The Journal of General Physiology
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Published online
doi:10.1085/jgp.200709872
The Journal of General Physiology, Vol. 130, No. 5, 525-540
The Rockefeller University Press, 0022-1295 $30.00
© Yamashita et al.
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ARTICLE

 Orai1 Mutations Alter Ion Permeation and Ca2+-dependent Fast Inactivation of CRAC Channels: Evidence for Coupling of Permeation and Gating



Megumi Yamashita, Laura Navarro-Borelly, Beth A. McNally, and Murali Prakriya

Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL 60611

Correspondence to Murali Prakriya: m-prakriya{at}northwestern.edu

Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels is an essential trigger for lymphocyte activation and proliferation. The recent identification of Orai1 as a key CRAC channel pore subunit paves the way for understanding the molecular basis of Ca2+ selectivity, ion permeation, and regulation of CRAC channels. Previous Orai1 mutagenesis studies have indicated that a set of conserved acidic amino acids in trans membrane domains I and III and in the I–II loop (E106, E190, D110, D112, D114) are essential for the CRAC channel's high Ca2+ selectivity. To further dissect the contribution of Orai1 domains important for ion permeation and channel gating, we examined the role of these conserved acidic residues on pore geometry, properties of Ca2+ block, and channel regulation by Ca2+. We find that alteration of the acidic residues lowers Ca2+ selectivity and results in striking increases in Cs+ permeation. This is likely the result of enlargement of the unusually narrow pore of the CRAC channel, thus relieving steric hindrance for Cs+ permeation. Ca2+ binding to the selectivity filter appears to be primarily affected by changes in the apparent on-rate, consistent with a rate-limiting barrier for Ca2+ binding. Unexpectedly, the mutations diminish Ca2+-mediated fast inactivation, a key mode of CRAC channel regulation. The decrease in fast inactivation in the mutant channels correlates with the decrease in Ca2+ selectivity, increase in Cs+ permeability, and enlargement of the pore. We propose that the structural elements involved in ion permeation overlap with those involved in the gating of CRAC channels.

Abbreviations used in this paper: 2-APB, 2-aminoethyldiphenyl borate; CRAC, calcium release-activated Ca2+; DVF, divalent free; NMDG, N-methyl-D-glucamine; TG, thapsigargin; TM, transmembrane; TRP, transient receptor potential; WT, wild type.


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