Az ischaemiás agykárosodásban szerepet játszó TrpM2 kationcsatorna szerkezet-funkció vizsgálata = Structure-function studies of the TrpM2 cation channel involved in ischaemic brain damage.

Megállapítottuk, hogy a csatornát 4 Ca2+ ion kötődése aktiválja. Az aktiváció a Monod-Wymann-Changeux mechanizmust követi, 1 Ca2+ kötődése ~33-szorosra, a 4 ion összesen ~10^6-szorosra, növeli a nyitott-csukott egyensúlyi állandót. A Ca2+ kötőhelyei a kaputól intracelluláris irányban találhatók egy védett üregben, közel a pórus nyílásához. A nyitott póruson beáramló Ca2+ telítésben tartja az aktiváló helyeket, ezért intakt sejtekben, ADPR jelenlétében, egy rövid Ca2+ szignál is elnyújtott TRPM2 aktivitást válthat ki. Megállapítottuk, hogy az ADPR nagy affinitással (K1/2=1uM) aktivál, de az ADPR-hidrolízisnek a csatorna csukódásában játszott szerepét nem tudtuk tisztázni. Az ADPRázok konzervált ""Nudix-box"" motívuma (REFXEE) a TRPM2 NUDT9-H doménjében atípusos (RILRQE). A NUDT9 enzimben az EF->IL mutáció 1%-ára csökkenti, az EE->KK mutáció felfüggeszti az ADPRáz aktivitást. Létrehoztunk egy ""inaktív"" QE->KK és egy ""hiperaktív"" IL->EF TRPM2 mutánst, de e 4 egyedi és 2 dupla mutáció egyike sem befolyásolta az ADPR iránti affinitást illetve a csukódási sebességet. Intakt sejtekben az AMP gátolta, míg a H2O2, a ciklikus ADPR (cADPR), és a nikotinsav-adenin-dinukleotid-foszfát (NAADP) aktiválták a TRPM2-t, és fokozták ADPR iránti érzékenységét. Izolált membrán patch-ben megállapítottuk, hogy a H2O2, az AMP, és a cADPR közvetlenül nem hatnak a TRPM2-re, míg az NAADP és az NAAD kis affinitású parciális agonisták. Tehát intakt sejtekben e modulátorok hatásai közvetettek. | We have revealed that the channel is activated by binding of 4 Ca2+ ions, following the Monod-Wymann-Changeux mechanism. Binding of 1 Ca2+ increases the closed-open equilibrium constant by ~33-fold, the 4 ions altogether by ~10^6-fold. The Ca2+ binding sites are found intracellularly of the gate, in a protected crevice, near the pore entrance, and are kept saturated by Ca2+ flowing through the open pore. Thus, in intact cells, in the presence of ADPR, a single brief Ca2+ spark can elicit prolonged TRPM2 channel activity. We have shown that ADPR activates the channel with high affinity (K1/2=1 uM), but could not clarify the role of ADPR hydrolysis in channel closure. The conserved ADPRase ""Nudix-box"" motif (REFXEE) is atypical in the NUDT9-H domain of TRPM2 (RILRQE). The EF->IL mutation decreases ADPRase activity of the NUDT9 enzyme to ~1%, while the EE->KK mutation completely abolishes it. We constructed an ""inactive"" QE?KK and a ""hiperactive"" IL->EF TRPM2 mutant, but neither of the 4 single and 2 double mutants affected ADPR affinity or channel closing rate. In intact cells AMP inhibits, while H2O2, cyclic ADPR (cADPR), and nicotinic acid-adenin-dinucleotide-phosphate (NAADP) activate TRPM2, and enhance its sensitivity to ADPR. We have found that direct application of H2O2, AMP, and cADPR in isolated patches does not affect the channels, while NAADP and NAAD are low-affinity partial agonists. Thus, in intact cells the effects of these modulators are indirect

Antagonistic Regulation of Native Ca2+- and ATP-sensitive Cation Channels in Brain Capillaries by Nucleotides and Decavanadate

Regulation by cytosolic nucleotides of Ca2+- and ATP-sensitive nonselective cation channels (CA-NSCs) in rat brain capillary endothelial cells was studied in excised inside-out patches. Open probability (Po) was suppressed by cytosolic nucleotides with apparent KI values of 17, 9, and 2 μM for ATP, ADP, and AMP, as a consequence of high-affinity inhibition of channel opening rate and low-affinity stimulation of closing rate. Cytosolic [Ca2+] and voltage affected inhibition of Po, but not of opening rate, by ATP, suggesting that the conformation of the nucleotide binding site is influenced only by the state of the channel gate, not by that of the Ca2+ and voltage sensors. ATP inhibition was unaltered by channel rundown. Nucleotide structure affected inhibitory potency that was little sensitive to base substitutions, but was greatly diminished by 3′-5′ cyclization, removal of all phosphates, or complete omission of the base. In contrast, decavanadate potently (K1/2 = 90 nM) and robustly stimulated Po, and functionally competed with inhibitory nucleotides. From kinetic analyses we conclude that (a) ATP, ADP, and AMP bind to a common site; (b) inhibition by nucleotides occurs through simple reversible binding, as a consequence of tighter binding to the closed-channel relative to the open-channel conformation; (c) the conformation of the nucleotide binding site is not directly modulated by Ca2+ and voltage; (d) the differences in inhibitory potency of ATP, ADP, and AMP reflect their different affinities for the closed channel; and (e) though decavanadate is the only example found to date of a compound that stimulates Po with high affinity even in the presence of millimolar nucleotides, apparently by competing for the nucleotide binding site, a comparable mechanism might allow CA-NSC channels to open in living cells despite physiological levels of nucleotides. Decavanadate now provides a valuable tool for studying native CA-NSC channels and for screening cloned channels

Putative chanzyme activity of TRPM2 cation channel is unrelated to pore gating

Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel expressed in immune cells of phagocytic lineage, pancreatic β cells, and brain neurons and is activated under oxidative stress. TRPM2 activity is required for immune cell activation and insulin secretion and is responsible for postischemic neuronal cell death. TRPM2 is opened by binding of ADP ribose (ADPR) to its C-terminal cytosolic nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain, which, when expressed in isolation, cleaves ADPR into AMP and ribose-5-phosphate. A suggested coupling of this enzymatic activity to channel gating implied a potentially irreversible gating cycle, which is a unique feature of a small group of channel enzymes known to date. The significance of such a coupling lies in the conceptually distinct pharmacologic strategies for modulating the open probability of channels obeying equilibrium versus nonequilibrium gating mechanisms. Here we examine the potential coupling of TRPM2 enzymatic activity to pore gating. Mutation of several residues proposed to enhance or eliminate NUDT9-H catalytic activity all failed to affect channel gating kinetics. An ADPR analog, α-β-methylene-ADPR (AMPCPR), was shown to be entirely resistant to hydrolysis by NUDT9, but nevertheless supported TRPM2 channel gating, albeit with reduced apparent affinity. The rate of channel deactivation was not slowed but, rather, accelerated in AMPCPR. These findings, as well as detailed analyses of steady-state gating kinetics of single channels recorded in the presence of a range of concentrations of ADPR or AMPCPR, identify TRPM2 as a simple ligand-gated channel that obeys an equilibrium gating mechanism uncoupled from its enzymatic activity

Sulfonylurea Receptors Type 1 and 2A Randomly Assemble to Form Heteromeric KATP Channels of Mixed Subunit Composition

ATP-sensitive potassium (KATP) channels play important roles in regulating insulin secretion, controlling vascular tone, and protecting cells against metabolic stresses. KATP channels are heterooctamers of four pore-forming inwardly rectifying (Kir6.2) subunits and four sulfonylurea receptor (SUR) subunits. KATP channels containing SUR1 (e.g. pancreatic) and SUR2A (e.g. cardiac) display distinct metabolic sensitivities and pharmacological profiles. The reported expression of both SUR1 and SUR2 together with Kir6.2 in some cells raises the possibility that heteromeric channels containing both SUR subtypes might exist. To test whether SUR1 can coassemble with SUR2A to form functional KATP channels, we made tandem constructs by fusing SUR to either a wild-type (WT) or a mutant N160D Kir6.2 subunit. The latter mutation greatly increases the sensitivity of KATP channels to block by intracellular spermine. We expressed, individually and in combinations, tandem constructs SUR1-Kir6.2 (S1-WT), SUR1-Kir6.2[N160D] (S1-ND), and SUR2A-Kir6.2[N160D] (S2-ND) in Xenopus oocytes, and studied the voltage dependence of spermine block in inside-out macropatches over a range of spermine concentrations and RNA mixing ratios. Each tandem construct expressed alone supported macroscopic K+ currents with pharmacological properties indistinguishable from those of the respective native channel types. Spermine sensitivity was low for S1-WT but high for S1-ND and S2-ND. Coexpression of S1-WT and S1-ND generated current components with intermediate spermine sensitivities indicating the presence of channel populations containing both types of Kir subunits at all possible stoichiometries. The relative abundances of these populations, determined by global fitting over a range of conditions, followed binomial statistics, suggesting that WT and N160D Kir6.2 subunits coassemble indiscriminately. Coexpression of S1-WT with S2-ND also yielded current components with intermediate spermine sensitivities, suggesting that SUR1 and SUR2A randomly coassemble into functional KATP channels. Further pharmacological characterization confirmed coassembly of not only S1-WT and S2-ND, but also of coexpressed free SUR1, SUR2A, and Kir6.2 into functional heteromeric channels

Bioenergetikai változások glutamát excitotoxicitásban és oxidatív stresszben = Bioenergetical changes in glutamate excitotoxicity and oxidative stress

Munkánk egyfelől az ischemia és számos neurodegenerativ betegség pathomechanizmusában meghatározó szerepet játszó oxidativ stressz akut hatásaival, másfelől az oxidativ stressz keletkezésével kapcsolatban hozott uj eredményeket. Megállapitások: 1) Enyhe oxidativ stressz potencirozza a Na-terhelésre bekövetkező glutamat felszabadulást és csökkenti a gluthation mennyiségét izolált idegvégződésekben. 2) Az oxidativ stressz gátolja a pH regulációt és a mitokondriumok kalcium háztartást reguláló szerepét az agy-vér gátat alkotó kapillárisokból tenyésztett endothel sejtekben. 3) A mitokondriális légzési lánc komponensei közül a ROS keletkezésében a meghatározó a komplex I gátlása, amely már kis mértékű gátlás esetén olyan mennyiségű ROS képzéssel jár, hogy gátolja az endogén akonitázt. 4) A légési lánc mellett fontos általunk leirt új forrása a ROS keletkezésnek az alpha-ketoglutarát enzim, amely normál katalitikus működése során szuperoxidot és hidrogén peroxidot termel, amit a NADH/NAD arány regulál. 5) Agykéregből származó sejttenyészeten a glutamat excitotoxikus hatásában szerepet játszanak a transient receptor potential csatornák és a pathomechanizmus fontos része a sejtek NADH szintjének csökkenése. 6) Megtörtént az eddig ismeretlen funkciójú kalcium-aktiválta nem-szelektiv kation csatornák részletes jellemzése agyi kapilláris endothel sejt tenyészetben. | The findings of this research project show, on one hand, sensitive processes that are impaired by oxidative stress in neurons and rat brain capillary endothelial cells (RBCE), on the other hand, mechanisms involved in the generation of oxidative stress. Major findings: i) Hydrogen peroxide potentiate the release of glutamate induced by a sodium load in isolated nerve terminals. ii) Oxidative stress impairs the intracellular pH regulation and the mitochondrial calcium regulation in RBCE cells. iii) Physiologically the most relevant site of reactive oxygen species (ROS) production in the respiratory chain is at complex I; when this complex is inhibited by 25-30 %, relevant to Parkinson?s disease, ROS is generated in sufficient amount to inhibit endogenous mitochondrial aconitase. iv) In addition to the respiratory chain, alpha-ketoglutarate dehydrogenase could be a significant source of ROS generation in mitochondria under conditions when the NADH/NAD ratio is increased (ischemia, Parkinson?s disease). v) Transient receptor potential (TRP) channels are involved in the delayed calcium deregulation (DCD) associated with glutamate excitotoxicity in cortical cell cultures. DCD is accompanied by a significant drop in the intracellular NADH level. vi) Calcium-activated non-selective cation channels, which are abundant in RBCE cells, have been characterized; the gating characteristics as well as the regulation by nucleotides have been described in details

Thermodynamics of CFTR Channel Gating: A Spreading Conformational Change Initiates an Irreversible Gating Cycle

CFTR is the only ABC (ATP-binding cassette) ATPase known to be an ion channel. Studies of CFTR channel function, feasible with single-molecule resolution, therefore provide a unique glimpse of ABC transporter mechanism. CFTR channel opening and closing (after regulatory-domain phosphorylation) follows an irreversible cycle, driven by ATP binding/hydrolysis at the nucleotide-binding domains (NBD1, NBD2). Recent work suggests that formation of an NBD1/NBD2 dimer drives channel opening, and disruption of the dimer after ATP hydrolysis drives closure, but how NBD events are translated into gate movements is unclear. To elucidate conformational properties of channels on their way to opening or closing, we performed non-equilibrium thermodynamic analysis. Human CFTR channel currents were recorded at temperatures from 15 to 35°C in inside-out patches excised from Xenopus oocytes. Activation enthalpies(ΔH‡) were determined from Eyring plots. ΔH‡ was 117 ± 6 and 69 ± 4 kJ/mol, respectively, for opening and closure of partially phosphorylated, and 96 ± 6 and 73 ± 5 kJ/mol for opening and closure of highly phosphorylated wild-type (WT) channels. ΔH‡ for reversal of the channel opening step, estimated from closure of ATP hydrolysis–deficient NBD2 mutant K1250R and K1250A channels, and from unlocking of WT channels locked open with ATP+AMPPNP, was 43 ± 2, 39 ± 4, and 37 ± 6 kJ/mol, respectively. Calculated upper estimates of activation free energies yielded minimum estimates of activation entropies (ΔS‡), allowing reconstruction of the thermodynamic profile of gating, which was qualitatively similar for partially and highly phosphorylated CFTR. ΔS‡ appears large for opening but small for normal closure. The large ΔH‡ and ΔS‡ (TΔS‡ ≥ 41 kJ/mol) for opening suggest that the transition state is a strained channel molecule in which the NBDs have already dimerized, while the pore is still closed. The small ΔS‡ for normal closure is appropriate for cleavage of a single bond (ATP's beta-gamma phosphate bond), and suggests that this transition state does not require large-scale protein motion and hence precedes rehydration (disruption) of the dimer interface