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Functional Analysis of Orai1 Concatemers Supports a Hexameric Stoichiometry for the CRAC Channel.
Biophys J. 2016 Nov 01; 111(9):1897-1907.BJ

Abstract

Store-operated Ca2+ entry occurs through the binding of the endoplasmic reticulum (ER) Ca2+ sensor STIM1 to Orai1, the pore-forming subunit of the Ca2+ release-activated Ca2+ (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, Ca2+-dependent inactivation, open probability), permeation (ion selectivity, affinity for Ca2+ block, La3+ 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 Ca2+ 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.

Authors+Show Affiliations

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California; Graduate Program in Immunology, Stanford University School of Medicine, Stanford, California.Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California.Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California; Graduate Program in Immunology, Stanford University School of Medicine, Stanford, California. Electronic address: rslewis@stanford.edu.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

27806271

Citation

Yen, Michelle, et al. "Functional Analysis of Orai1 Concatemers Supports a Hexameric Stoichiometry for the CRAC Channel." Biophysical Journal, vol. 111, no. 9, 2016, pp. 1897-1907.
Yen M, Lokteva LA, Lewis RS. Functional Analysis of Orai1 Concatemers Supports a Hexameric Stoichiometry for the CRAC Channel. Biophys J. 2016;111(9):1897-1907.
Yen, M., Lokteva, L. A., & Lewis, R. S. (2016). Functional Analysis of Orai1 Concatemers Supports a Hexameric Stoichiometry for the CRAC Channel. Biophysical Journal, 111(9), 1897-1907. https://doi.org/10.1016/j.bpj.2016.09.020
Yen M, Lokteva LA, Lewis RS. Functional Analysis of Orai1 Concatemers Supports a Hexameric Stoichiometry for the CRAC Channel. Biophys J. 2016 Nov 1;111(9):1897-1907. PubMed PMID: 27806271.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Functional Analysis of Orai1 Concatemers Supports a Hexameric Stoichiometry for the CRAC Channel. AU - Yen,Michelle, AU - Lokteva,Ludmila A, AU - Lewis,Richard S, PY - 2016/04/06/received PY - 2016/08/29/revised PY - 2016/09/12/accepted PY - 2016/11/3/pubmed PY - 2017/6/29/medline PY - 2016/11/3/entrez SP - 1897 EP - 1907 JF - Biophysical journal JO - Biophys. J. VL - 111 IS - 9 N2 - Store-operated Ca2+ entry occurs through the binding of the endoplasmic reticulum (ER) Ca2+ sensor STIM1 to Orai1, the pore-forming subunit of the Ca2+ release-activated Ca2+ (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, Ca2+-dependent inactivation, open probability), permeation (ion selectivity, affinity for Ca2+ block, La3+ 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 Ca2+ 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. SN - 1542-0086 UR - https://www.unboundmedicine.com/medline/citation/27806271/Functional_Analysis_of_Orai1_Concatemers_Supports_a_Hexameric_Stoichiometry_for_the_CRAC_Channel_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0006-3495(16)30820-7 DB - PRIME DP - Unbound Medicine ER -