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Determination of K A values by controlled receptor expression in Xenopus oocytes
Author(s) -
Murakoshi Hideyuki,
Nunoki Kazuo,
Ishii Kuniaki,
Taira Norio
Publication year - 1995
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1995.tb16412.x
Subject(s) - receptor , xenopus , oocyte , chemistry , biology , endocrinology , medicine , microbiology and biotechnology , biochemistry , embryo , gene
1 In the present study we estimated the K A value of endothelin‐1 (ET‐1) for ET A ‐receptors by a new method in which the level of expression of ET A ‐receptors in Xenopus oocytes was altered in a controlled way. 2 Kvl.2 (a delayed rectifier type K channel) cRNA at the fixed concentration of 0.2 μg μl −1 was mixed with ET A ‐receptor cRNA at various concentration ratios (10 −3 ‐3). Oocytes were examined 2–4 days after the injection of the cRNA mixtures. 3 In these oocytes, ET‐1 suppressed the amplitude of Kvl.2 current in a dose‐dependent manner in the range of 0.1–100 nM; the maximum inhibition produced by ET‐1 was larger and the EC 50 value for the inhibition by ET‐1 was smaller as the mixture ratio was increased. Double‐reciprocal plots of equiactive concentrations of ET‐1 in 1/1‐ and 1/30‐injected oocytes yielded a K A for ET‐1 of 7.4 nM. The number of ET A ‐receptors in 1/30‐injected oocytes was 13% of that in 1/1‐injected oocytes, whereas the inhibition of the current in 1/30‐injected oocytes was about 60% of that in 1/1‐injected oocytes. This suggests the presence of spare receptors of ET A in the latter. 4 A saturation binding experiment estaimated a K D value of 0.1 nM for ET‐1 at ET A ‐receptors and the number of ET A ‐receptors in 1/30‐injected oocytes was 23% of that in 1/1‐injected ones. This value was not significantly different from that estimated by the above new method. However, there was a discrepancy between K A and 1 K D , which could be due to factors unique to the expression system employed in the present study.