High-affinity Metal-binding Site Research Articles

Albumin at the intersection between antioxidant and pro-oxidant in patients on peritoneal dialysis

Albumin (HSA) is a multifunctional protein and due to its free Cys34 thiol group, represents a main source of free thiols in the circulation. This property of HSA, combined with its ability to sequester redox active Cu(II) ions, makes HSA a dominant circulatory antioxidant. End stage kidney disease (ESRD) is a condition accompanied by elevated oxidative stress. The aim of the present study was to examine changes in the antioxidative capacity of HSA and Cu(II) binding affinity in patients on peritoneal dialysis (PD), and relate it to the Cys34 thiol group content and other structural changes of this molecule.HSA molecules are modified in ESRD patients subjected to PD, having significantly lower thiol group and bound Cu(II) content, reduced antioxidant capacity, an increased content of advanced glycation end-products and altered conformation. Also, Cu(II) binding capacity of HSA in these patients is impaired, since a significant portion of the high-affinity metal-binding site is unable to interact with Cu(II).Taking into account that the concentration of Cu(II) in the circulation of ESRD patients is much higher than in healthy persons and that Cu(II) binding capacity of HSA in these patients is significantly impaired, HSA may be considered as a novel circulatory pro-oxidant, thus exacerbating oxidative stress.

Delineating an extracellular redox-sensitive module in T-type Ca2+ channels

T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing a high-affinity metal-binding site formed by the extracellular IS1–IS2 and IS3–IS4 loops of domain I and a cluster of extracellular cysteines in the IS1–IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide, as well as a reactive oxygen species–producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His191 in Cav3.2 reduced the channel's sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1–IS2 loop of Cav3.2 reduced its sensitivity to MTSES and SP. We hypothesize that oxidative modification of IS1–IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2 so that it becomes sensitive to ambient zinc.

Intracerebroventricular administration of histidine reduces kainic acid-induced convulsive seizures in mice.

Kainic acid (KA)-induced seizures and other experimental models of epilepsy have been proven to be instrumental in identifying novel targets that could be responsible for human icto- and epileptogenesis. We have previously shown that the ablation of pharmacoresistant voltage-gated Ca2+ channels with Cav2.3 as central ion-conducting pore (R-type Ca2+ channel) reduces the sensitivity towards KA-induced epilepsy in mice. In vivo, Cav2.3 channels are thought to be under tight allosteric control by endogenous loosely bound trace metal cations (Zn2+ and Cu2+) that suppress channel gating via a high-affinity trace metal-binding site. Metal dyshomeostasis in the brain, which is a common feature of (KA-induced) seizures, could therefore alter the normal function of Cav2.3 channels and may shift hippocampal and neocortical signaling towards hyperexcitation. To investigate the role of loosely bound metal ions for KA-induced hyperexcitation in vivo, we examined the effects of manipulating brain trace metal homeostasis in mice. To this end, we developed a murine system for intracerebroventricular administration of trace metal ions and/or histidine (His), which can bind Zn2+ and Cu2+ and is involved in their transendothelial transport at the blood-brain barrier. Unexpectedly, our preliminary findings indicate that application of His alone but not in the presence of Zn2+ has substantial beneficial effects on the outcome of KA-induced epilepsy in mice. As such, our results emphasize previous findings on the complex, two-sided role of loosely bound metal ions with regard to neuronal excitation and degeneration under pathophysiological conditions.

Mechanistic Insights into the Metal-Dependent Activation of ZnII-Dependent Metallochaperones.

Members of the COG0523 subfamily of candidate GTPase metallochaperones function in bacterial transition-metal homeostasis, but the nature of the cognate metal, mechanism of metal transfer, and identification of target protein(s) for metal delivery remain open questions. Here, we explore the multifunctionality of members of the subfamily linked to delivering ZnII to apoprotein targets under conditions of host-imposed transition-metal depletion. We examine two zinc-uptake repressor (Zur)-regulated COG0523 family members, each from a major human pathogen, Acinetobacter baumannii (AbZigA) and Staphylococcus aureus (SaZigA), in an effort to develop a model for ZnII metallochaperone activity. ZnII chelator competition experiments reveal one high-affinity (KZn1 ≈ 1010-1011 M-1) metal-binding site in each GTPase, while AbZigA and SaZigA are characterized by an additional one and two (lower-affinity) metal-binding sites, respectively. CoII titrations reveal that both metallochaperones have similar electronic absorption characteristics that indicate the presence of two tetrahedral metal coordination sites. High-affinity metal binding at the CXCC motif activates the GTPase activity of both enzymes, with ZnII more effective than CoII. Both GTPases bind the product, GDP, more tightly in the apoprotein than the ZnII-bound state and exhibit what is best described as a "locked" conformation around the GTP substrate. Negative thermodynamic linkage is observed between nucleotide binding and metal binding, leading to a new mechanistic model for COG0523-catalyzed metal delivery.

Copper-assisted interaction between amyloid-β and prion: Ternary metal complexes with Aβ N-terminus and octarepeat

Alzheimer and prion diseases are neurodegenerative disorders in which amyloid-beta (Aβ) and prion protein (PrP), respectively, failed their normal functions inducing cellular apoptosis. Histidine-based high-affinity metal-binding sites in the N-terminus domains, which favor the binding of transition metals such as copper and zinc, feature both Aβ and PrP. Recently, the biological role of copper(II) on the two protein interaction has been revealed. Many studies have been focused on copper(II) binding affinity for both Aβ or prion protein while ternary complexes formed by this metal with both proteins have not been investigated. Here we report a combined ESI MS, potentiometric and spectroscopic (CD, UV-Vis) study on ternary complexes formed by copper(II) ion with Aβ and PrP peptides fragments containing potential metal binding sites. In particular, Aβ(1-4), Aβ(1-6), Ac-Aβ(1-6), Aβ(1-16)PEG and the octarepeat (PHGGGWGQ) of prion protein were studied. ESI-MS results indicate that the formation of 1:1:1 ternary complexes occurred at the physiological pH range. Potentiometric and spectroscopic data demonstrate that in a large pH range, ternary species prevail over binary species with the histidine-driven amide deprotonation.

Structural Basis for Copper-Oxygen Mediated C-H Bond Activation by the Formylglycine-Generating Enzyme.

The formylglycine-generating enzyme (FGE) is a unique copper protein that catalyzes oxygen-dependent C-H activation. We describe 1.66 Å- and 1.28 Å-resolution crystal structures of FGE from Thermomonospora curvata in complex with either AgI or CdII providing definitive evidence for a high-affinity metal-binding site in this enzyme. The structures reveal a bis-cysteine linear coordination of the monovalent metal, and tetrahedral coordination of the bivalent metal. Similar coordination changes may occur in the active enzyme as a result of CuI/II redox cycling. Complexation of copper atoms by two cysteine residues is common among copper-trafficking proteins, but is unprecedented for redox-active copper enzymes or synthetic copper catalysts.

Regulation of CNGA1 Channel Gating by Interactions with the Membrane

Cyclic nucleotide-gated (CNG) channels are expressed in rod photoreceptors and open in response to direct binding of cyclic nucleotides. We have previously shown that potentiation of CNGA1 channels by transition metals requires a histidine in the A' helix following the S6 transmembrane segment. Here, we used transition metal ion FRET and patch clamp fluorometry with a fluorescent, noncanonical amino acid (3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap)) to show that the potentiating transition metal Co(2+) binds in or near the A' helix. Adding high-affinity metal-binding sites to the membrane (stearoyl-nitrilotriacetic acid (C18-NTA)) increased potentiation for low Co(2+) concentrations, indicating that the membrane can coordinate metal ions with the A' helix. These results suggest that restraining the A' helix to the plasma membrane potentiates CNGA1 channel opening. Similar interactions between the A' helix and the plasma membrane may underlie regulation of structurally related hyperpolarization-activated cyclic nucleotide-gated (HCN) and voltage-gated potassium subfamily H (KCNH) channels by plasma membrane components.

Regulation of CNGA1 Channel Gating by Interactions with the Membrane

We have previously shown that a histidine (H420) in the first intracellular helix (A’ helix) of cyclic nucleotide-gated (CNGA1) channels is involved in potentiation of CNGA1 currents by transition metal ions. The binding of transition metals stabilizes the open state of the channel, so understanding the details of metal ion coordination will provide insight into the structural changes that occur after ligand binding to promote channel opening. Because structural studies suggest that the A’ helix is proximal to the membrane, we tested the hypothesis that a metal ion is coordinated between H420 and the intracellular leaflet of the bilayer with transition metal FRET (tmFRET) and patch-clamp fluorometry. To label CNGA1 channels, the amber codon suppression strategy was used to incorporate a fluorescent, noncanonical amino acid, L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (L-Anap) near H420. We chose Co2+ (1-30 μM) to potentiate CNGA1 currents and serve as a FRET acceptor for L-Anap in inside-out patches from Xenopus oocytes. We found that Co2+ quenched Anap fluorescence by ∼50%, and the H420Q mutation significantly attenuated quenching, providing further evidence that Co2+ binds to H420. Next, to test whether the membrane provides a second Co2+ ligand, we added high-affinity metal binding sites to the membrane using a novel, metal-chelating fatty acid (C18-NTA). We found that metal potentiation was increased for low (1-3 μM) Co2+ concentrations with C18-NTA-Co2+, and this effect required H420. These results indicate that membrane-bound metal ions interact with the A’ helix, suggesting that the mechanism for metal potentiation is metal ion coordination between H420 and a lipid with an anionic headgroup with lower affinity for Co2+ compared to C18-NTA. This arrangement of the A’ helix relative to the membrane potentiates channel opening and may be an important structural transition that occurs with cyclic nucleotide regulation.

Ins and outs of T-channel structure function

We review the ins and outs of T-channel structure, focusing on the extracellular high-affinity metal-binding site and intracellular loops. The high-affinity metal-binding site was localized to repeat I of Cav3.2. Interestingly, a similar binding site was found in the high voltage-activated Cav2.3 channel where it controls the channels' voltage dependence. Histidine at position 191 has a particularly interesting role in the high-affinity binding site, and its modification plays an important role in channel regulation by pharmacological agents that alter redox reactions. The intracellular loop connecting repeats I and II plays two important roles in Cav3.2 properties: one, its gating; and two, its surface expression. These studies have also identified a highly conserved intracellular gating brake that is predicted to form a helix-loop-helix structure. We conclude that the gating brake establishes important contacts with the gating machinery, thereby stabilizing a closed state of T-channels. This interaction is disrupted by depolarization, allowing the S6 segments to open and allowing Ca(2+) ions to flow through. Studies in cultured hippocampal neurons provided novel insights into how mutations found in idiopathic generalized epilepsy patients increase seizure susceptibility by both altering T-current pacemaker currents and by activating Ca-activated transcription factors that regulate dendritic arborization. These studies reveal novel roles for T-channels to control cellular physiology.

Selectivity of Ni(II) and Zn(II) binding to Sporosarcina pasteurii UreE, a metallochaperone in the urease assembly: a calorimetric and crystallographic study

Urease is a nickel-dependent enzyme that plays a critical role in the biogeochemical nitrogen cycle by catalyzing the hydrolysis of urea to ammonia and carbamate. This enzyme, initially synthesized in the apo form, needs to be activated by incorporation of two nickel ions into the active site, a processdriven by the dimeric metallochaperone UreE. Previous studies reported that this protein can bind different metal ions in vitro, beside the cognate Ni(II). This study explores the metal selectivity and affinity of UreE from Sporosarcina pasteurii (Sp,formerly known as Bacillus pasteurii) for cognate [Ni(II)] and noncognate [Zn(II)] metal ions. In particular, the thermodynamic parameters of SpUreE Ni(II) and Zn(II) binding have been determined using isothermal titration calorimetry. These experiments show that two Ni(II) ions bind to the protein dimer with positive cooperativity. The high-affinity site involves the conserved solvent-exposed His(100) and theC-terminal His(145), whereas the low-affinity site comprises also the C-terminalHis(147). Zn(II) binding to the protein, occurring in the same proteinregions and with similar affinityas compared to Ni(II), causes metal-driven dimerization of the protein dimer. The crystal structure of the protein obtained in the presence of equimolar amounts of both metal ions indicates that the high-affinity metal binding site binds Ni(II) preferentially over Zn(II). The ability of the protein to select Ni(II) over Zn(II) was confirmed by competition experiments in solution as well as by analysis of X-ray anomalous dispersion data. Overall, the thermodynamics and structural parameters that modulate the metal ion specificity of thedifferent binding sites on the protein surface of SpUreEhave been established.

Cobalt speciation assay for human serum, Part I. Method for measuring large and small molecular cobalt and protein-binding capacity using size exclusion chromatography with inductively coupled plasma-mass spectroscopy detection

A method utilizing size exclusion liquid chromatography (SEC) was developed to separate and quantify large molecular cobalt (Co) (e.g., albumin-Co) from cyanocobalamin (vitamin B12) and small molecular Co (e.g., glutathione-Co and free Co) in human serum. Highly selective and sensitive detection using inductively coupled plasma–mass spectrometry was coupled with SEC to provide a method with reliable accuracy, precision, recoveries, stability, and a detection limit of 0.037 μg/L in undiluted serum. Other divalent metal cations known to compete with Co(II) for serum albumin-binding sites (such as iron, zinc, manganese, cadmium, copper, nickel, and lead) did not significantly alter Co(II) quantification. Co–protein binding capacity determination of individual serum samples indicated that addition of 2500 μg Co/L to undiluted human serum resulted in approximately 90% distribution to the large molecular Co peak, consistent with Co binding to high-affinity divalent metal binding sites on albumin. Since serum albumin binding partially sequesters biologically active Co(II) ions, this method provides an important tool for better understanding the kinetics and toxicology of Co compounds. Thus, the proposed method might play an important role in establishing Co dose–response relationships that affect the equilibrium concentrations of free ionic Co(II).

A<i>β</i>-Like Peptide Displayed on Bacteriophage T7 Catalyzes Chromate and Uranyl Reduction

In order to discover genes capable of catalyzing the reductive immobilization of toxic chromate and uranyl ions, we have created a T7 bacteriophage library containing cDNA from environmental microbes (i.e., Geobacter sulfurreducens and Shewanella oneidensis MR-1) that are known to mediate the reduction of chromate and uranyl ions. After three rounds of screening, ten bacteriophage mutants were found to mediate the NADH-dependent reduction of chromate and uranyl ions whose cDNA encodes polypeptide chains ranging 14 to 73 amino acids in length. All identified sequences contain disordered structural motifs similar to the β-amyloid peptide (Aβ) known to promote aggregation and formation of high-affinity metal binding sites. Confirmation of this structural similarity involved phage display of the 42 amino-acid Aβ-peptides that have been found to catalyze the NADH-dependent reduction of both chromate and uranyl ions. Transmission electron microscopy (TEM) and X-ray absorption near edge structure (XANES) measurements confirm that reduced uranium is present on the surface of bacteriophage expressing the Aβ-peptide. The surface-displayed Aβ-like peptide on bacteriophage has the potential to couple naturally occurring electron transfer shuttles present in soils to promote economically viable remediation of contaminated sites containing toxic chromate and uranyl ions.

The metal selectivity of a short peptide maquette imitating the high-affinity metal-binding site of E. coli HypB

A seven-residue peptide based on the high-affinity metal-binding site of E. coli HypB maintains the nickel-binding activity of the full-length protein. The ability of the peptide to bind transition metals other than nickel was explored, and is discussed in the context of the function of HypB in hydrogenase biosynthesis.

Structural Determinants of the High Affinity Extracellular Zinc Binding Site on Cav3.2 T-type Calcium Channels

Ca(v)3.2 T-type channels contain a high affinity metal binding site for trace metals such as copper and zinc. This site is occupied at physiologically relevant concentrations of these metals, leading to decreased channel activity and pain transmission. A histidine at position 191 was recently identified as a critical determinant for both trace metal block of Ca(v)3.2 and modulation by redox agents. His(191) is found on the extracellular face of the Ca(v)3.2 channel on the IS3-S4 linker and is not conserved in other Ca(v)3 channels. Mutation of the corresponding residue in Ca(v)3.1 to histidine, Gln(172), significantly enhances trace metal inhibition, but not to the level observed in wild-type Ca(v)3.2, implying that other residues also contribute to the metal binding site. The goal of the present study is to identify these other residues using a series of chimeric channels. The key findings of the study are that the metal binding site is composed of a Asp-Gly-His motif in IS3-S4 and a second aspartate residue in IS2. These results suggest that metal binding stabilizes the closed conformation of the voltage-sensor paddle in repeat I, and thereby inhibits channel opening. These studies provide insight into the structure of T-type channels, and identify an extracellular motif that could be targeted for drug development.

Probing the Cu2+ and Zn2+ binding affinity of histidine-rich glycoprotein

The zinc(II) and copper(II) binding ability of two oligopeptide fragments, Ac-HHPHG-NH(2) and Ac-HHPHGHHPHG-NH(2), derived from the repeat-region of the His-Pro-rich domain of histidine-rich glycoprotein (HRG) and the structure of the formed complexes have been investigated by potentiometry, NMR-, UV-visible-, CD-, SRCD- and EPR spectroscopy. Exclusive coordination of the side-chain imidazoles of the peptides has been observed with both metal ions in the acidic and neutral pH range. While the three His units of the pentapeptide resulted in a modest stability of the ML complexes, the decapeptide with its increased number of His residues offered a high-affinity metal binding site for both metal ions with the participation of at least four nitrogen donors. Due to the high number of potential donor groups, the formation of binding isomers of the protonated and parent complexes is very likely. Both peptides show a synchrotron radiation (SR) CD-pattern resembling to that of the polyproline II structure, similarly to that of the His-Pro-rich domain of the HRG protein. The longer sequence was shown to bind a second metal ion in the slightly acidic pH-range. The determined stability data suggest a remarkable extra stabilization emerging in the decapeptide for the coordination of the second metal ions, as compared to the ML complexes of the pentapeptide. Whether the observed cooperativity has similarities to the cooperative metal binding feature of HRG or the two phenomena have different sources is a question yet to be clarified.

Severe Zinc Depletion of Escherichia coli: ROLES FOR HIGH AFFINITY ZINC BINDING BY ZinT, ZINC TRANSPORT AND ZINC-INDEPENDENT PROTEINS

Zinc ions play indispensable roles in biological chemistry. However, bacteria have an impressive ability to acquire Zn(2+) from the environment, making it exceptionally difficult to achieve Zn(2+) deficiency, and so a comprehensive understanding of the importance of Zn(2+) has not been attained. Reduction of the Zn(2+) content of Escherichia coli growth medium to 60 nm or less is reported here for the first time, without recourse to chelators of poor specificity. Cells grown in Zn(2+)-deficient medium had a reduced growth rate and contained up to five times less cellular Zn(2+). To understand global responses to Zn(2+) deficiency, microarray analysis was conducted of cells grown under Zn(2+)-replete and Zn(2+)-depleted conditions in chemostat cultures. Nine genes were up-regulated more than 2-fold (p < 0.05) in cells from Zn(2+)-deficient chemostats, including zinT (yodA). zinT is shown to be regulated by Zur (zinc uptake regulator). A mutant lacking zinT displayed a growth defect and a 3-fold lowered cellular Zn(2+) level under Zn(2+) limitation. The purified ZinT protein possessed a single, high affinity metal-binding site that can accommodate Zn(2+) or Cd(2+). A further up-regulated gene, ykgM, is believed to encode a non-Zn(2+) finger-containing paralogue of the Zn(2+) finger ribosomal protein L31. The gene encoding the periplasmic Zn(2+)-binding protein znuA showed increased expression. During both batch and chemostat growth, cells "found" more Zn(2+) than was originally added to the culture, presumably because of leaching from the culture vessel. Zn(2+) elimination is shown to be a more precise method of depleting Zn(2+) than by using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

Deciphering interactions of the aminoglycoside phosphotransferase(3′)‐IIIa with its ligands

Aminoglycoside phosphotransferase(3')-IIIa (APH) is the enzyme with broadest substrate range among the phosphotransferases that cause resistance to aminoglycoside antibiotics. In this study, the thermodynamic characterization of interactions of APH with its ligands are done by determining dissociation constants of enzyme-substrate complexes using electron paramagnetic resonance and fluorescence spectroscopy. Metal binding studies showed that three divalent cations bind to the apo-enzyme with low affinity. In the presence of AMPPCP, binding of the divalent cations occurs with 7-to-37-fold higher affinity to three additional sites dependent on the presence and absence of different aminoglycosides. Surprisingly, when both ligands, AMPPCP and aminoglycoside, are present, the number of high affinity metal binding sites is reduced to two with a 2-fold increase in binding affinity. The presence of divalent cations, with or without aminoglycoside present, shows only a small effect (<3-fold) on binding affinity of the nucleotide to the enzyme. The presence of metal-nucleotide, but not nucleotide alone, increases the binding affinity of aminoglycosides to APH. Replacement of magnesium (II) with manganese (II) lowered the catalytic rates significantly while affecting the substrate selectivity of the enzyme such that the aminoglycosides with 2'-NH(2) become better substrates (higher V(max)) than those with 2'-OH.

High affinity cation-binding sites in Complex I from Escherichia coli

Studies on the activity of Complex I from Escherichia coli in the presence of different metal cations revealed at least two high affinity metal-binding sites. Membrane-bound or isolated Complex I was activated by K + (apparent binding constant ∼ 125 μM) and inhibited by La 3+ ( IC 50 = 1 μM). K + and La 3+ do not occupy the same site. Possible localization of these metal-binding sites and their implication in catalysis are discussed.

Metal Preference At The Second Metal Binding Site Of E. coli NikR

Escherichia coli NikR (EcNikR) regulates cellular nickel uptake by binding free nickel ions at high affinity metal binding sites in the protein, which induces EcNikR binding to the nik operon - a process that leads to suppression of the gene encoding the nickel uptake transporter, NikABCDE. A structure of the EcNikR-DNA complex suggests that a second metal binding site is present in addition to the high affinity sites, and raises the question of which metal occupies this second site under physiologic conditions: K+, which is present in the crystal structure, or Ni+2. To determine which ion is preferred at the second metal binding site, and the reason for any preference of one ion over another, we calculated the electrostatic free energy of EcNikR binding to DNA with either K+ or Ni+2 in the second site. While the interaction between EcNikR and DNA is more favorable when the second site contains Ni+2, the large desolvation penalty associated with moving Ni+2 from solution to the relatively buried second site offsets this favorable interaction. Consequently, our data suggest that EcNikR binding to DNA is more favorable when the second site contains a K+ ion. Moreover, additional calculations indicate that the second site is best suited for an ion having the size of K+ and not Ni+2, suggesting that the second site is optimized for K+. Taken together, our data suggest that the second metal binding site contains K+ and explain why K+ is preferred over Ni+2 at this site.

Molecular Basis of Luminal Ca2+ Gating of the Cardiac Ryanodine Receptor

It has long been known that Ca2+ release from the sarcoplasmic reticulum (SR) can occur spontaneously when the SR Ca2+ content reaches a critical threshold, and that this spontaneous SR Ca2+ release can lead to lethal cardiac arrhythmias. Although it is clear that spontaneous SR Ca2+ release results from the activation of the cardiac ryanodine receptor (RyR2) by luminal Ca2+, the molecular basis of luminal Ca2+ activation of RyR2 remains undefined and controversial. An increasing body of evidence indicates that the RyR2 channel contains a luminal Ca2+ sensor distinct from its cytosolic Ca2+ sensor. To localize this luminal Ca2+ sensor, we have systematically mutated each of the negatively charged residues in the predicted pore-forming region of RyR2 that is likely to be accessible to luminal Ca2+. We found that alanine-mutations within and near the COOH-terminal end of the TM10 transmembrane helix (the pore inner helix) abolish the luminal, but not the cytosolic, Ca2+ activation of single RyR2 channels. HEK293 cells expressing these alanine-mutants display caffeine-induced Ca2+ release, but show no store-overload-induced Ca2+ release. Interestingly, introducing histidines into this region creates a high affinity metal binding site. The histidine-mutant channels are blocked by Ni2+ (<1 uM), while 100 uM Ni2+ does not block the RyR2 wt channels. Molecular modeling of the RyR2 channel pore based on the three-dimensional structure of the KcsA potassium channel reveals a cluster of negatively charged residues located within the internal pore at the helix bundle crossing, which is thought to form the ion gate of the RyR2 channel. Collectively, our results suggest that luminal Ca2+ opens the RyR2 channel by binding to the intracellular gate.