The Na /Ca 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

Na /Ca exchanger (NCX) is a plasma membrane transporter that moves Ca in or out of the cell, depending on membrane potential and transmembrane ion gradients. NCX is the main pathway for Ca 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 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 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 M) reversibly attenuates electrically evoked Ca transients in FDB and caffeine-induced Ca 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 [H]ryanodine to RyR1 (IC50 5.1 0.9 M) and the cardiac isoform RyR2 (IC50 13.4 1.8 M). 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 [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. The Na /Ca exchanger (NCX) is a Ca extruder that is powered by the energy of the Na gradient across the cell membrane, which is ultimately derived from the Na /K ATPase activity. Depending on ionic concentration and membrane potential, NCX can mediate Ca efflux (forward mode) or Ca influx (reverse mode) (Blaustein and Lederer, 1999). In cardiac muscle under normal physiological conditions, NCX is mainly responsible for Ca extrusion from the myocyte. The influx component may occur either during the initial phase of the action potential or under pathological conditions such as ischemia and reperfusion injury (Lytton, 2007). NCX also plays a significant role in maintaining skeletal muscle Ca homeostasis (Fraysse et al., 2001; Sokolow et al., 2004). Three NCX isoforms have been identified, and two of these, NCX1 and NCX3, are expressed in skeletal muscle (Fraysse et al., 2001), and their activity contributes to fatigue resistance (Sokolow et al., 2004; Germinario et al., 2008). Reverse operation of NCX has also been proposed as a contributor to neuronal Ca overload during anoxia and ischemia (Stys et al., 1992). KB-R7943 is an inhibitor that is frequently used as an experimental tool to assess the contribution of all three members of the NCX family (NCX1, NCX2, and NCX3) to physiological and pathophysiological processes. Concentrations ranging from 1 to 5 M produce a half-maximal block (IC50) of the NCX reverse mode (inhibition of Ca entry), and This work was supported by the National Institutes of Health National Institute of Arthritis and Musculoskeletal and Skin Disease [Grants 1R01AR43140, 1P01-AR52354]. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.109.057265. ABBREVIATIONS: NCX, Na /Ca exchanger; FDB, flexor digitorum brevis; RyR, ryanodine receptor; ECC, excitation-contraction coupling; KB-R7943, 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate; SN-6, 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-4-thiazoli dinecarboxylic acid ethyl ester; HEK, human embryonic kidney; BLM, bilayer lipid membrane; Ry, ryanodine; FDB, flexor digitorum brevis; wt, wild type. 0026-895X/09/7603-560–568$20.00 MOLECULAR PHARMACOLOGY Vol. 76, No. 3 Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics 57265/3503546 Mol Pharmacol 76:560–568, 2009 Printed in U.S.A. 560 at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from inhibition is not selective among the three NCX isoforms (Iwamoto, 2004). KB-R7943 has been used to selectively inhibit the reverse mode of NCX to understand its contribution to cardiac ischemia and injury (Schröder et al., 1999; Seki et al., 2002; Amran et al., 2003; Baczkó et al., 2003; Iwamoto, 2004; Stys, 2004; Dietz et al., 2007; MacDonald and Howlett, 2008; O’Rourke, 2008). However, besides its effect on NCX, KB-R7943 also has off-target effects, including binding to the norepinephrine transporter, (IC50 3 M) (Matsuda et al., 2001) and block of Ba currents through Cav1.2 with inhibitory constants similar to those needed to inhibit NCX (IC50 6.8 M) (Ouardouz et al., 2005). KB-R7943 also inhibits currents through 3 4 and 7 nicotinic acetylcholine receptors (IC50 0.4 M) (Pintado et al., 2000) and inhibits members of transient receptor potential channels with an IC50 ranging from 0.5 to 1.4 M (Kraft, 2007). More recently, KB-R7943 has been also shown to inhibit the mitochondrial Ca uniporter (IC50 5.5 M) (Santo-Domingo et al., 2007). While performing short-term experiments with KB-R7943 in an attempt to pharmacologically block NCX in skeletal muscle fibers, we discovered another potent pharmacological activity of the drug: inhibition of excitation-contraction coupling (ECC). ECC in skeletal muscle is a cascade of events that is initiated by the depolarization of T-tubule membrane and activation of dihydropyridine receptor, which is necessary for activation of RyR1, intracellular Ca release channels anchored within the sarcoplasmic reticulum (Flucher and Franzini-Armstrong, 1996; Bindokas et al., 2003). Here we show that KB-R7943 interferes with ECC by directly blocking RyR1 and that its inhibitory activity extends to another broadly expressed isoform, RyR2. Materials and Methods Materials. KB-R7943, SN-6 (obtained from Tocris Bioscience, Ellisville, MO), and 4-nitrobenzylphenylether (Fig. 1) (obtained from Sigma-Aldrich, St. Louis, MO) were dissolved in dimethyl sulfoxide, giving a stock solution of 50 mM. [H]Ryanodine ([H]Ry; 50–60 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). The nominal free Ca in all basal buffers (before the addition of CaCl2 or EGTA) was measured by Ca 2 electrode calibrated to National Bureau of Standards stocks. Free Ca concentrations stated in the text were arrived based on the nominal Ca plus added CaCl2 and EGTA using the Bound and Determined Software (Brooks and Storey, 1992), which accounts for buffer composition (e.g., ATP), pH, and temperature in calculating the free Ca . Preparation of Isolated Fiber Cultures from Adult Mouse Skeletal Muscle. Flexor digitorum brevis (FDB) were dissected from adult mice (C57BL/6), and single intact FDB myofibers were enzymatically isolated as described previously (Brown et al., 2007). After isolation, the fibers were plated on Matrigel-coated plates (BD Biosciences, San Jose, CA) and maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% bovine serum albumin and 0.1 mg/ml penicillin/streptomycin (Sigma). Fibers were kept overnight in a 5% CO2 incubator, and experiments were conducted within 12 to 24 h of plating. Photometric Analysis of Ca Transients in Dissociated Fibers. FDB fibers were loaded with Fluo-4 acetoxymethyl ester (10 M, 40 min) (Invitrogen) in normal Ringer’s solution containing 146 mM NaCl, 4.7 mM KCl, 0.6 mM MgSO4, 6 mM glucose, 25 mM HEPES, 2 mM CaCl2, and 0.02% Pluronic F-127 (Invitrogen). The cells were then washed three times with imaging buffer and transferred to the stage of a Nikon Diaphot inverted microscope (Nikon, Tokyo, Japan) and illuminated at 494 nm to excite Fluo-4 with a Delta Ram wavelength-selectable light source (Photon Technology International, Lawrenceville, NJ). Fluorescence emission at 510 nm was captured from individual fibers. Electrical field stimuli were applied using two platinum electrodes fixed to opposite sides of the well and connected to a Master 8 stimulator (A.M.P.I., Jerusalem, Israel) set at a 4-V, 1-ms bipolar pulse duration over a range of frequencies (0.05–20 Hz; 10-s pulse train duration). Fluo-4 fluorescence emission from individual fibers was measured at 200 Hz using digital photometry (model D 104; Photon Technology International). Ca Imaging of HEK 293 Cells. HEK 293 cells stably expressing the wtRyR1 (wtRyR1-HEK 293) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 2 mM glutamine, 100 g/ml streptomycin, 100 U/ml penicillin, 1 mM sodium pyruvate, and 10% fetal bovine serum at 37°C under 5% CO2 (Pessah et al., 2009). wtRyR1-HEK 293 cells were loaded with 5 M Fluo-4 acetoxymethyl ester at 37°C for 30 min to measure Ca transients in an imaging buffer consisting of 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose, pH 7.4, supplemented with 0.05% bovine serum albumin. The cells were washed three times with imaging buffer and additionally incubated for 20 min at room temperature. Dye-loaded cells were washed three times with imaging buffer and imaged with a charge-coupled device camera (model 512B; Photometrics, Tucson, AZ) with a 40 objective lens attached to an IX-71 microscope (Olympus, Center Valley, PA). The sequence of images was captured and monitored using EasyRatioPro (Photon Technologies International, Birmingham, NJ). Caffeine dissolved in the imaging buffer was focally applied for 15 s using AutoMate Scientific (Berkeley, CA). KB-R7943 was dissolved in the imaging buffer, and wtRyR1-HEK 293 cells were incubated for 10 min before the application of caffeine. Preparation of RyR-Enriched Microsomes. Microsomal membrane vesicles (junctional sarcoplasmic reticulum) were prepared as described previously from New Zealand White rabbit (Mack et al., 1992) or C57BL/6J mouse (Buck et al., 1997) skeletal muscle (RyR1) and New Zealand White rabbit and mouse cardiac muscle (RyR2) (Pessah et al., 1990; Zimányi and Pessah, 1991). The preparations were stored in 10% sucrose and 10 mM HEPES, pH 7.4, at 80°C until needed. [H]Ry Binding Analysis. [H]Ry binds with high affinity and specificity to the open state of RyR1 and RyR2 and therefore provides a convenient measure of ligands that influence channel conformation (Pessah et al., 1985, 1987; Zimányi and Pessah, 1991). Microsomal membrane vesicles enriched in RyR1 (50 g protein/ml) were incubated in the presence or absence of the different NCX inhibitors in a Fig. 1. Chemical structures of the pharmacological agents used in this study. KB-R7943 Blocks Ryanodine Receptors 561 at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from solution containing 20 mM HEPES, pH 7.1, 140 mM KCl, 15 mM NaCl, 0.1 mM free Ca , and 2 nM [H]Ry. [H]Ry binding to RyR2 was measured under the same conditions described for RyR1 except that the protein concentration was 0.1 mg/ml. The binding reactions were quenched by filtration through GF/B glass fiber filters and washed twice with ice-cold harvest buffer (20 mM Tris-HCl, 140 mM KCl, 15 mM NaCl, and 0.05 M CaCl2, pH 7.1). Nonspecific binding was determined by incubating microsomes with 1000-fold excess unlabeled ryanodine. Binding curves were fitted using a nonlinear least-squares regression model with Origin 7.0 software (OriginLab Corp, Northampton, MA) to calculate EC50. Reconstitution of RyR1 Single Channels in Planar Lipid Bilayer. Single-channel kinetics in bilayer lipid membranes (BLMs) involved reconstitution of RyR1 and RyR2 from skeletal and cardiac SR membranes, respectively, and recording of channel activity was performed as reported previously (Feng et al., 2008). RyR1 channels were reconstituted into planar lipid bilayer [phosphatidylethanolamine/phosphatidylserine/phosphatidylcholine, 5:3:2 (w/w); Avanti Polar Lipids, Inc., Alabaster, AL]. Incorporation of RyR1 channel into BLM was induced by introducing SR vesicles to the cis chamber, which had a 10-fold higher Cs concentration relative to the trans chamber. The cis chamber (virtually grounded) contained 0.8 ml of 500 mM CsCl, a defined concentration of free Ca buffered with EGTA (Brooks and Storey, 1992) and 10 mM HEPES, pH 7.4, whereas the trans side (voltage input was applied) contained 50 mM CsCl, 0.1 to 3 mM CaCl2, and 10 mM HEPES, pH 7.4. Upon the fusion of SR vesicle into bilayer, cis chamber was perfused to prevent more SR fusion. Single-channel activity was measured using a patchclamp amplifier (Bilayer Clamp BC 525C; Warner Instruments, Hampden, CT) at a holding potential of 40 mV applied to the trans chamber. The amplified current signals, filtered at 1 kHz (Low-Pass Bessel Filter 8 Pole; Warner Instruments) were digitized and acquired at a sampling rate of 10 kHz (Digidata 1320A; Molecular Devices, Sunnyvale, CA). All of the recordings were made for at least 2 to 30 min under each experimental condition. The channel open probability (Po), mean open times, and mean closed-dwell times (to and tc) were calculated without additional filtering by using Clampfit, pClamp software 9.0 (Molecular Devices). KB-R7943 was added to the cis chamber (cytoplasmic side of the channel) to test its influence on channel-gating parameters.