensor, Orai1 channels 
      possess high selectivity for calcium but extremely small conductance that has 
      precluded direct recording of single-channel currents. We have developed an 
      approach to visualize Orai1 activity by fusing Orai1 to fluorescent, genetically 
      encoded calcium indicators (GECIs). The GECI-Orai1 probes reveal local Ca(2+) 
      influx at STIM1-Orai1 puncta. By whole cell recording, these fusions are fully 
      functional as CRAC channels. When GECI-Orai1 and the CRAC-activating domain (CAD) 
      of STIM1 were coexpressed at low levels and imaged using a total internal 
      reflectance fluorescence microscope, cells exhibited sporadic fluorescence 
      transients the size of diffraction-limited spots and the brightness of a few 
      activated GECI proteins. Transients typically rose rapidly and fell into two 
      classes according to duration: briefer "flickers" lasting only a few hundred 
      milliseconds, and longer "pulses" lasting one to several seconds. The size, 
      intensity, trace shape, frequency, distribution, physiological characteristics, 
      and association with CAD binding together demonstrate that GECI-Orai1 
      fluorescence transients correspond to single-channel Orai1 responses. Single 
      Orai1 channels gated by CAD, and small Orai1 puncta gated by STIM1, exhibit 
      repetitive fluctuations in single-channel output. CAD binding supports a role in 
      open state maintenance and reveals a second phase of CAD/STIM1 binding after 
      channel opening. These first recordings of single-channel Orai1 currents reveal 
      unexpected dynamics, and when paired with CAD association, support multiple 
      single-channel states.
FAU - Dynes, Joseph L
AU  - Dynes JL
AD  - Department of Physiology and Biophysics, University of California, Irvine, CA 
      92697.
FAU - Amcheslavsky, Anna
AU  - Amcheslavsky A
AD  - Department of Physiology and Biophysics, University of California, Irvine, CA 
      92697.
FAU - Cahalan, Michael D
AU  - Cahalan MD
AD  - Department of Physiology and Biophysics, University of California, Irvine, CA 
      92697 Institute for Immunology, University of California, Irvine, CA 92697 
      mcahalan@uci.edu.
LA  - eng
GR  - R01 AI121945/AI/NIAID NIH HHS/United States
GR  - R01 NS014609/NS/NINDS NIH HHS/United States
GR  - UL1 TR000153/TR/NCATS NIH HHS/United States
GR  - R01 NS-14609/NS/NINDS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20151228
PL  - United States
TA  - Proc Natl Acad Sci U S A
JT  - Proceedings of the National Academy of Sciences of the United States of America
JID - 7505876
RN  - 0 (Calcium Channels)
RN  - 0 (ORAI1 Protein)
RN  - 0 (ORAI1 protein, human)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (enhanced green fluorescent protein)
RN  - 147336-22-9 (Green Fluorescent Proteins)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Calcium/*metabolism
MH  - Calcium Channels/chemistry/*metabolism
MH  - *Calcium Signaling
MH  - Cell Membrane/metabolism
MH  - Fluorescence
MH  - Green Fluorescent Proteins/metabolism
MH  - HEK293 Cells
MH  - Humans
MH  - *Ion Channel Gating
MH  - ORAI1 Protein
MH  - Optogenetics/*methods
MH  - Protein Structure, Tertiary
MH  - Recombinant Fusion Proteins/metabolism
MH  - Time Factors
MH  - Transfection
PMC - PMC4720334
OTO - NOTNLM
OT  - CRAC channel
OT  - Orai
OT  - Stim
OT  - local Ca2+
OT  - store-operated Ca2+ entry
COIS- The authors declare no conflict of interest.
EDAT- 2015/12/30 06:00
MHDA- 2016/06/15 06:00
PMCR- 2016/07/12
CRDT- 2015/12/30 06:00
PHST- 2015/12/30 06:00 [entrez]
PHST- 2015/12/30 06:00 [pubmed]
PHST- 2016/06/15 06:00 [medline]
PHST- 2016/07/12 00:00 [pmc-release]
AID - 1523410113 [pii]
AID - 201523410 [pii]
AID - 10.1073/pnas.1523410113 [doi]
PST - ppublish
SO  - Proc Natl Acad Sci U S A. 2016 Jan 12;113(2):440-5. doi: 10.1073/pnas.1523410113. 
      Epub 2015 Dec 28.