Exploring the biophysical evidence that mammalian two pore channels are NAADP-activated calcium-permeable channels

Nicotinic acid adenine dinucleotide phosphate (NAADP) potently releases Ca2+ from acidic intracellular endo-lysosomal Ca2+-stores. It is widely accepted that two types of two pore channels, termed TPC1 and TPC2, are responsible for the NAADP-mediated Ca2+-release but the underlying mechanisms regulating their gating appear to be different. For example, although both TPC1 and TPC2 are activated by NAADP, TPC1 appears to be additionally regulated by cytosolic Ca2+. Ion conduction and permeability also differ markedly. TPC1 and TPC2 are permeable to a range of cations although biophysical experiments suggest that TPC2 is slightly more selective for Ca2+ over K+ than TPC1 and hence capable of releasing greater quantities of Ca2+ from acidic stores. TPC1 is also permeable to H+ and therefore may play a role in regulating lysosomal and cytosolic pH, possibly creating localised acidic domains. The significantly different gating and ion conducting properties of TPC1 and TPC2 suggest that these two ion channels may play complementary physiological roles as Ca2+ release channels of the endo-lysosomal system.PostprintPeer reviewe

Dysregulated Zn2+ homeostasis impairs cardiac type-2 ryanodine receptor and mitsugumin 23 functions, leading to sarcoplasmic reticulum Ca2+ leakage

SJP is supported by a Royal Society of Edinburgh Biomedical Fellowship. Benedict Reilly-O’Donnell is supported by a University of St Andrews 600th Anniversary Scholarship. This work was supported by the British Heart Foundation (grant no: FS/14/69/31001 to SJP) and the Japan Society for the Promotion of Science (Core-to-Core Program awarded to HT).Aberrant Zn2+ homeostasis is associated with dysregulated intracellular Ca2+ release, resulting in chronic heart failure. In the failing heart a small population of cardiac ryanodine receptors (RyR2) displays sub-conductance-state gating leading to Ca2+ leakage from sarcoplasmic reticulum (SR) stores, which impairs cardiac contractility. Previous evidence suggests contribution of RyR2-independent Ca2+ leakage through an uncharacterized mechanism. We sought to examine the role of Zn2+ in shaping intracellular Ca2+ release in cardiac muscle. Cardiac SR vesicles prepared from sheep or mouse ventricular tissue were incorporated into phospholipid bilayers under voltage-clamp conditions, and the direct action of Zn2+ on RyR2 channel function was examined. Under diastolic conditions, the addition of pathophysiological concentrations of Zn2+ (≥2 nm) caused dysregulated RyR2-channel openings. Our data also revealed that RyR2 channels are not the only SR Ca2+-permeable channels regulated by Zn2+. Elevating the cytosolic Zn2+ concentration to 1 nm increased the activity of the transmembrane protein mitsugumin 23 (MG23). The current amplitude of the MG23 full-open state was consistent with that previously reported for RyR2 sub-conductance gating, suggesting that in heart failure in which Zn2+ levels are elevated, RyR2 channels do not gate in a sub-conductance state, but rather MG23-gating becomes more apparent. We also show that in H9C2 cells exposed to ischemic conditions, intracellular Zn2+ levels are elevated, coinciding with increased MG23 expression. In conclusion, these data suggest that dysregulated Zn2+ homeostasis alters the function of both RyR2 and MG23 and that both ion channels play a key role in diastolic SR Ca2+ leakage.Publisher PDFPeer reviewe

Investigating the role of Zn²⁺ in regulating the function of intracellular Ca²⁺-release channels

The tightly regulated openings of the cardiac ryanodine receptor (RyR2) help to ensure that intracellular Ca²⁺- release from the sarcoplasmic reticulum (SR) can only occur when heart contractions are required. Usually this process is self-regulatory, where Ca²⁺ both activates and inhibits release of further Ca²⁺ from the SR. In the progression of heart failure some of this control is lost and in rest periods Ca²⁺ can leak from the SR into the cytosol. Recent evidence has suggested that Zn²⁺- dyshomeostasis may contribute to SR Ca²⁺- leak but the underlying mechanism is unclear. Using single channel electrophysiological studies in combination with live cell imaging of HEK 293 and fibroblasts, this study reveals that Zn²⁺, along with Ca²⁺ and the inhibitor Mg²⁺, plays a physiological role in the grading of Ca²⁺- release via RyR2. Importantly the data reveal that pathophysiological concentrations of Zn²⁺ (> 100pM) within the cytosol remove the requirement of Ca²⁺ to activate RyR2, resulting in irregular channel activity even in the presence of Mg²⁺. This increase in channel open probability due to Zn²⁺ is known to be associated with increased Ca²⁺- release events such as Ca²⁺ sparks suggesting that Zn²⁺ is a regulator of the SR Ca²⁺-leak current. A potential source of releasable Zn²⁺, which could modulate RyR2 activity in cardiomyocytes, are the acidic organelles (endosomes and lysosomes). This study provides key evidence that the two pore channels (TPCs), which are expressed on the surface of these organelles, are candidate channels for ligand-gated release of Zn²⁺. Importantly this research demonstrates that dysregulated Zn²⁺ homeostasis, resulting in elevated Zn²⁺ within the lysosome, has severe consequences upon cellular Ca²⁺- release from fibroblasts, which is primarily the result of Zn²⁺ acting as a pore blocker of TPC2. Together these data reveal a key role of Zn²⁺ as a second messenger which can regulate intracellular Ca²⁺- release in both health and disease

Exploring a novel role for zinc in modulation of sarcoplasmic reticulum calcium release in skeletal muscle

Aberrant Ca2+ release from sarcoplasmic reticulum Ca2+ stores due to dysfunction in the type-1 ryanodine receptor (RyR1) is implicated in the pathophysiology of muscular dystrophy (1). Recently it has been suggested that dynamic alterations in intracellular Zn2+ and Ca2+ homeostasis play a key role in the pathogenesis of dystrophies (2). The role of physiological levels of Zn2+ in modulating RyR1-channel gating has never been considered. We therefore set out to study the effects of Zn2+ on RyR1-channel function. RyR1 channels were prepared from guinea pig skeletal muscle and incorporated into planar phosphatidylethanolamine lipid bilayers under voltage-clamp conditions using previously described techniques (3). Single channel recordings were acquired under standard experimental conditions of 250 mM HEPES, 80 mM Tris, pH 7.2 (free [Ca2+] 10 µM) at the cytoplasmic face (cis-chamber) and 250 mM Glutamic acid, 10 mM HEPES, pH 7.2 with Ca(OH)2 (free [Ca2+] ≈50 mM) at the luminal (trans-chamber) face of the channel. The cis-chamber was held at 0 mV relative to ground. Channel open probability (Po) was determined over 3 minutes, and a Student's t-test used to determine statistical significance between mean values. Sequential addition of cytosolic Zn2+ in the range from 1 nM to 1 µM increased RyR1 Po in a dose dependent manner. Given that low concentrations of Zn2+ (10 nM) significantly increased channel Po from 0.018 ± 0.014 to 0.162 ± 0.038 (SEM, n=3, P<0.05) this indicates that Zn2+ has high affinity for RyR1. At concentrations above 1 µM the effect of Zn2+ appeared to inhibit channel activity. Regarding the mechanism of how Zn2+ modulates RyR1 function, we show that Zn2+ primarily increases Po by increasing the frequency of channel openings. The mean channel open time was 1.7 ± 0.5 in the absence and 2.1 ± 0.1, 2.3 ± 0.3, and 2.7 ± 0.5 ms (SEM, n = 3) in the presence of 1 nM, 10 nM and 100 nM Zn2+ respectively, suggesting that Zn2+ increases Po by sensitising the channel to cytosolic Ca2+. We also found that the presence of cytosolic Ca2+ (10 µM) was an absolute requirement for channel activation. At all Zn2+ doses that we tested (≤100 nM) lowering cytosolic Ca2+ to a sub-activating concentration (≈4 nM) by the addition of 1 mM BAPTA, reduced channel Po to zero (n=3). Our data reveal that Zn2+ is a high affinity effector of RyR1 that increases channel Po by modulating the sensitivity of the channel to cytosolic Ca2+. We suggest that perturbation of Zn2+-homeostasis will lead to aberrant Ca2+-release through inappropriate activation of RyR1 and that this will contribute to the pathophysiology of debilitating muscular wasting disorders such as muscular dystrophy. Where applicable, experiments conform with Society ethical requirement