Visualizing Calcium Influx through Single Orai1 Channels

Abstract

Orai is the pore-forming subunit of two-component channels that mediate store-operated calcium entry (SOCE). When activated by the ER resident calcium sensor STIM, Orai channels possess high selectivity for calcium but an extremely small conductance (∼10 fS in 2 mM Ca2+ from noise analysis) that has precluded direct recording of single channel currents. We have developed an approach to visualize the activity of single Orai1 channels by fusing human Orai1 to a fluorescent, genetically-encoded calcium indicator (GECI). Whole-cell recording in HEK 293 cells co-transfected with STIM1 showed that GECI-Orai1 is fully functional as a CRAC channel, with normal activation rates, current amplitudes, ion selectivity and rectification, and Ca2+-dependent inactivation. When GECI-Orai1 and the CRAC-activating domain (CAD) of STIM1 were co-expressed at low levels and imaged using a TIRF microscope, cells exhibited sporadic fluorescence transients the size of diffraction-limited spots and the brightness of a few activated GECI proteins. Transients typically rose to a maximum within 100-200 milliseconds and fell into two classes according to duration: briefer “flickers” lasting only a few hundred milliseconds, and longer “pulses” lasting ∼1 to several seconds. The frequency of both classes increased as CAD expression levels were increased. GECI-Orai1 transients exhibited physiological characteristics expected for single Orai1 channels in the plasma membrane, including rapid response to changes in extracellular calcium, enhancement by valinomycin-induced hyperpolarization, and block by trivalent cations. When single molecule GECI-Orai1 traces were aligned by the rise in green fluorescence, a corresponding rise in mCherry-CAD fluorescence, indicative of binding, was observed. These data indicate that GECI-Orai1 transients correspond to the opening of single Ora1 channels gated by CAD. These first recordings of single channel Orai1 currents reveal unexpected dynamics, and when paired with CAD association, support multiple single channel states.