Functional Analysis of Orai1 Concatemers Supports a Hexameric Stoichiometry for the CRAC Channel

Abstract

Store-operated Ca entry occurs through the binding of the endoplasmic reticulum (ER) Ca sensor STIM1 to Orai1, the pore-forming subunit of the Ca release-activated Ca (CRAC) channel. Although the essential steps leading to channel opening have been described, fundamental questions remain, including the functional stoichiometry of the CRAC channel. The crystal structure of Drosophila Orai indicates a hexameric stoichiometry, while studies of linked Orai1 concatemers and single-molecule photobleaching suggest that channels assemble as tetramers. We assessed CRAC channel stoichiometry by expressing hexameric concatemers of human Orai1 and comparing in detail their ionic currents to those of native CRAC channels and channels generated from monomeric Orai1 constructs. Cell surface biotinylation results indicated that Orai1 channels in the plasma membrane were assembled from intact hexameric polypeptides and not from truncated protein products. In addition, the L273D mutation depressed channel activity equally regardless of which Orai1 subunit in the concatemer carried the mutation. Thus, functional channels were generated from intact Orai1 hexamers in which all subunits contributed equally. These hexameric Orai1 channels displayed the biophysical fingerprint of native CRAC channels, including the distinguishing characteristics of gating (store-dependent activation, Ca-dependent inactivation, open probability), permeation (ion selectivity, affinity for Ca block, La sensitivity, unitary current magnitude), and pharmacology (enhancement and inhibition by 2-aminoethoxydiphenyl borate). Because permeation characteristics depend strongly on pore geometry, it is unlikely that hexameric and tetrameric pores would display identical Ca affinity, ion selectivity, and unitary current magnitude. Thus, based on the highly similar pore properties of the hexameric Orai1 concatemer and native CRAC channels, we conclude that the CRAC channel functions as a hexamer of Orai1 subunits.

The STIM-Orai Pathway: Conformational Coupling Between STIM and Orai in the Activation of Store-Operated Ca2+ Entry

“…Thus, function of the concatemeric hexamer is sensitive to replacement of each of the six residues with the pore-dead E106A mutation indicating that all the residues are required for channel function. Interestingly, the function of the other shorter concatemers (pentamers, tetramers, trimers, and dimers) resulted from the first two N-terminal subunits being unexpectedly fed into a hexameric structure composed of a “trimer of dimers.” Our results strongly suggested that the functional Orai1 channel is indeed a hexamer (Cai et al 2016) agreeing with other studies published at the same time (Yen et al 2016)…”

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

“…The conclusion from the crystal structure that Orai is a hexamer was reinforced by determination of the molecular mass of the purified channel complex using light scattering/UV absorbance/refractive index measurements and by protein cross-linking of Drosophila Orai expressed in mammalian HEK293 cell membranes that produced a clearly resolvable ladder of multimers up to the hexamer (Hou et al 2012). Two careful new studies examining Orai1 concatemers explain the earlier concatemer results and support a hexameric structure (Yen et al 2016; Cai et al 2016). …”

ER-plasma membrane junctions: Why and how do we study them?

“…Consistently, atomic force microscopy revealed that human Orai1 assembles as a hexamer and STIM1 binds to Orai1 hexamer with six-fold symmetry [84]. A recent study using Orai1 concatemers further supported a hexameric stoichiometry for the SOC channel [85]. It has been postulated that Orai1 diffuses passively into ER-PM junctions, and is then trapped and activated by prior translocated STIM1 to elicit SOCE.…”