Resveratrol Directly Controls the Activity of Neuronal Ryanodine Receptors at the Single-Channel Level

Abstract

Calcium ion dyshomeostasis contributes to the progression of many neurodegenerative diseases and represents a target for the development of neuroprotective therapies, as reported by Duncan et al. (Molecules 15(3):1168-95, 2010), LaFerla (Nat Rev Neurosci 3(11):862-72, 2002), and Niittykoshi et al. (Invest Ophthalmol Vis Sci 51(12):6387-93, 2010). Dysfunctional ryanodine receptors contribute to calcium ion dyshomeostasis and potentially to the pathogenesis of neurodegenerative diseases by generating abnormal calcium ion release from the endoplasmic reticulum, according to Bruno et al. (Neurobiol Aging 33(5):1001 e1-6, 2012) and Stutzmann et al. (J Neurosci 24(2):508-13, 2004). Since ryanodine receptors share functional and structural similarities with potassium channels, as reported by Lanner et al. (Cold Spring Harb Perspect Biol 2(11):a003996, 2010), and small molecules with anti-oxidant properties, such as resveratrol (3,5,4'-trihydroxy-trans-stilbene), directly control the activity of potassium channels, according to Wang et al. (J Biomed Sci 23(1):47, 2016), McCalley et al. (Molecules 19(6):7327-40, 2014), Novakovic et al. (Mol Hum Reprod 21(6):545-51, 2015), Li et al. (Cardiovasc Res 45(4):1035-45, 2000), Gopalakrishnan et al. (Br J Pharmacol 129(7):1323-32, 2000), and Hambrock et al. (J Biol Chem 282(5):3347-56, 2007), we hypothesized that trans-resveratrol can modulate intracellular calcium signaling through direct binding and functional regulation of ryanodine receptors. The goal of our study was to identify and measure the control of ryanodine receptor activity by trans-resveratrol. Mechanisms of calcium signaling mediated by the direct interaction between trans-resveratrol and ryanodine receptors were identified and measured with single-channel electrophysiology. Addition of trans-resveratrol to the cytoplasmic face of the ryanodine receptor increased single-channel activity at physiological and elevated pathophysiological cytoplasmic calcium ion concentrations. The open probability of the channel increases after interacting with the small molecule in a dose-dependent manner, but remains also dependent on the concentration of its physiological ligand, cytoplasmic-free calcium ions. This study provides the first evidence of a direct functional interaction between trans-resveratrol and ryanodine receptors. Such functional control of ryanodine receptors by trans-resveratrol as a novel mechanism of action could provide additional rationales for the development of novel therapeutic strategies to treat and prevent neurodegenerative diseases.

Keywords: 3,5,4′-Trihydroxy-trans-stilbene; Ca2+; Calcium; Electrophysiology; Neurodegeneration; Neuroprotection; Neuroprotective therapies; Pharmacology; RyR; Trans-resveratrol.

Figure 1.. Representative traces of mouse brain ryanodine receptor activity.

Sample 30 s current traces at resting (A, 10 nM, pCa 8) and elevated cytosolic Ca²⁺ concentrations (B, 1 μM, pCa 6; C, 10 μM, pCa 5). Channel openings are represented by downward deflections from the closed state at zero current baseline to a 2 pA subconductance state (S2) and to the fully open state (S4) at 4 pA.

Figure 2.. Channel amplitude histograms from mouse brain ryanodine receptor single channel activity recorded at varying cytosolic Ca²⁺ concentrations.

Channels were activated by 10 μM (pCa 5; black), 1 μM (pCa 6; red), and 10 nM (pCa 8; grey) free intracellular calcium concentrations. In response to increased cytosolic Ca²⁺ concentrations, the frequency of events at both the 2 pA subconductance state (S2) and the 4 pA fully open state (S4) is increased.

Figure 3.. Channel open probability of mouse brain ryanodine receptors increases in the presence of trans-resveratrol at elevated cytosolic Ca²⁺ concentrations.

(A) Representative current traces (30 s) illustrating channel openings before (top trace) and after (bottom trace) the addition of 100 μM trans-resveratrol. (B) Short (2 sec) current traces illustrate channel dwell time at baseline (top) and in the presence of 100 μM trans-resveratrol (bottom). (C) Representative amplitude histogram showing a broadening of the S2 subconductance level and an increase in fully open S4 events, indicating an increase in the number of ryanodine receptor openings in the presence of 100 μM trans-resveratrol (red) compared to baseline (black) at 10 μM Ca²⁺ / pCa 5. A fifth order polynomial distribution curve was fitted for baseline conditions (R² = 0.899; pCa 5) and for channels recorded in the presence of 100 μM trans-resveratrol (R² = 0.952; pCa 5) showing an upward shift indicative of elevated channel opening frequency. (D) Gaussian fitting (baseline, R² = 0.937; 100 μM trans-resveratrol, R² = 0.928) of the channel dwell time data from the same representative data set as in C indicates that average channel dwell time remains unchanged in the presence of trans-resveratrol (red).

Figure 4.. Quantification of brain ryanodine receptor channel open frequency, dwell time and open probability in the presence of varying concentrations of trans-resveratrol at pCa 5 / 10 μM Ca²⁺.

Normalized channel open frequency (A) and normalized average dwell time (B) for subconductance level S2 indicating no change over baseline at all concentrations of trans-resveratrol tested. 100 μM trans-resveratrol increases the channel open frequency (C) and average dwell time (D) of the fully open conductance state S4 by 134.9% and 90.2% respectively. (E) The open probability of the brain ryanodine receptor increases by 49.3% in the presence of 100 μM trans-resveratrol. All values were normalized to baseline and are averages ± standard error of the mean. One-way ANOVA with Dunnett’s post hoc test; #, p < 0.05; ##, p < 0.01; ###, p < 0.001; n=3.

Figure 5.. Trans-resveratrol potentiates brain ryanodine receptor activity through increased channel opening frequency and dwell time at low cytosolic Ca²⁺ levels.

Representative 30 s (A) and 2 s (B) ryanodine receptor channel recordings at baseline conditions (10 nM Ca²⁺, pCa 8, top) and after addition of 10 pM trans-resveratrol (bottom). (C) Representative amplitude histograms at pCa 8 (black) and pCa 8 in the presence of 10 pM trans-resveratrol (red) indicating that trans-resveratrol increases the number of fully open S4 events and 2 pA subconductance state S2 events. A fourth order polynomial distribution was fitted for baseline conditions (pCa 8; R² = 0.782) and for channels recorded in the presence of 10 pM trans-resveratrol (pCa 8; R² = 10.922) displaying an upward shift for both S2 and S4 states indicative of elevated channel opening frequency. (D) Gaussian fitting (baseline, R² = 0.946; 10 pM trans-resveratrol, R² = 0.895) of the channel dwell time data from the same representative data set as in C shows that average channel dwell time increases significantly after trans-resveratrol administration as indicated by a rightward shift of the curve.

Figure 6.. Quantification of brain ryanodine receptor channel open frequency, dwell time and open probability in the presence of varying concentrations of trans-resveratrol at pCa 8 / 10 nM Ca²⁺.

Normalized channel open frequency (A) and normalized average dwell time (B) for subconductance level S2 indicating potentiation by 10 pM trans-resveratrol (78% and 60% respectively) of a representative response. 10 pM trans-resveratrol significantly increases the channel open frequency (C) and average dwell time (D) of the fully open conductance state, S4, of a representative channel by approximately 2100% and 1200% respectively. (E) The open probability of a representative brain ryanodine receptor increases by approximately 400% in the presence of 10 pM trans-resveratrol.

Figure 7.. Potential mechanisms of action underlying trans-resveratrol binding to and control of brain ryanodine receptors.

(A) A single trans-resveratrol (RV) binding domain is accessible at low cytosolic Ca²⁺concentrations (left) resulting in minimal RyR conformational change (dark blue subunit) allowing low concentrations of trans-resveratrol to potentiate RyR activity. Higher concentrations of trans-resveratrol are needed to potentiate RyR activity when significant conformational changes due to Ca²⁺ binding at high cytosolic Ca²⁺concentrations (right). (B) The bottom panel illustrates regulation of the RyR by two separate binding domains for trans-resveratrol. The high affinity site (red, left) is accessible at resting Ca²⁺ concentrations, but is blocked when Ca²⁺ binds the receptor and induces a conformational change. The conformational change of the RyR at high cytosolic Ca²⁺concentrations uncovers a low affinity binding site for trans-resveratrol (pink, right), requiring higher concentrations of trans-resveratrol for binding and subsequent potentiation of RyR activity. The locations of each site are hypothetical and do not imply identified structural domains.