Enantiomeric specificity of (-)-2,2',3,3',6,6'-hexachlorobiphenyl toward ryanodine receptor types 1 and 2

Abstract

Polychlorinated biphenyls (PCBs) with unsymmetrical chlorine substitutions and multiple orthosubstitutions that restrict rotation around the biphenyl bond may exist in two stable enantiomeric forms.Stereospecific binding and functional modification of specific biological signaling targets have not been previously described for PCB atropisomers. We report that (-)-2,2',3,3',6,6'-hexachlorobiphenyl [(-)-PCB 136] enhances the binding of [3H]ryanodine to high-affinity sites on ryanodine receptors type 1(RyR1) and type 2 (RyR2) (EC50 values ~0.95 microM), whereas (+)-PCB 136 is inactive at < or =10 microM.(-)-PCB 136 induces a rapid release of Ca2+ from microsomal vesicles by selective sensitization of RyRs, an effect not antagonized by (+)-PCB 136. (-)-PCB 136 (500nM) enhances the activity of reconstituted RyR1 channels 3-fold by stabilizing the open and destabilizing the closed conformational states. The enantiomeric specificity is also demonstrated in intact HEK 293 cells expressing RyR1 where exposure to (-)-PCB 136 (100 nM; 12 h) sensitizes responses to caffeine, whereas (+)-PCB 136 does not. These data show enantiomeric specificity of (-)-PCB 136 toward a broadly expressed family of microsomal Ca2+ channels that may extend to other chiral noncoplanar PCBs and related structures.Evidence for enantioselective enrichment of PCBs in biological tissues that express RyR1 and RyR2channels may provide new mechanistic leads about their toxicological impacts on human health

Figure 1. (-)-PCB 136, but not (+)-PCB 136, enhances the binding of [3H]ryanodine

PCB 136 atropisomers (A) possess differential biological activities toward the junctional sarcoplasmic reticulum Ca²⁺ channel complexes RyR1 isolated from rabbit or mouse skeletal muscle (B) and RyR2 isolated from rat cardiac ventricle muscle (C) assessed by [³H]ryanodine binding analysis. PCB 136 atropisomers were tested at a concentration range between 0.1 and 10 μM at 37°C for 3 hr or 26°C for 16hr, respectively. The graphs are representatives of a total n=9 independent assays, each individual assay had triplicate samples for every data point.

Figure 2. (-)-PCB 136, but not (+)-PCB 136, triggers Ca²⁺ efflux from microsomal vesicles by selective activation of RyR1

Each cuvette contained 1500μl assay buffer including 50μg/ml rabbit junctional sarcoplasmic reticulum proteins without or with 1μM ruthenium red (RR) pre-incubated for 2 min at 37°C. After the sequential additions of 70 μM CaCl₂ and completion of active loading of extravesicular Ca²⁺ was complete (downward deflections), the indicated concentration of PCB 136 or solvent dimethyl sulfoxide (DMSO) was introduced into the assay. Upward deflections are caused by the release of sequestered Ca²⁺ released to the extravesicular medium. Approximately 10 min later, either 1μM RR, a RyR channel blocker or 200 nM thapsigargan (TG), a SR/ER Ca²⁺-ATPase blocker, was added to the transport buffer as denoted in each trace. All the experiments shown were replicated 2 or 3 times with the same results.

Figure 3. (+)-PCB 136 does not compete with (-)-PCB 136 for RyR1 activation

(-)-PCB 136 elicited rapid Ca²⁺ release from actively loaded rabbit junctional sarcoplasmic reticulum vesicles and these effects were not affected by the presence of molar excess of (+)-PCB 136. Upon the completion of the Ca²⁺ loading phase with two bolus additions of 70 μM Ca²⁺ (downward deflections), DMSO (trace a), 5μM (+)-PCB 136 (trace b), 1μM (-)-PCB 136 (trace c) or 5μM (+)-PCB 136 plus 1μM (-)-PCB 136 (trace d) was added to the respective assays. Addition of 200 nM TG approximately12 min later dissipated the Ca²⁺ gradients. Two additional independent measurements were performed with the same results.

Figure 4. Enantiospecific sensitization of RyR1 channel activity by (-)-PCB 136

Junctional SR isolated from mouse skeletal sarcoplasmic reticulum was used to reconstitute single RyR1 channels were in BLM. After recording basal channel gating activity, (+)- or (-)-PCB 136 was introduced into the cytoplasmic (cis) side of the channel. Single channel activity was recorded at a holding potential of -40mV relative to the luminal (trans) side of the channel. The open (current conducting) state of the channel is an upward deflection with “O” indicating the maximal open current amplitude. Open probability (Po), mean open (τ_o) and closed (τ_o) dwell times are denoted above the representative traces. The displayed RyR1 channels are representative of a total number of 8 independent bilayer measurements tested with (+)- or (-)-PCB 136 concentrations of 0.2 to 5μM.

Figure 5. (-)-PCB 136 selectively sensitizes caffeine responses in RyR1-expressing HEK 293 cells

(A) In-cell western analysis using monoclonal antibody (mAb) 34C that recognizes RyR1 and RyR3. A clone selected for stable expression of RyR1 binds mAb 34C whereas RyR-null HEK 293 cells do not show detectable binding of the mAb. (B) Fluo-4 fluorescence emission traces showing Ca²⁺ transient (arbitrary units) responses of individual cells to focal 10 sec application of caffeine with or without 12 hour pre-treatment to PCB 136. (C) Summary data of dose-response relationship for caffeine in control (DMSO) and PCB pretreated HEK 293 cells. Data represents dose-response relationships averaged from ≥10 individual cells. Pretreatment of RyR1-null cells with DMSO, (-)-PCB 136, or (+)-PCB 136 had no influence on responses to caffeine.