Structural Domains Influencing Sensitivity to Isothiourea Derivative Inhibitor KB-R7943 in Cardiac Na+/Ca2+ Exchanger
- Takahiro Iwamoto1,
- Satomi Kita1,
- Akira Uehara2,
- Yutaka Inoue1,
- Yuki Taniguchi1,
- Issei Imanaga2 and
- Munekazu Shigekawa1
- 1Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka, Japan (T.I., S.K., Y.I., Y.T., M.S.); and2Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (A.U., I.I.)
Abstract
KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) is a potent and selective Na+/Ca2+ exchange (NCX) inhibitor that is 3-fold more inhibitory to NCX3 than to NCX1 or NCX2. Here we searched for amino acid residues that may form the KB-R7943 receptor in the exchanger by analyzing the function of chimeras between NCX1 and NCX3 as well as of their site-directed mutants. We found that the highly conserved α-2 repeat of the exchanger is almost exclusively responsible for the difference in drug response of the isoforms. Such difference was mostly reproduced by single substitutions of residues in the α-2 repeat (V820G or Q826V in NCX1 and A809V or A809I in NCX3), suggesting their importance in drug sensitivity. Cysteine scanning mutagenesis of the α-2 repeat of NCX1 identified one residue (Gly833) that caused a large (≥ 30-fold) reduction in drug sensitivity. We found that the Gly-to-Thr substitution caused even larger reduction in drug sensitivity. Interestingly, extracellularly applied KB-R7943 at 0.8 μM markedly inhibited the whole-cell outward exchange current, whereas the drug applied intracellularly at 30 μM did not. These results suggest that KB-R7943 inhibits the exchanger from the external side in intact cells and that a region of the α-2 repeat of NCX1 containing Gly833 may participate in the formation of the drug receptor. Because we suggested previously that Gly833 is accessible from the inside of a cell, the results raised an interesting possibility that this residue may alter its position during Na+/Ca2+ exchange in such a way that it becomes accessible to external drug.
Footnotes
-
Send reprint requests to: Dr. Munekazu Shigekawa, Department of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5, Suita, Osaka 565-8565, Japan. E-mail: shigekaw{at}ri.ncvc.go.jp
-
This work was supported by Grants-in-Aid for Scientific Research [10470013 (M.S.) and 12670102 (T.I.)] from the Ministry of Education, Science and Culture of Japan; Research Grant for Cardiovascular Diseases (11-C) from the Ministry of Health and Welfare (M.S.); and grants from the Uehara Memorial Foundation (M.S.), the Mochida Memorial Foundation (T.I.), and the Japan Cardiovascular Research Foundation (T.I.).
- Abbreviations:
- NCX
- Na+/Ca2+exchanger
- TM
- transmembrane
- NCKX
- Na+/Ca2+/K+ exchanger
- KB-R7943
- (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate)
- Na+i
- intracellular Na+
- Ca2+o
- extracellular Ca2+
- BSS
- balanced salt solution
-
- Received September 28, 2000.
- Accepted November 22, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
