title: Orai1 mutations alter ion permeation and Ca2+-dependent fast inactivation of CRAC channels: evidence for coupling of permeation and gating.
abstract: Ca(2+) entry through store-operated Ca(2+) release-activated Ca(2+) (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 Ca(2+) 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 Ca(2+) 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 Ca(2+) block, and channel regulation by Ca(2+). We find that alteration of the acidic residues lowers Ca(2+) 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. Ca(2+) binding to the selectivity filter appears to be primarily affected by changes in the apparent on-rate, consistent with a rate-limiting barrier for Ca(2+) binding. Unexpectedly, the mutations diminish Ca(2+)-mediated fast inactivation, a key mode of CRAC channel regulation. The decrease in fast inactivation in the mutant channels correlates with the decrease in Ca(2+) 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.
authors.affiliation: Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
authors.forename: Megumi
authors.initials: M
authors.lastname: Yamashita
authors.forename: Laura
authors.initials: L
authors.lastname: Navarro-Borelly
authors.forename: Beth A
authors.initials: BA
authors.lastname: McNally
authors.forename: Murali
authors.initials: M
authors.lastname: Prakriya
mesh_terms: D001671:Biochemistry / Q000379:methods; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D015220:Calcium Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology; D002586:Cesium / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015640:Ion Channel Gating; D007477:Ions; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D009154:Mutation; D000071740:ORAI1 Protein; D018408:Patch-Clamp Techniques; D010539:Permeability; D014162:Transfection
chemical_list: D015220:Calcium Channels; D007477:Ions; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D002586:Cesium; D002118:Calcium