Aequorin targeted to the endoplasmic reticulum reveals heterogeneity in luminal Ca++ concentration and reports agonist- or IP3-induced release of Ca++.
A chimeric protein (ERaeq) comprised of the invariant chain (Ii) of class II major histocompatability complex (MHC-II) and aequorin was localized in the endoplasmic reticulum (ER) of transfected human embryonal kidney 293 cells. The targeted aequorin resided in the lumen of the ER membrane system, including the nuclear cistern, and following addition of the chromophore coelenterazine underwent Ca(++)-activated chemiluminescence. The majority of chemiluminescence produced by coelenterazine treatment of ERaeq-expressing 293 cells was consumed rapidly (within 2-4 min) upon re-addition of Ca++ to coelenterazine-loaded cells, a finding consistent with very high Ca++ concentrations (approximately 10(-5)-10(-3) M Ca++ ion) inside the ER. However, following the initial rapid consumption of ERaeq chemiluminescence, the activity that remained (10-30% of total sample luminescence of permeabilized cells or 50-70% of total sample luminescence of intact cells) was found to produce a stable baseline corresponding to a Ca++ ion concentration < or = 1-2 microM. The stable baseline of luminescence observed following rapid consumption of the majority of the sample's activity was not derived from re-binding of fresh chromophore to spent photoprotein, suggesting that a minority fraction of the ER membrane system within which the ERaeq chimera was distributed contained a relatively low Ca++ concentration. Addition of IP3 to digitonin-permeabilized cells, or agonist treatment of intact cells decreased this residual signal. Luminescence recordings from cells expressing an ER-targeted aequorin with relatively high affinity for Ca++ thus reveal heterogeneity in luminal ER Ca++ concentration and permit observation of receptor- and IP3-activated release of Ca++ from the ER membrane system.
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Laboratory of Cell Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA.
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D000331:Aequorin / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015870:Gene Expression; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D000331:Aequorin; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium