Functional stoichiometry of the unitary calcium-release-activated calcium channel

Abstract

Two proteins, STIM1 in the endoplasmic reticulum and Orai1 in the plasma membrane, are required for the activation of Ca 2؉ releaseactivated Ca 2؉ (CRAC) channels at the cell surface. How these proteins interact to assemble functional CRAC channels has remained uncertain. Here, we determine how many Orai1 and STIM1 molecules are required to form a functional CRAC channel. We engineered several genetically expressed fluorescent Orai1 tandem multimers and a fluorescent, constitutively active STIM1 mutant. The tandem multimers assembled into CRAC channels, as seen by rectifying inward currents and by cytoplasmic calcium elevations. CRAC channels were visualized as fluorescent puncta in total internal reflection microscopy. With single-molecule imaging techniques, it was possible to observe photo-bleaching of individual fluorophores and to count the steps of bleaching as a measure of the stoichiometry of each CRAC channel complex. We conclude that the subunit stoichiometry in an active CRAC channel is four Orai1 molecules and two STIM1 molecules. Fluorescence resonance energy transfer experiments also showed that four Orai1 subunits form the assembled channel. From the fluorescence intensity of single fluorophores, we could estimate that our transfected HEK293 cells had almost 400,000 CRAC channels and that, when intracellular Ca 2؉ stores were depleted, the channels clustered in aggregates containing Ϸ1,300 channels, amplifying the local Ca 2؉ entry.endoplasmic reticulum ͉ fluorescence resonance energy transfer

Store-Operated Calcium Channels: New Perspectives on Mechanism and Function

“…The CAD fragment forms a tetramer in solution ), whereas the longer STIM1 cytosolic fragments generally appear as dimers (Ji et al 2008;Penna et al 2008;Muik et al 2009;Yuan et al 2009;Zhou et al 2010a) that may need to unfold to expose the CAD and activate Orai1 (Korzeniowski et al 2010;Muik et al 2011). These findings raise the intriguing possibility that on store depletion, STIM1 undergoes a conformational change that results in formation of a tetramer through interactions among multiple CADs (Covington et al 2010), an idea that remains to be tested.…”

Monomeric type I and type III transforming growth factor-β receptors and their dimerization revealed by single-molecule imaging

“…It has been demonstrated that the statistical analysis of bleaching steps of GFP-fused membrane proteins is a new way to determine the subunit stoichiometry of membranebound proteins [10,11]. From the distribution of the photobleaching steps of TβRI-GFP spots on HeLa cell membranes (from 13 cells), we found 82.1% (487 of 589 spots) bleached in one step, and 16.1% (92 of 589) bleached in two steps ( Figure 1B, 1C and Supplementary information, Figure S2).…”

“…Recent advances in single-molecule fluorescence imaging have offered a new way to analyze membrane proteins with ultrasensitivity and probe their stoichiometry in intact cells [10][11][12][13][14]. Using green fluorescent protein (GFP) to label the membrane proteins, stepwise photobleaching curves of individual proteins are counted, and a binomial distribution of photobleaching steps is obtained to investigate protein stoichiometry.…”

Grafting voltage and pharmacological sensitivity in potassium channels

“…The mEGFP tags were fused to the C-terminus of TASK3 channels and the N-terminus of the VSDs in KT channels, respectively ( Figure 2C and 2D). To maintain the density of channels low enough to minimize the chance of incidental overlap of two or more channels within a diffraction-limited area, the cells expressing TASK3 or KT channels were fixed only 3-5 h after transfection and examined by TIRF microcopy, as previously described [26,27]. In the quiescent cells, the exogenously expressed channels exhibited a punctate appearance of fluorescence on the membrane surface.…”

“…For each identified trace, the bleaching steps were determined manually by one investigator and rescored blindly by another. Other criteria were as described in [26].…”