The Na⁺/Ca²⁺Exchange Inhibitor 2-(2-(4-(4-Nitrobenzyloxy)phenyl)ethyl)isothiourea Methanesulfonate (KB-R7943) Also Blocks Ryanodine Receptors Type 1 (RyR1) and Type 2 (RyR2) Channels

Abstract

Naϩ /Ca 2ϩ exchanger (NCX) is a plasma membrane transporter that moves Ca 2ϩ in or out of the cell, depending on membrane potential and transmembrane ion gradients. NCX is the main pathway for Ca 2ϩ extrusion from excitable cells. NCX inhibitors can ameliorate cardiac ischemia-reperfusion injury and promote high-frequency fatigue of skeletal muscle, purportedly by inhibiting the Ca 2ϩ inward mode of NCX. Here we tested two known NCX inhibitors, 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)-isothiourea methanesulfonate (KB-R7943) and the structurally related 2-[[4-[(4-Nitrophenyl)methoxy]phenyl]methyl]-4-thiazoli dinecarboxylic acid ethyl ester (SN-6), for their influence on electrically or caffeine-evoked Ca 2ϩ transients in adult dissociated flexor digitorum brevis (FDB) skeletal muscle fibers and human embryonic kidney (HEK) 293 cells that have stable expression of type 1 ryanodine receptor (RyR1). KB-R7943 (Յ10 M) reversibly attenuates electrically evoked Ca 2ϩ transients in FDB and caffeine-induced Ca 2ϩ release in HEK 293, whereas the structurally related NCX inhibitor SN-6 does not, suggesting that KB-R7943 directly inhibits RyR1. In support of this interpretation, KB-R7943 inhibits high-affinity binding of [ 3 H]ryanodine to RyR1 (IC 50 ϭ 5.1 Ϯ 0.9 M) and the cardiac isoform RyR2 (IC 50 ϭ 13.4 Ϯ 1.8 M). KB-R7943 interfered with the gating of reconstituted RyR1 and RyR2 channels, reducing open probability (P o ), shortening mean open time, and prolonging mean closed time. KB-R7943 was more effective at blocking RyR1 with cytoplasmic conditions favoring high P o compared with those favoring low P o . SN-6 has negligible activity toward altering [ 3 H]ryanodine binding of RyR1 and RyR2. Our results identify that KB-R7943 is a reversible, activity-dependent blocker of the two most broadly expressed RyR channel isoforms and contributes to its pharmacological and therapeutic activities.

Tetanic Ca2+ transient differences between slow- and fast-twitch mouse skeletal muscle fibres: a comprehensive experimental approach

“…This is suggested by: (i) a similar effect of KB-R7943 to that of FCCP and Ru360, and (ii) the concentration of KB-R7943 used can inhibit MCU in some cells (Santo-Domingo et al 2007). In fact, KB-R7943 has been shown not to be as specific as expected (Iwamoto et al 1996;Linck et al 1998;Arakawa et al 2000;Santo-Domingo et al 2007; Barrientos et al 2009) and our results and other's ( Barrientos et al 2009) discourage its use in intact skeletal muscle fibres.…”

Gene Dose Influences Cellular and Calcium Channel Dysregulation in Heterozygous and Homozygous T4826I-RYR1 Malignant Hyperthermia-susceptible Muscle

“…Single intact myofibers were enzymatically isolated as described previously (19,33). In order to reduce stress-activated SR Ca 2ϩ release during isolation, especially in T4826I-RYR1 fibers, 10 M dantrolene was included in the initial dissociation medium.…”

Crystal structure of (Z)-3-(adamantan-1-yl)-1-(3-chlorophenyl)-S-benzylisothiourea, C24H27ClN2S

“…Several substituted isothiourea derivatives were reported to possess significant cardiovascular properties as nitric oxide synthase inhibitors [1][2][3][4][5], and calcium channel blockers [6]. Isothiourea derivatives were also reported to display potent antimicrobial [7,8], anticancer [9,10], and anti-anthelmintic [11] activities.…”