Genetically targeted single-channel optical recording reveals multiple Orai1 gating states and oscillations in calcium influx.
Proc Natl Acad Sci U S A
; 113(2): 440-5, 2016 Jan 12.
Article
em En
| MEDLINE
| ID: mdl-26712003
ABSTRACT
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Canais de Cálcio
/
Ativação do Canal Iônico
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Cálcio
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Sinalização do Cálcio
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Optogenética
Limite:
Humans
Idioma:
En
Revista:
Proc Natl Acad Sci U S A
Ano de publicação:
2016
Tipo de documento:
Article