Minding the calcium store: Ryanodine receptor activation as a convergent mechanism of PCB toxicity

Abstract

Chronic low-level polychlorinated biphenyl (PCB) exposures remain a significant public health concern since results from epidemiological studies indicate that PCB burden is associated with immune system dysfunction, cardiovascular disease, and impairment of the developing nervous system. Of these various adverse health effects, developmental neurotoxicity has emerged as a particularly vulnerable endpoint in PCB toxicity. Arguably the most pervasive biological effects of PCBs could be mediated by their ability to alter the spatial and temporal fidelity of Ca2+ signals through one or more receptor-mediated processes. This review will focus on our current knowledge of the structure and function of ryanodine receptors (RyRs) in muscle and nerve cells and how PCBs and related non-coplanar structures alter these functions. The molecular and cellular mechanisms by which non-coplanar PCBs and related structures alter local and global Ca2+ signaling properties and the possible short and long-term consequences of these perturbations on neurodevelopment and neurodegeneration are reviewed.

Fig 1

Coplanar structure of dioxin and two examples of dioxin-like PCBs. Non-dioxin-like PCBs have ≥ 2 chlorine substitutions in the ortho-position that introduce steric hindrance thereby promoting non-coplanar geometry, as typified by PCB 95 and PCB 153. 3-D projections were calculated using the Molecular Dynamics Tool of ChemIDplus Advanced (Nat. Lib. Med.).

Fig 2

Relative concentration of 28 non-coplanar PCBs needed to double [³H]ryanodine binding to ryanodine receptor type 1 (RyR1; black bars) and their corresponding occurrence in Fox River fish, marsh sediments, and human serum (red bars). PCBs in parentheses are co-eluting congeners.

Fig 3

Several proteins interact with RyRs to regulate their function as high conductance Ca²⁺ channels in striated muscle. The large cytoplasmic assembly (“junctional foot”) interacts with ion channels in the plasma membrane, cytoplasmic signaling proteins, and cytoplasmic enzymes that regulate phosphorylation and redox sensing. The transmembrane assembly that anchors RyRs to the ER/SR interacts with proteins that fine tune communication with the Ca²⁺ stores within the SR/ER lumen. For clarity, interactions have been left out of the schematic.

Fig 4

(A) Electron micrograph of the T-tubule/SR junction of negatively stained skeletal myotubes. Arrows indicate the position of densely staining “junctional feet” that are the large cytoplasmic domain of a row of RyR1s that span the junctional space between the two membranes (adapted from (Protasi, Franzini-Armstrong, & Allen, 1998)). (B) 3-D model of the relative orientation of four Ca_V1.1 (i.e., α1s) L-type Ca²⁺ channel subunits (brown) and RyR1 (green) based on cyroEM reconstruction studies (adapted from (Wolf, Eberhart, Glossmann, Striessnig, & Grigorieff, 2003).

Fig 5

Skeletal myotubes acutely exposed to 5μM PCB 95 exhibited significantly higher Ca²⁺ transient amplitudes evoked by low frequency (0.1Hz) electrical pulse trains (A&B; p<0.05) and a failure to recover their original baseline (i.e., resting Ca²⁺ level) compared to the corresponding control period when solvent (DMSO) alone was perfused (Ctrl). (C) Responses to 10 s electrical pulse trains of 2.5 or 5Hz resulted in significantly higher transient amplitudes compared to the corresponding control (Ctrl) period. Ectopic Ca²⁺ transients (red arrows) are frequently observed soon after electrical stimuli ceased in the PCB exposed myotubes and were not observed in control. Adapted from (Cherednichenko, 2009).

Fig 6

(A) 3-D structure of RyR1 in the closed state at 10Å resolution showing the location of the FKBP12 docking site near domain 9 (adapted from (Samso, Feng, Pessah, & Allen, 2009). (B) PCB 95 triggered Ca²⁺ release from skeletal junctional SR is inhibited by pre-incubating with rapamycin that disrupts the FKBP12/RyR1 complex (adapted from (Wong & Pessah, 1997)

Fig 7

PCB 95 directly stabilizes the fully open (conducting) conformation of the RyR1/FKBP12 channel complex reconstituted in the bilayer lipid membrane preparation, whereas EGTA fully closes the channel (left and middle panels). Right panels show the corresponding structural shifts calculated from cryoEM reconstruction of single hydrated particles showing the main constrictions along the ion pathway in the closed and open states. The cytosolic constriction (cc) relaxes and the inner branches (ib) become more separated in the open state. Adapted from (Samso et al., 2009)

Fig 8

Bastadin 5 showing “eastern: and “western” dibromocatechol ethers (red boxes) that are the putative pharmacophores for RyR1. The structure of the antibacterial triclosan is shown in the lower right.

Fig 9

Bastadin 5 in the presence of a ryanodine concentration that blocks RyR1 channels reduces resting Ca²⁺ to a greater extent in cells expressing missense mutations that confer MH susceptibility to humans than in cell expressing wild type RyR1 (Wt). *p<0.05 adapted from (T. Yang et al., 2007)

Fig 10

Dose response relationship of selected PCBs towards enhancing the binding of [³H]Ry to RyR1 showing the importance of ortho-substitutions (non-coplanarity (left panel) and substitutions at the para position.

Fig 11

Correlation between the PCB concentration needed to double [³H]Ry binding to RyR1 and the initial rate of PCB-induced Ca²⁺ efflux from SR vesicles (data for each congener were normalized to respective parameters obtained with PCB 95). BZ numbers are given, Adapted from (Pessah et al., 2006).

Fig 12

(A) PCB 136 is chiral because the asymmetric distribution of chlorines prevents interconversion of its (+) and (−) atropisomers. Upon separation, (−) PCB 136 was found to be active towards enhancing the activity of RyR1 (B) and RyR2 (C), whereas (+) PCB 136 was not active. Adapted from (Pessah et al., 2009)

Fig 13

Proposed relationship between intracellular Ca2+ concentration and dendritic growth. Adapted from (Segal, Korkotian, & Murphy, 2000).

Fig 14. Developmental A1254 exposure interferes with normal dendritic growth and experience-dependent dendritic plasticity

Dendritic morphology was analyzed among P31 rats trained in the Morris water maze (Maze) and among littermates identically housed and exposed but not trained (Non-Maze). As seen in representative camera lucida drawings of the basilar dendritic arbor of cortical neurons, developmental exposure to A1254 at 1 or 6 mg/kg/d in the dam’s diet throughout gestation and lactation significantly increased dendritic arborization relative to vehicle controls (0 mg/kg/d A1254). Maze training significantly increased dendritic complexity among animals in the control group but this experience-dependent plasticity was blocked among animals in the A1254 treatment groups with a more pronounced effect observed in the lower treatment group. Data are presented as the mean±SEM (N=17–21 neurons per group). The percent changes in dendritic length were calculated using data obtained from 17–21 neurons per treatment group. The percent change in dendritic length as a function of maze training was calculated as the difference in dendritic length of neurons in maze-trained animals versus non-maze-trained animals divided by the dendritic length of neurons in maze-trained animals multiplied by 100. *p<0.05; **p<0.01; ***p<0.001. From (D. Yang et al., 2009)

Figure 15

Activity increases intracellular Ca2+ via NMDA receptor activation and calcium-induced calcium release, which alters dendritic growth via transcriptional or translational mechanisms. The former involves CaMK I activation and enhanced CREB-dependent Wnt transcription; Wnt binds the Frizzled receptor to activate downstream effector molecules β-catenin, JNK and Rac. The latter involves Ca2+-dependent activation of mTOR, which relieves repression of initiation factor-4E by 4EBP1. mTOR is regulated by FKBP12, which is targeted by non-coplanar PCBs.

Fig 16. Mechanisms by which non-coplanar PCBs might induce apoptotic DNA fragmentation

Specific noncoplanar PCBs may directly activate the ryanodine receptor (RyR) causing release of Ca²⁺ from endoplasmic reticular (ER) stores. Increased cytoplasmic Ca²⁺ activates caspases resulting in apoptosis. Increased cytoplasmic Ca²⁺ may also cause increased mitochondrial Ca²⁺ influx, which increases generation of reactive oxygen species (ROS) thereby promoting caspase-dependent apoptosis. Alternatively, or in addition, PCBs may generate ROS directly, which then increase cytoplasmic levels of Ca²⁺ via activation of RyRs. Blocking the L-type voltage-sensitive Ca²⁺ channel with verapamil or the NMDA receptor with APV does not have any effect on PCB-induced DNA fragmentation, suggesting that, in this model system, extracellular calcium is not involved in the apoptotic signaling pathway.

Figure 17

3-D projections of Bisphenol A, triclosan, and 2,2,′,4,4′-tetrabromodiphenylether (BDE-47). 3-D projections were calculated with the Molecular Dynamics Tool of ChemIDplus Advanced (Nat. Lib. Med.).