Phenotype of a recombinant store-operated channel: highly selective permeation of Ca2+.
1. Genes related to trp (transient receptor potential) are proposed to encode store-operated channels. We examined the ionic permeation of recombinant channels formed by stable and transient expression of the TRP homologue bCCE1 in Chinese hamster ovary (CHO) cells (CHO(CCE1)) and rat basophilic leukaemia (RBL) cells, respectively. 2. Store-operated currents were activated in CHO(CCE1) cells by internal dialysis of IP3 under strong buffering of intracellular Ca2+. The action of IP3 was mimicked by thapsigargin but not by IP4. 3. With extracellular Ca2+, Na+ and Mg2+, the store-operated currents of CHO(CCE1) rectified inwardly in the presence of internal Cs+. Outward currents were not detected below +80 mV. Identical currents were recorded with external Ba2+ and also with no external Na+ and Mg2+. In the absence of external Mg2+, the inward currents showed an anomalous mole fraction behaviour between Ca2+ and Na+. Half-maximal inhibition of Na+ currents was observed with approximately 100 nM and full block with 2-5 microM external Ca2+. 4. In the parental CHO(-) cells, IP3 dialysis evoked inward currents that also displayed anomalous mole fraction behaviour between Ca2+ and Na+. However, half-maximal block of Na+ currents required 5 times higher Ca2+ concentrations in CHO(-) cells. Additionally, the density of Ca2+ and Na+ currents at -80 mV was 5 and 2 times larger in CHO(CCE1) cells, respectively. 5. In RBL cells, dialysis of IP3 evoked store-operated currents that showed 1.4-fold larger densities at -80 mV in cells expressing bCCE1. 6. The enhanced density of store-operated currents in CHO(CCE1) cells and in bCCE1-transfected RBL cells probably reflects the phenotype of CCE1. These results suggest a highly selective permeation of Ca2+ through recombinant channels formed by CCE1 either alone or in combination with endogenous channel proteins.
Institut für Pharmakologie und Toxikologie, Universität des Saarlandes, D-66421 Homburg/Saar, Germany.
J
J
Warnat
S
S
Philipp
S
S
Zimmer
V
V
Flockerzi
A
A
Cavalié
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D006224:Cricetinae; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D015640:Ion Channel Gating / Q000502:physiology; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D010641:Phenotype; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; D011994:Recombinant Proteins; D015544:Inositol 1,4,5-Trisphosphate; D012964:Sodium; D008274:Magnesium; D002118:Calcium