Dataset Information

Tetrameric Orai1 is a teardrop-shaped molecule with a long, tapered cytoplasmic domain.

ABSTRACT: The Ca(2+) release-activated Ca(2+) channel is a principal regulator of intracellular Ca(2+) rise, which conducts various biological functions, including immune responses. This channel, involved in store-operated Ca(2+) influx, is believed to be composed of at least two major components. Orai1 has a putative channel pore and locates in the plasma membrane, and STIM1 is a sensor for luminal Ca(2+) store depletion in the endoplasmic reticulum membrane. Here we have purified the FLAG-fused Orai1 protein, determined its tetrameric stoichiometry, and reconstructed its three-dimensional structure at 21-A resolution from 3681 automatically selected particle images, taken with an electron microscope. This first structural depiction of a member of the Orai family shows an elongated teardrop-shape 150A in height and 95A in width. Antibody decoration and volume estimation from the amino acid sequence indicate that the widest transmembrane domain is located between the round extracellular domain and the tapered cytoplasmic domain. The cytoplasmic length of 100A is sufficient for direct association with STIM1. Orifices close to the extracellular and intracellular membrane surfaces of Orai1 seem to connect outside the molecule to large internal cavities.

SUBMITTER: Maruyama Y

PROVIDER: S-EPMC2679469 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Tetrameric Orai1 is a teardrop-shaped molecule with a long, tapered cytoplasmic domain.

Maruyama Yuusuke Y Ogura Toshihiko T Mio Kazuhiro K Kato Kenta K Kaneko Takeshi T Kiyonaka Shigeki S Mori Yasuo Y Sato Chikara C

The Journal of biological chemistry 20090316 20

The Ca(2+) release-activated Ca(2+) channel is a principal regulator of intracellular Ca(2+) rise, which conducts various biological functions, including immune responses. This channel, involved in store-operated Ca(2+) influx, is believed to be composed of at least two major components. Orai1 has a putative channel pore and locates in the plasma membrane, and STIM1 is a sensor for luminal Ca(2+) store depletion in the endoplasmic reticulum membrane. Here we have purified the FLAG-fused Orai1 pr ...[more]

PMID: 19289460

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Project description:BackgroundClC-Kb and ClC-Ka are homologous chloride channels that facilitate chloride homeostasis in the kidney and inner ear. Disruption of ClC-Kb leads to Bartter's Syndrome, a kidney disease. A point mutation in ClC-Kb, R538P, linked to Bartter's Syndrome and located in the C-terminal cytoplasmic domain was hypothesized to alter electrophysiological properties due to its proximity to an important membrane-embedded helix.Methodology/principal findingsTwo-electrode voltage clamp experiments were used to examine the electrophysiological properties of the mutation R538P in both ClC-Kb and ClC-Ka. R538P selectively abolishes extracellular calcium activation of ClC-Kb but not ClC-Ka. In attempting to determine the reason for this specificity, we hypothesized that the ClC-Kb C-terminal domain had either a different oligomeric status or dimerization interface than that of ClC-Ka, for which a crystal structure has been published. We purified a recombinant protein corresponding to the ClC-Kb C-terminal domain and used multi-angle light scattering together with a cysteine-crosslinking approach to show that the dimerization interface is conserved between the ClC-Kb and ClC-Ka C-terminal domains, despite the fact that there are several differences in the amino acids that occur at this interface.ConclusionsThe R538P mutation in ClC-Kb, which leads to Bartter's Syndrome, abolishes calcium activation of the channel. This suggests that a significant conformational change--ranging from the cytoplasmic side of the protein to the extracellular side of the protein--is involved in the Ca(2+)-activation process for ClC-Kb, and shows that the cytoplasmic domain is important for the channel's electrophysiological properties. In the highly similar ClC-Ka (90% identical), the R538P mutation does not affect activation by extracellular Ca(2+). This selective outcome indicates that ClC-Ka and ClC-Kb differ in how conformational changes are translated to the extracellular domain, despite the fact that the cytoplasmic domains share the same quaternary structure.

Dataset Information

Tetrameric Orai1 is a teardrop-shaped molecule with a long, tapered cytoplasmic domain.

Publications

Tetrameric Orai1 is a teardrop-shaped molecule with a long, tapered cytoplasmic domain.

Similar Datasets

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