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

Genaro Barrientos, Diptiman D. Bose, Wei Feng, Isela Padilla, Isaac N Pessah

Research output: Contribution to journalArticle

35 Citations (Scopus)

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 (Po), shortening mean open time, and prolonging mean closed time. KB-R7943 was more effective at blocking RyR1 with cytoplasmic conditions favoring high Po compared with those favoring low Po. 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.

Original languageEnglish (US)
Pages (from-to)560-568
Number of pages9
JournalMolecular Pharmacology
Volume76
Issue number3
DOIs
StatePublished - Sep 2009

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Ryanodine Receptor Calcium Release Channel
Ryanodine
Caffeine
Inhibitory Concentration 50
Protein Isoforms
2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate
Kidney
Membrane Transport Proteins
Skeletal Muscle Fibers
Reperfusion Injury
Membrane Potentials
Fatigue
Esters
Skeletal Muscle
Cell Membrane
Pharmacology
Ions

ASJC Scopus subject areas

  • Pharmacology
  • Molecular Medicine

Cite this

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. / Barrientos, Genaro; Bose, Diptiman D.; Feng, Wei; Padilla, Isela; Pessah, Isaac N.

In: Molecular Pharmacology, Vol. 76, No. 3, 09.2009, p. 560-568.

Research output: Contribution to journalArticle

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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 (Po), shortening mean open time, and prolonging mean closed time. KB-R7943 was more effective at blocking RyR1 with cytoplasmic conditions favoring high Po compared with those favoring low Po. 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.",
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T1 - 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

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AU - Feng, Wei

AU - Padilla, Isela

AU - Pessah, Isaac N

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N2 - 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 (Po), shortening mean open time, and prolonging mean closed time. KB-R7943 was more effective at blocking RyR1 with cytoplasmic conditions favoring high Po compared with those favoring low Po. 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.

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