Abstract

Na⁺/Ca²⁺ exchanger (NCX) is a plasma membrane transporter that moves Ca²⁺ in or out of the cell, depending on membrane potential and transmembrane ion gradients. NCX is the main pathway for Ca²⁺ extrusion from excitable cells. NCX inhibitors can ameliorate cardiac ischemia-reperfusion injury and promote high-frequency fatigue of skeletal muscle, purportedly by inhibiting the Ca²⁺ 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 Ca²⁺ 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 (≤10 μM) reversibly attenuates electrically evoked Ca²⁺ transients in FDB and caffeine-induced Ca²⁺ 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 [³H]ryanodine to RyR1 (IC₅₀ = 5.1 ± 0.9 μM) and the cardiac isoform RyR2 (IC₅₀ = 13.4 ± 1.8 μM). 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 [³H]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.