RT Journal Article
SR Electronic
T1 Structural Domains Influencing Sensitivity to Isothiourea Derivative Inhibitor KB-R7943 in Cardiac Na<sup>+</sup>/Ca<sup>2+</sup> Exchanger
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 524
OP 531
DO 10.1124/mol.59.3.524
VO 59
IS 3
A1 Takahiro Iwamoto
A1 Satomi Kita
A1 Akira Uehara
A1 Yutaka Inoue
A1 Yuki Taniguchi
A1 Issei Imanaga
A1 Munekazu Shigekawa
YR 2001
UL http://molpharm.aspetjournals.org/content/59/3/524.abstract
AB 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.