Genetically targeted single-channel optical recording reveals multiple Orai1 gating states and oscillations in calcium influx

Abstract

Orai1 comprises the pore-forming subunit of the Ca2+ release-activated Ca2+ (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 Ca2+ 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.

Genetically Encoded Fluorescent Indicators for Organellar Calcium Imaging

“…Another group successfully detected nanodomain Ca 2þ signals in the surrounding region of Ca V 2.2, a voltage-dependent Ca 2þ channel, by fusion with a FRET-typed GECI, TN-XL (66). A single-FP-typed GECI, G-GECO, was also used by attaching it to Orai1, a Ca 2þ release-activated Ca 2þ channel on the plasma membrane, and results from studies with G-GECO revealed that Ca 2þ influx through Orai1 makes several characteristic patterns, such as rapid transients and periodic oscillation (67).…”

The STIM-Orai Pathway: Orai, the Pore-Forming Subunit of the CRAC Channel

“…Recently developed alternative methods, such as detecting the gating conformational change with Tb 3+ or other fluorescent probes (Gudlur et al 2014) or recording optically from single Orai1 channels tagged with genetically encoded calcium indicators (Dynes et al 2016), may offer a way around these limitations.…”

Integration of nicotinic acid adenine dinucleotide phosphate (NAADP)‐dependent calcium signalling

“…Recently, Dynes et al . () generated a ORAI1‐GECO construct and thereby were able to measure optical single‐channel recordings of ORAI1 in a TIRF microscope set‐up. Furthermore, using a TPC2‐GECO construct (TPC‐GCaMP6), Ambrosio et al .…”