Samantha J. Pitt

TPC2 Is a Novel NAADP-sensitive Ca2+ Release Channel, Operating as a Dual Sensor of Luminal pH and Ca2+

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca2+ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca2+ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca2+ release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca2+ that will enable it to act as a Ca2+ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca2+] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca2+ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca2+ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μm but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.

Reconstituted Human TPC1 Is a Proton-Permeable Ion Channel and Is Activated by NAADP or Ca2+

Stimuli that increase calcium or NAADP may promote proton release from the endosomes and lysosomes by activating TPC1. Showing a Preference for Protons Protons (H+) and calcium (Ca2+) produce a variety of different effects in cells. One way to determine which channels allow each of these ions to pass is to isolate the proteins and incorporate them into artificial bilayers. With this approach, Pitt et al. found that H+ was the preferred ion that passed through the human two-pore channel 1 (TPC1), which in cells is located in acidic membrane-bound compartments called endosomes and lysosomes. They also identified intracellular signaling messengers that stimulated TPC1 and signals that changed the relative ability of different positively charged ions to flow through the channel. The exact function of the released H+ remains an open question. NAADP potently triggers Ca2+ release from acidic lysosomal and endolysosomal Ca2+ stores. Human two-pore channels (TPC1 and TPC2), which are located on these stores, are involved in this process, but there is controversy over whether TPC1 and TPC2 constitute the Ca2+ release channels. We therefore examined the single-channel properties of human TPC1 after reconstitution into bilayers of controlled composition. We found that TPC1 was permeable not only to Ca2+ but also to monovalent cations and that permeability to protons was the highest (relative permeability sequence: H+ >> K+ > Na+ ≥ Ca2+). NAADP or Ca2+ activated TPC1, and the presence of one of these ligands was required for channel activation. The endolysosome-located lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] had no effect on TPC1 open probability but significantly increased the relative permeability of Na+ to Ca2+ and of H+ to Ca2+. Furthermore, our data showed that, although both TPC1 and TPC2 are stimulated by NAADP, these channels differ in ion selectivity and modulation by Ca2+ and pH. We propose that NAADP triggers H+ release from lysosomes and endolysomes through activation of TPC1, but that the Ca2+-releasing ability of TPC1 will depend on the ionic composition of the acidic stores and may be influenced by other regulators that affect TPC1 ion permeation.

High Intracellular Chloride Slows the Decay of Glycinergic Currents

The time course of currents mediated by native and recombinant glycine receptors was examined with a combination of rapid agonist applications to outside-out patches and single-channel recording. The deactivation time constant of currents evoked by brief, saturating pulses of glycine is profoundly affected by the chloride concentration on the intracellular side of the cell membrane. Deactivation was threefold slower when intracellular chloride was increased from a low level (10 mm), similar to that observed in living mature neurons, to 131 mm (“symmetrical” chloride, often used in pipette internal solutions). Single-channel analysis revealed that high chloride has its greatest effect on the channel closing rate, slowing it by a factor of 2 compared with the value we estimated in the cell-attached mode (in which the channels are at physiological intracellular chloride concentrations). The same effect of chloride was observed when glycinergic evoked synaptic currents were recorded from juvenile rat spinal motoneurons in vitro, because the decay time constant was reduced from ∼7 ms to ∼3 ms when cells were dialyzed with 10 mm chloride intracellular recording solution. Our results indicate that the time course of glycinergic synaptic inhibition in intact neurons is much faster than is estimated by measurements in symmetrical chloride and can be modulated by changes in intracellular chloride concentration in the range that can occur in physiological or pathological conditions.

FKBP12 Activates the Cardiac Ryanodine Receptor Ca2+-Release Channel and Is Antagonised by FKBP12.6

Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca2+-homeostasis in heart failure but there is controversy over the functional effects of FKBP12.6 on RyR2 channel gating. We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca2+-induced Ca2+-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca2+, whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 µM) increased Ca2+-wave frequency and decreased the SR Ca2+-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12. We provide a biophysical analysis of the mechanisms by which FK-binding proteins can regulate RyR2 single-channel gating. Our data indicate that FKBP12, in addition to FKBP12.6, may be important in regulating RyR2 function in the heart. In heart failure, it is possible that an alteration in the dual regulation of RyR2 by FKBP12 and FKBP12.6 may occur. This could contribute towards a higher RyR2 open probability, ‘leaky’ RyR2 channels and Ca2+-dependent arrhythmias.

Charade of the SR K+-Channel: Two Ion-Channels, TRIC-A and TRIC-B, Masquerade as a Single K+-Channel

The presence of a sarcoplasmic reticulum (SR) K+-selective ion-channel has been known for >30 years yet the molecular identity of this channel has remained a mystery. Recently, an SR trimeric intracellular cation channel (TRIC-A) was identified but it did not exhibit all expected characteristics of the SR K+-channel. We show that a related SR protein, TRIC-B, also behaves as a cation-selective ion-channel. Comparison of the single-channel properties of purified TRIC-A and TRIC-B in symmetrical 210 mM K+ solutions, show that TRIC-B has a single-channel conductance of 138 pS with subconductance levels of 59 and 35 pS, whereas TRIC-A exhibits full- and subconductance open states of 192 and 129 pS respectively. We suggest that the K+-current fluctuations observed after incorporating cardiac or skeletal SR into bilayers, can be explained by the gating of both TRIC-A and TRIC-B channels suggesting that the SR K+-channel is not a single, distinct entity. Importantly, TRIC-A is regulated strongly by trans-membrane voltage whereas TRIC-B is activated primarily by micromolar cytosolic Ca2+ and inhibited by luminal Ca2+. Thus, TRIC-A and TRIC-B channels are regulated by different mechanisms, thereby providing maximum flexibility and scope for facilitating monovalent cation flux across the SR membrane.

Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release *

Background: In heart failure, the release of calcium becomes erratic leading to the generation of arrhythmias. Dysregulated Zn2+ homeostasis occurs in chronic heart failure. Results: Zn2+ can directly activate RyR2, removing the dependence of Ca2+ for channel activation. Conclusion: Zn2+ shapes Ca2+ dynamics by directly interacting with and modulating RyR2 function. Significance: This highlights a new role for Zn2+ in cardiac excitation-contraction coupling. Aberrant Zn2+ homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn2+ modulates cardiac ryanodine receptor gating and Ca2+ dynamics in isolated cardiomyocytes. We reveal that Zn2+ is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn2+ concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca2+. At concentrations of free Zn2+ >1 nm, Zn2+ is the main activating ligand, and the dependence on Ca2+ is removed. Zn2+ is therefore a higher affinity activator of RyR2 than Ca2+. Millimolar levels of free Zn2+ were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn2+ modulates both the frequency and amplitude of Ca2+ waves in a concentration-dependent manner and that physiological levels of Zn2+ elicit Ca2+ release in the absence of activating levels of cytosolic Ca2+. This highlights a new role for intracellular Zn2+ in shaping Ca2+ dynamics in cardiomyocytes through modulation of RyR2 gating.

In vivo effects of 17β-estradiol on cardiac Na(+)/K(+)-ATPase expression and activity in rat heart.

In this study the in vivo effects of estradiol in regulating Na(+)/K(+)-ATPase function in rat heart was studied. Adult male Wistar rats were treated with estradiol (40μg/kg, i.p.) and after 24h the animals were sacrificed and the heart excised. Following estradiol administration, cardiac Na(+)/K(+)-ATPase activity, expression of the α1 subunit, and phosphorylation of the α1 subunit were significantly increased. These animals also had significantly decreased levels of digoxin-like immunoreactive factor(s). Na(+) levels were also significantly reduced but to a level that was still within the normal physiological range, highlighting the ability of the Na(+)/K(+)-ATPase to balance the ionic composition following treatment with estradiol. Estradiol treated rats also showed increased phosphorylation of protein kinase B (Akt), and extracellular-signal-regulated kinase 1/2 (ERK1/2). We therefore suggest a role for Akt and/or ERK1/2 in estradiol-mediated regulation of cardiac Na(+)/K(+)-ATPase expression and activity in rat heart.

Ca2+-Dependent Phosphorylation of RyR2 Can Uncouple Channel Gating from Direct Cytosolic Ca2+ Regulation

Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca2+ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca2+. We show that hyperphosphorylation by exogenous PKA increases open probability (Po) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca2+; lowering [Ca2+] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca2+-sensitive mode of gating. We additionally found that cytosolic incubation with Mg2+/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca2+. We show that the endogenous kinase is both Ca2+-dependent and sensitive to inhibitors of PKC. Moreover, the Ca2+-dependent, endogenous kinase–induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca2+-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca2+ regulation.

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 endolysosomal 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 endolysosomal system.

A high fat diet induces sex-specific differences in hepatic lipid metabolism and nitrite/nitrate in rats

Men and women differ substantially with regard to the severity of insulin resistance (IR) but the underlying mechanism(s) of how this occurs is poorly characterized. We investigated whether a high fat (HF) diet resulted in sex-specific differences in nitrite/nitrate production and lipid metabolism and whether these variances may contribute to altered obesity-induced IR. Male and female Wistar rats were fed a standard laboratory diet or a HF diet for 10 weeks. The level of plasma nitrite/nitrate, as well as free fatty acid (FFA), in both plasma and liver lysates were assessed. The levels of inducible nitric oxide (NO) synthase (iNOS), p65 subunit of NFκB, total and phosphorylated forms of Akt, mTOR and PDK-1 in lysates, and the levels of glucose transporter 2 (Glut-2) and fatty acid translocase/cluster of differentiation 36 (FAT/CD36) in plasma membrane fractions of liver were assessed. HF-fed male rats exhibited a significant increase in plasma nitrite/nitrate, and hepatic FFA and FAT/CD36 levels compared with controls. They also displayed a relative decrease in iNOS and Glut-2 levels in the liver. Phosphorylation of Akt (at Ser(473) and Thr(308)), mTOR and PDK-1 was also reduced. HF-fed female rats exhibited increased levels of NFκB-p65 in liver compared with controls, while levels of Glut-2, FAT/CD36 and Akt phosphorylation at Thr(308) and PDK-1 were decreased. Our results reveal that altered lipid and glucose metabolism in obesity, lead to altered iNOS expression and nitrite/nitrate production. It is likely that this mechanism contributes to sex-specific differences in the development of IR.