TY - JOUR AU - Simon, Márton AU - Iordanov, Iordan AU - Szöllősi, András AU - Csanády, László TI - Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein. JF - ELIFE J2 - ELIFE VL - 12 PY - 2023 PG - 19 SN - 2050-084X DO - 10.7554/eLife.90736 UR - https://m2.mtmt.hu/api/publication/34232792 ID - 34232792 AB - CFTR, the anion channel mutated in cystic fibrosis patients, is a model ABC protein whose ATP-driven conformational cycle is observable at single-molecule level in patch-clamp recordings. Bursts of CFTR pore openings are coupled to tight dimerization of its two nucleotide-binding domains (NBDs) and in wild-type (WT) channels are mostly terminated by ATP hydrolysis. The slow rate of non-hydrolytic closure - which determines how tightly bursts and ATP hydrolysis are coupled - is unknown, as burst durations of catalytic site mutants span a range of ~200-fold. Here, we show that Walker A mutation K1250A, Walker B mutation D1370N, and catalytic glutamate mutations E1371S and E1371Q all completely disrupt ATP hydrolysis. True non-hydrolytic closing rate of WT CFTR approximates that of K1250A and E1371S. That rate is slowed ~15-fold in E1371Q by a non-native inter-NBD H-bond, and accelerated ~15-fold in D1370N. These findings uncover unique features of the NBD interface in human CFTR. LA - English DB - MTMT ER - TY - JOUR AU - Szöllősi, András AU - Almássy, János TI - Functional characterization of the transient receptor potential melastatin 2 (TRPM2) cation channel from Nematostella vectensis reconstituted into lipid bilayer JF - SCIENTIFIC REPORTS J2 - SCI REP VL - 13 PY - 2023 IS - 1 PG - 10 SN - 2045-2322 DO - 10.1038/s41598-023-38640-6 UR - https://m2.mtmt.hu/api/publication/34064647 ID - 34064647 N1 - Department of Biochemistry, Semmelweis University, Tuzolto u. 37-47, Budapest, 1094, Hungary Department of Physiology, Semmelweis University, Tuzolto u. 37-47, Budapest, 1094, Hungary ELKH-SE Ion Channel Research Group, Semmelweis University, Tuzolto u. 37-47, Budapest, 1094, Hungary HCEMM-SE Molecular Channelopathies Research Group, Semmelweis University, Tuzolto u. 37-47, Budapest, 1094, Hungary Export Date: 4 September 2023 Correspondence Address: Szollosi, A.; Department of Biochemistry, Tuzolto u. 37-47, Hungary; email: szollo5@gmail.com Chemicals/CAS: adenosine diphosphate ribose, 20762-30-5; calcium, 7440-70-2, 14092-94-5; Adenosine Diphosphate Ribose; Calcium; Cations; Lipid Bilayers; TRPM Cation Channels Funding details: 739593 Funding details: Nemzeti Kutatási, Fejlesztési és Innovaciós Alap, NKFIA, KKP 144199 Funding text 1: This work was supported by Semmelweis Science and Innovation Fund STIA_18_M; EU Horizon 2020 Research and Innovation Program grant 739593 and National Research, Development and Innovation Fund grant KKP 144199. We thank László Csanády for funding and thorough reading and comments on the manuscript. We thank Balázs Tóth for help with poly- l -lysine measurements and stimulating discussions. AB - Transient receptor potential melastatin 2 (TRPM2) cation channel activity is required for insulin secretion, immune cell activation and body heat control. Channel activation upon oxidative stress is involved in the pathology of stroke and neurodegenerative disorders. Cytosolic Ca 2+ , ADP-ribose (ADPR) and phosphatidylinositol-4,5-bisphosphate (PIP 2 ) are the obligate activators of the channel. Several TRPM2 cryo-EM structures have been resolved to date, yet functionality of the purified protein has not been tested. Here we reconstituted overexpressed and purified TRPM2 from Nematostella vectensis (nvTRPM2) into lipid bilayers and found that the protein is fully functional. Consistent with the observations in native membranes, nvTRPM2 in lipid bilayers is co-activated by cytosolic Ca 2+ and either ADPR or ADPR-2′-phosphate (ADPRP). The physiological metabolite ADPRP has a higher apparent affinity than ADPR. In lipid bilayers nvTRPM2 displays a large linear unitary conductance, its open probability (P o ) shows little voltage dependence and is stable over several minutes. P o is high without addition of exogenous PIP 2 , but is largely blunted by treatment with poly- l -Lysine, a polycation that masks PIP 2 headgroups. These results indicate that PIP 2 or some other activating phosphoinositol lipid co-purifies with nvTRPM2, suggesting a high PIP 2 binding affinity of nvTRPM2 under physiological conditions. LA - English DB - MTMT ER - TY - JOUR AU - Szöllősi, András TI - Two decades of evolution of our understanding of the transient receptor potential melastatin 2 (Trpm2) cation channel JF - LIFE-BASEL J2 - LIFE-BASEL VL - 11 PY - 2021 IS - 5 PG - 23 SN - 2075-1729 DO - 10.3390/life11050397 UR - https://m2.mtmt.hu/api/publication/32040119 ID - 32040119 N1 - Department of Medical Biochemistry, Semmelweis University, Budapest, 1085, Hungary MTA-SE Lendület Ion Channel Research Group, Semmelweis University, Budapest, 1085, Hungary HCEMM-SE Molecular Channelopathies Research Group, Semmelweis University, Budapest, 1085, Hungary Cited By :2 Export Date: 9 March 2022 Correspondence Address: Szollosi, A.; Department of Medical Biochemistry, Hungary; email: szollosi.andras@med.semmelweis-univ.hu Funding details: 739593 Funding details: LP2017-14/2017 Funding text 1: Supported by MTA Lend?let grant LP2017-14/2017 and EU Horizon 2020 Research and Innovation Program grant 739593. Funding text 2: Funding: Supported by MTA Lendület grant LP2017-14/2017 and EU Horizon 2020 Research and Innovation Program grant 739593. AB - The transient receptor potential melastatin (TRPM) family belongs to the superfamily of TRP ion channels. It consists of eight family members that are involved in a plethora of cellular functions. TRPM2 is a homotetrameric Ca2+-permeable cation channel activated upon oxidative stress and is important, among others, for body heat control, immune cell activation and insulin secretion. Invertebrate TRPM2 proteins are channel enzymes; they hydrolyze the activating ligand, ADP-ribose, which is likely important for functional regulation. Since its cloning in 1998, the understanding of the biophysical properties of the channel has greatly advanced due to a vast number of structure– function studies. The physiological regulators of the channel have been identified and characterized in cell-free systems. In the wake of the recent structural biochemistry revolution, several TRPM2 cryo-EM structures have been published. These structures have helped to understand the general features of the channel, but at the same time have revealed unexplained mechanistic differences among channel orthologues. The present review aims at depicting the major research lines in TRPM2 structure-function. It discusses biophysical properties of the pore and the mode of action of direct channel effectors, and interprets these functional properties on the basis of recent three-dimensional structural models. © 2021 by the author. Licensee MDPI, Basel, Switzerland. LA - English DB - MTMT ER - TY - JOUR AU - Iordanov, Iordan AU - Tóth, Balázs AU - Szöllősi, András AU - Csanády, László TI - Enzyme activity and selectivity filter stability of ancient TRPM2 channels were simultaneously lost in early vertebrates JF - ELIFE J2 - ELIFE VL - 8 PY - 2019 IS - 2019 PG - 23 SN - 2050-084X DO - 10.7554/eLife.44556 UR - https://m2.mtmt.hu/api/publication/30637149 ID - 30637149 N1 - Funding Agency and Grant Number: Howard Hughes Medical InstituteHoward Hughes Medical Institute; Magyar Tudomanyos Akademia [LP2017-14/2017]; Ministry of Human Capacities of Hungary [UNKP 17-4-I-SE-61, UNKP 18-4-SE-132]; Magyar Tudomanyos Akademia Funding text: Howard Hughes Medical Institute International Early Career Scientist Award Laszlo Csanady; Magyar Tudomanyos Akademia LP2017-14/2017 Laszlo Csanady; Ministry of Human Capacities of Hungary UNKP 17-4-I-SE-61 Balazs Toth; Magyar Tudomanyos Akademia Bolyai Research Fellowship Balazs Toth; Ministry of Human Capacities of Hungary UNKP-FIKP Laszlo Csanady; Ministry of Human Capacities of Hungary UNKP 18-4-SE-132 Balazs Toth; The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Export Date: 7 January 2020 Correspondence Address: Csanády, L.; Department of Medical Biochemistry, Semmelweis UniversityHungary; email: csanady.laszlo@med.semmelweis-univ.hu Export Date: 8 January 2020 Correspondence Address: Csanády, L.; Department of Medical Biochemistry, Semmelweis UniversityHungary; email: csanady.laszlo@med.semmelweis-univ.hu AB - Transient Receptor Potential Melastatin 2 (TRPM2) is a cation channel important for the immune response, insulin secretion, and body temperature regulation. It is activated by cytosolic ADP ribose (ADPR) and contains a nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain homologous to ADPR phosphohydrolases (ADPRases). Human TRPM2 (hsTRPM2) is catalytically inactive due to mutations in the conserved Nudix box sequence. Here, we show that TRPM2 Nudix motifs are canonical in all invertebrates but vestigial in vertebrates. Correspondingly, TRPM2 of the cnidarian Nematostella vectensis (nvTRPM2) and the choanoflagellate Salpingoeca rosetta (srTRPM2) are active ADPRases. Disruption of ADPRase activity fails to affect nvTRPM2 channel currents, reporting a catalytic cycle uncoupled from gating. Furthermore, pore sequence substitutions responsible for inactivation of hsTRPM2 also appeared in vertebrates. Correspondingly, zebrafish (Danio rerio) TRPM2 (drTRPM2) and hsTRPM2 channels inactivate, but srTRPM2 and nvTRPM2 currents are stable. Thus, catalysis and pore stability were lost simultaneously in vertebrate TRPM2 channels. LA - English DB - MTMT ER - TY - JOUR AU - Zhang, Zhe AU - Tóth, Balázs AU - Szöllősi, András AU - Chen, Jue AU - Csanády, László TI - Structure of a TRPM2 channel in complex with Ca2+ explains unique gating regulation JF - ELIFE J2 - ELIFE VL - 7 PY - 2018 PG - 22 SN - 2050-084X DO - 10.7554/eLife.36409 UR - https://m2.mtmt.hu/api/publication/30465914 ID - 30465914 AB - Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel required for immune cell activation, insulin secretion, and body heat control. TRPM2 is activated by cytosolic Ca2+, phosphatidyl-inositol-4,5-bisphosphate and ADP ribose. Here, we present the 3 A resolution electron cryo-microscopic structure of TRPM2 from Nematostella vectensis, 63% similar in sequence to human TRPM2, in the Ca2+-bound closed state. Compared to other TRPM channels, TRPM2 exhibits unique structural features that correlate with its function. The pore is larger and more negatively charged, consistent with its high Ca2+ selectivity and larger conductance. The intracellular Ca2+ binding sites are connected to the pore and cytosol, explaining the unusual dependence of TRPM2 activity on intra- and extracellular Ca2+. In addition, the absence of a post filter motif is likely the cause of the rapid inactivation of human TRPM2. Together, our cryo-EM and electrophysiology studies provide a molecular understanding of the unique gating mechanism of TRPM2. LA - English DB - MTMT ER - TY - JOUR AU - Szöllősi, András AU - Vieira-Pires, Ricardo S AU - Teixeira-Duarte, Celso M AU - Rocha, R AU - Morais-Cabral, Joao H TI - Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter JF - PLOS BIOLOGY J2 - PLOS BIOL VL - 14 PY - 2016 IS - 1 SN - 1544-9173 DO - 10.1371/journal.pbio.1002356 UR - https://m2.mtmt.hu/api/publication/3110723 ID - 3110723 AB - KtrAB belongs to the Trk/Ktr/HKT superfamily of monovalent cation (K+ and Na+) transport proteins that closely resemble K+ channels. These proteins underlie a plethora of cellular functions that are crucial for environmental adaptation in plants, fungi, archaea, and bacteria. The activation mechanism of the Trk/Ktr/HKT proteins remains unknown. It has been shown that ATP stimulates the activity of KtrAB while ADP does not. Here, we present X-ray structural information on the KtrAB complex with bound ADP. A comparison with the KtrAB-ATP structure reveals conformational changes in the ring and in the membrane protein. In combination with a biochemical and functional analysis, we uncover how ligand-dependent changes in the KtrA ring are propagated to the KtrB membrane protein and conclude that, despite their structural similarity, the activation mechanism of KtrAB is markedly different from the activation mechanism of K+ channels. © 2016 Szollosi et al. LA - English DB - MTMT ER - TY - JOUR AU - Csanády, László AU - Mihályi, Csaba AU - Szöllősi, András AU - Törőcsik, Beáta AU - Vergani, P TI - Conformational changes in the catalytically inactive nucleotide-binding site of CFTR JF - JOURNAL OF GENERAL PHYSIOLOGY J2 - J GEN PHYSIOL VL - 142 PY - 2013 IS - 1 SP - 61 EP - 73 PG - 13 SN - 0022-1295 DO - 10.1085/jgp.201210954 UR - https://m2.mtmt.hu/api/publication/2333381 ID - 2333381 AB - A central step in the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the association of its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric adenosine triphosphate (ATP)-binding cassette protein in which the two interfacial-binding sites (composite sites 1 and 2) are functionally different. During gating, the canonical, catalytically active nucleotide-binding site (site 2) cycles between dimerized prehydrolytic (state O1), dimerized post-hydrolytic (state O2), and dissociated (state C) forms in a preferential C-->O1-->O2-->C sequence. In contrast, the catalytically inactive nucleotide-binding site (site 1) is believed to remain associated, ATP-bound, for several gating cycles. Here, we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1.Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N6-(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2x) transition O1-->O2 and decreases the nonhydrolytic closing rate (transition O1-->C) of CFTR mutants K1250A ( approximately 4x) and E1371S ( approximately 3x). Mutation H1348A also slowed ( approximately 3x) the O1-->O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A ( approximately 3x) and E1371S ( approximately 3x) background mutants. Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. The marked effect that different structural perturbations at site 1 have on both steps O1-->C and O1-->O2 suggests that the overall conformational changes that CFTR undergoes upon opening and coincident with hydrolysis at the active site 2 include significant structural rearrangement at site 1. LA - English DB - MTMT ER - TY - JOUR AU - Vieira-Pires, RS AU - Szöllősi, András AU - Morais-Cabral, JH TI - The structure of the KtrAB potassium transporter JF - NATURE J2 - NATURE VL - 496 PY - 2013 IS - 7445 SP - 323 EP - 328 PG - 6 SN - 0028-0836 DO - 10.1038/nature12055 UR - https://m2.mtmt.hu/api/publication/2329285 ID - 2329285 AB - In bacteria, archaea, fungi and plants the Trk, Ktr and HKT ion transporters are key components of osmotic regulation, pH homeostasis and resistance to drought and high salinity. These ion transporters are functionally diverse: they can function as Na + or K + channels and possibly as cation/K + symporters. They are closely related to potassium channels both at the level of the membrane protein and at the level of the cytosolic regulatory domains. Here we describe the crystal structure of a Ktr K + transporter, the KtrAB complex from Bacillus subtilis. The structure shows the dimeric membrane protein KtrB assembled with a cytosolic octameric KtrA ring bound to ATP, an activating ligand. A comparison between the structure of KtrAB-ATP and the structures of the isolated full-length KtrA protein with ATP or ADP reveals a ligand-dependent conformational change in the octameric ring, raising new ideas about the mechanism of activation in these transporters. © 2013 Macmillan Publishers Limited. All rights reserved. LA - English DB - MTMT ER - TY - JOUR AU - Szöllősi, András AU - Muallem, DR AU - Csanády, László AU - Vergani, P TI - Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating JF - JOURNAL OF GENERAL PHYSIOLOGY J2 - J GEN PHYSIOL VL - 137 PY - 2011 IS - 6 SP - 549 EP - 562 PG - 14 SN - 0022-1295 DO - 10.1085/jgp.201110608 UR - https://m2.mtmt.hu/api/publication/1690126 ID - 1690126 LA - English DB - MTMT ER - TY - JOUR AU - Szöllősi, András AU - Nenquin, M AU - Henquin, JC TI - Pharmacological stimulation and inhibition of insulin secretion in mouse islets lacking ATP-sensitive K plus channels JF - BRITISH JOURNAL OF PHARMACOLOGY J2 - BR J PHARMACOL VL - 159 PY - 2010 IS - 3 SP - 669 EP - 677 PG - 9 SN - 0007-1188 DO - 10.1111/j.1476-5381.2009.00588.x UR - https://m2.mtmt.hu/api/publication/1504152 ID - 1504152 LA - English DB - MTMT ER -