Andras Szollosi | Semmelweis University (original) (raw)

Papers by Andras Szollosi

Research paper thumbnail of Two Decades of Evolution of Our Understanding of the Transient Receptor Potential Melastatin 2 (TRPM2) Cation Channel

Life

The transient receptor potential melastatin (TRPM) family belongs to the superfamily of TRP ion c... more 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 chan...

Research paper thumbnail of Author response: Structure of a TRPM2 channel in complex with Ca2+ explains unique gating regulation

Research paper thumbnail of Enzyme activity and selectivity filter stability of ancient TRPM2 channels were simultaneously lost in early vertebrates

eLife

Transient Receptor Potential Melastatin 2 (TRPM2) is a cation channel important for the immune re... more 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 inactivat...

Research paper thumbnail of {"__content__"=>"Structure of a TRPM2 channel in complex with Ca explains unique gating regulation.", "sup"=>{"__content__"=>"2+"}}

eLife, Jan 10, 2018

Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca-permeable cation channel required for i... more Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca-permeable cation channel required for immune cell activation, insulin secretion, and body heat control. TRPM2 is activated by cytosolic Ca, phosphatidyl-inositol-4,5-bisphosphate and ADP ribose. Here we present the ~3Å resolution electron cryo-microscopy structure of TRPM2 from , 63% similar in sequence to human TRPM2, in the Ca-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 Ca selectivity and larger conductance. The intracellular Ca binding sites are connected to the pore and cytosol, explaining the unusual dependence of TRPM2 activity on intra- and extracellular Ca. 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...

Research paper thumbnail of Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter

PLOS Biology, 2016

KtrAB belongs to the Trk/Ktr/HKT superfamily of monovalent cation (K + and Na +) transport protei... more 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.

Research paper thumbnail of Regulation of biphasic and pulsatile insulin secretion

Research paper thumbnail of Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating

Journal of General Physiology, 2011

CFTR, whose failure causes cystic fibrosis (Riordan et al., 1989), belongs to the ATP-binding cas... more CFTR, whose failure causes cystic fibrosis (Riordan et al., 1989), belongs to the ATP-binding cassette (ABC) transporter superfamily. Although most ABC transporters use the energy derived from ATP binding and hydrolysis for active transport across membranes, in CFTR, the binding and hydrolysis of ATP drive channel gating (Muallem and Vergani, 2009). Like other ABC transporters, CFTR consists of two homologous halves, each containing a cytosolic ABC, otherwise known as a nucleotide-binding domain (NBD) and a transmembranespanning domain (Locher, 2009). NBDs are highly conserved among all ABC proteins, containing several conserved motifs, including the Walker A and B motifs (Walker et al., 1982) in a RecA-like subdomain ("head") and the signature sequence (LSGGQXXR) in a helical subdomain ("tail"; e.g., Hung et al., 1998; Karpowich et al., 2001). High resolution crystal structures of ABC transporters and isolated NBD dimers reveal that in ATP-bound crystals, NBDs can form tight "head-to-tail" dimers (e.g.,

Research paper thumbnail of Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2

The Journal of General Physiology, 2010

The chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) displays a ty... more The chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) displays a typical adenosine trisphosphate (ATP)-binding cassette (ABC) protein architecture comprising two transmembrane domains, two intracellular nucleotide-binding domains (NBDs), and a unique intracellular regulatory domain. Once phosphorylated in the regulatory domain, CFTR channels can open and close when supplied with cytosolic ATP. Despite the general agreement that formation of a head-to-tail NBD dimer drives the opening of the chloride ion pore, little is known about how ATP binding to individual NBDs promotes subsequent formation of this stable dimer. Structural studies on isolated NBDs suggest that ATP binding induces an intra-domain conformational change termed “induced fit,” which is required for subsequent dimerization. We investigated the allosteric interaction between three residues within NBD2 of CFTR, F1296, N1303, and R1358, because statistical coupling analysis suggests coevolut...

Research paper thumbnail of Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating

The Journal of General Physiology, 2011

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the... more Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic ana...

Research paper thumbnail of Overnight Culture Unmasks Glucose-induced Insulin Secretion in Mouse Islets Lacking ATP-sensitive K+ Channels by Improving the Triggering Ca2+ Signal

Journal of Biological Chemistry, 2007

2 The abbreviations used are: K ATP channel, ATP-sensitive potassium channel; SUR1, sulfonylurea ... more 2 The abbreviations used are: K ATP channel, ATP-sensitive potassium channel; SUR1, sulfonylurea receptor 1; [Ca 2ϩ ] c , cytosolic Ca 2ϩ concentration; KO, knockout .

Research paper thumbnail of Both Triggering and Amplifying Pathways Contribute to Fuel-induced Insulin Secretion in the Absence of Sulfonylurea Receptor-1 in Pancreatic  -Cells

Journal of Biological Chemistry, 2004

1 The abbreviations used are: K ATP , ATP-sensitive K ϩ ; SUR1, sulfonylurea receptor 1; Sur1KO, ... more 1 The abbreviations used are: K ATP , ATP-sensitive K ϩ ; SUR1, sulfonylurea receptor 1; Sur1KO, Sur1 knockout mice; K IR , potassium inward rectifier channel; [Ca 2ϩ ] i , cytoplasmic free Ca 2ϩ concentration; BCH, 2-amino-2-norbornanecarboxylic acid.

Research paper thumbnail of Glucose Stimulates Ca2+ Influx and Insulin Secretion in 2-Week-old beta-Cells Lacking ATP-sensitive K+ Channels

Journal of Biological Chemistry, 2007

2 The abbreviations used are: K ATP channel, ATP-sensitive potassium channel; SUR1, sulfonylurea ... more 2 The abbreviations used are: K ATP channel, ATP-sensitive potassium channel; SUR1, sulfonylurea receptor 1; [Ca 2ϩ ] c , cytosolic Ca 2ϩ concentration; DiBAC 4 (3), bis-(1,3-dibutylbarbituric acid)trimethine oxonol.

Research paper thumbnail of Insulin secretion in islets from mice with a double knockout for the dense core vesicle proteins islet antigen-2 (IA-2) and IA-2beta

The Journal of endocrinology, 2008

Islet antigen-2 (IA-2 or ICA 512) and IA-2b (or phogrin) are major autoantigens in type 1 diabete... more Islet antigen-2 (IA-2 or ICA 512) and IA-2b (or phogrin) are major autoantigens in type 1 diabetes. They are located in dense core secretory vesicles including insulin granules, but their role in b-cell function is unclear. Targeted disruption of either IA-2 or IA-2b, or both, impaired glucose tolerance, an effect attributed to diminution of insulin secretion. In this study, we therefore characterized the dynamic changes in cytosolic Ca 2C ([Ca 2C ] c ) and insulin secretion in islets from IA-2/IA-2b double knockout (KO) mice. High glucose (15 mM) induced biphasic insulin secretion in IA-2/IA-2b KO islets, with a similar first phase and smaller second phase compared with controls. Since the insulin content of IA-2/IA-2b KO islets was w45% less than that of controls, fractional insulin secretion (relative to content) was thus increased during first phase and unaffected during second phase. This peculiar response occurred in spite of a slightly smaller rise in [Ca 2C ] c , could not be attributed to an alteration of glucose metabolism (NADPH fluorescence) and also was observed with tolbutamide. The dual control of insulin secretion via the K ATP channel-dependent triggering pathway and K ATP channel-independent amplifying pathway was unaltered in IA-2/IA-2b KO islets, and so were the potentiations by acetylcholine or cAMP (forskolin). Intriguingly, amino acids, in particular the cationic arginine and lysine, induced larger fractional insulin secretion in IA-2/IA-2b KO than control islets. In conclusion, IA-2 and IA-2b are dispensable for exocytosis of insulin granules, but are probably more important for cargo loading and/or stability of dense core vesicles.

Research paper thumbnail of Conformational Changes in the Catalytically Inactive Nucleotide Binding Site of CFTR

Biophysical Journal, 2013

A central step in the gating of the CFTR chloride channel is the association of its two cytosolic... more A central step in the gating of the 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 ABC 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 O 1 ), dimerized posthydrolytic (state O 2 ), and dissociated (state C) forms in a preferential C→O 1 →O 2 →C sequence. In contrast, the catalytically inactive nucleotide binding site (site 1) is believed to remain associated, ATPbound, 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.

Research paper thumbnail of Functional Characterization of the KtrAB Potassium Transport System

Biophysical Journal, 2013

Research paper thumbnail of Studies on Correlated Pairs at the NBD-NBD and NBD-TMD Interfaces in CFTR

Biophysical Journal, 2011

Research paper thumbnail of A Triad of Residues F1296-N1303-R1358 in NBD2 of CFTR is Involved in ATP-driven Gating

Biophysical Journal, 2009

Research paper thumbnail of The Structure of the KtrAB Potassium Transporter

Biophysical Journal, 2013

In bacteria, archaea, fungi and plants the Trk, Ktr and HKT ion transporters are key components o... more 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.

Research paper thumbnail of Two Decades of Evolution of Our Understanding of the Transient Receptor Potential Melastatin 2 (TRPM2) Cation Channel

Life

The transient receptor potential melastatin (TRPM) family belongs to the superfamily of TRP ion c... more 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 chan...

Research paper thumbnail of Author response: Structure of a TRPM2 channel in complex with Ca2+ explains unique gating regulation

Research paper thumbnail of Enzyme activity and selectivity filter stability of ancient TRPM2 channels were simultaneously lost in early vertebrates

eLife

Transient Receptor Potential Melastatin 2 (TRPM2) is a cation channel important for the immune re... more 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 inactivat...

Research paper thumbnail of {"__content__"=>"Structure of a TRPM2 channel in complex with Ca explains unique gating regulation.", "sup"=>{"__content__"=>"2+"}}

eLife, Jan 10, 2018

Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca-permeable cation channel required for i... more Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca-permeable cation channel required for immune cell activation, insulin secretion, and body heat control. TRPM2 is activated by cytosolic Ca, phosphatidyl-inositol-4,5-bisphosphate and ADP ribose. Here we present the ~3Å resolution electron cryo-microscopy structure of TRPM2 from , 63% similar in sequence to human TRPM2, in the Ca-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 Ca selectivity and larger conductance. The intracellular Ca binding sites are connected to the pore and cytosol, explaining the unusual dependence of TRPM2 activity on intra- and extracellular Ca. 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...

Research paper thumbnail of Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter

PLOS Biology, 2016

KtrAB belongs to the Trk/Ktr/HKT superfamily of monovalent cation (K + and Na +) transport protei... more 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.

Research paper thumbnail of Regulation of biphasic and pulsatile insulin secretion

Research paper thumbnail of Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating

Journal of General Physiology, 2011

CFTR, whose failure causes cystic fibrosis (Riordan et al., 1989), belongs to the ATP-binding cas... more CFTR, whose failure causes cystic fibrosis (Riordan et al., 1989), belongs to the ATP-binding cassette (ABC) transporter superfamily. Although most ABC transporters use the energy derived from ATP binding and hydrolysis for active transport across membranes, in CFTR, the binding and hydrolysis of ATP drive channel gating (Muallem and Vergani, 2009). Like other ABC transporters, CFTR consists of two homologous halves, each containing a cytosolic ABC, otherwise known as a nucleotide-binding domain (NBD) and a transmembranespanning domain (Locher, 2009). NBDs are highly conserved among all ABC proteins, containing several conserved motifs, including the Walker A and B motifs (Walker et al., 1982) in a RecA-like subdomain ("head") and the signature sequence (LSGGQXXR) in a helical subdomain ("tail"; e.g., Hung et al., 1998; Karpowich et al., 2001). High resolution crystal structures of ABC transporters and isolated NBD dimers reveal that in ATP-bound crystals, NBDs can form tight "head-to-tail" dimers (e.g.,

Research paper thumbnail of Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2

The Journal of General Physiology, 2010

The chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) displays a ty... more The chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) displays a typical adenosine trisphosphate (ATP)-binding cassette (ABC) protein architecture comprising two transmembrane domains, two intracellular nucleotide-binding domains (NBDs), and a unique intracellular regulatory domain. Once phosphorylated in the regulatory domain, CFTR channels can open and close when supplied with cytosolic ATP. Despite the general agreement that formation of a head-to-tail NBD dimer drives the opening of the chloride ion pore, little is known about how ATP binding to individual NBDs promotes subsequent formation of this stable dimer. Structural studies on isolated NBDs suggest that ATP binding induces an intra-domain conformational change termed “induced fit,” which is required for subsequent dimerization. We investigated the allosteric interaction between three residues within NBD2 of CFTR, F1296, N1303, and R1358, because statistical coupling analysis suggests coevolut...

Research paper thumbnail of Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating

The Journal of General Physiology, 2011

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the... more Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic ana...

Research paper thumbnail of Overnight Culture Unmasks Glucose-induced Insulin Secretion in Mouse Islets Lacking ATP-sensitive K+ Channels by Improving the Triggering Ca2+ Signal

Journal of Biological Chemistry, 2007

2 The abbreviations used are: K ATP channel, ATP-sensitive potassium channel; SUR1, sulfonylurea ... more 2 The abbreviations used are: K ATP channel, ATP-sensitive potassium channel; SUR1, sulfonylurea receptor 1; [Ca 2ϩ ] c , cytosolic Ca 2ϩ concentration; KO, knockout .

Research paper thumbnail of Both Triggering and Amplifying Pathways Contribute to Fuel-induced Insulin Secretion in the Absence of Sulfonylurea Receptor-1 in Pancreatic  -Cells

Journal of Biological Chemistry, 2004

1 The abbreviations used are: K ATP , ATP-sensitive K ϩ ; SUR1, sulfonylurea receptor 1; Sur1KO, ... more 1 The abbreviations used are: K ATP , ATP-sensitive K ϩ ; SUR1, sulfonylurea receptor 1; Sur1KO, Sur1 knockout mice; K IR , potassium inward rectifier channel; [Ca 2ϩ ] i , cytoplasmic free Ca 2ϩ concentration; BCH, 2-amino-2-norbornanecarboxylic acid.

Research paper thumbnail of Glucose Stimulates Ca2+ Influx and Insulin Secretion in 2-Week-old beta-Cells Lacking ATP-sensitive K+ Channels

Journal of Biological Chemistry, 2007

2 The abbreviations used are: K ATP channel, ATP-sensitive potassium channel; SUR1, sulfonylurea ... more 2 The abbreviations used are: K ATP channel, ATP-sensitive potassium channel; SUR1, sulfonylurea receptor 1; [Ca 2ϩ ] c , cytosolic Ca 2ϩ concentration; DiBAC 4 (3), bis-(1,3-dibutylbarbituric acid)trimethine oxonol.

Research paper thumbnail of Insulin secretion in islets from mice with a double knockout for the dense core vesicle proteins islet antigen-2 (IA-2) and IA-2beta

The Journal of endocrinology, 2008

Islet antigen-2 (IA-2 or ICA 512) and IA-2b (or phogrin) are major autoantigens in type 1 diabete... more Islet antigen-2 (IA-2 or ICA 512) and IA-2b (or phogrin) are major autoantigens in type 1 diabetes. They are located in dense core secretory vesicles including insulin granules, but their role in b-cell function is unclear. Targeted disruption of either IA-2 or IA-2b, or both, impaired glucose tolerance, an effect attributed to diminution of insulin secretion. In this study, we therefore characterized the dynamic changes in cytosolic Ca 2C ([Ca 2C ] c ) and insulin secretion in islets from IA-2/IA-2b double knockout (KO) mice. High glucose (15 mM) induced biphasic insulin secretion in IA-2/IA-2b KO islets, with a similar first phase and smaller second phase compared with controls. Since the insulin content of IA-2/IA-2b KO islets was w45% less than that of controls, fractional insulin secretion (relative to content) was thus increased during first phase and unaffected during second phase. This peculiar response occurred in spite of a slightly smaller rise in [Ca 2C ] c , could not be attributed to an alteration of glucose metabolism (NADPH fluorescence) and also was observed with tolbutamide. The dual control of insulin secretion via the K ATP channel-dependent triggering pathway and K ATP channel-independent amplifying pathway was unaltered in IA-2/IA-2b KO islets, and so were the potentiations by acetylcholine or cAMP (forskolin). Intriguingly, amino acids, in particular the cationic arginine and lysine, induced larger fractional insulin secretion in IA-2/IA-2b KO than control islets. In conclusion, IA-2 and IA-2b are dispensable for exocytosis of insulin granules, but are probably more important for cargo loading and/or stability of dense core vesicles.

Research paper thumbnail of Conformational Changes in the Catalytically Inactive Nucleotide Binding Site of CFTR

Biophysical Journal, 2013

A central step in the gating of the CFTR chloride channel is the association of its two cytosolic... more A central step in the gating of the 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 ABC 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 O 1 ), dimerized posthydrolytic (state O 2 ), and dissociated (state C) forms in a preferential C→O 1 →O 2 →C sequence. In contrast, the catalytically inactive nucleotide binding site (site 1) is believed to remain associated, ATPbound, 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.

Research paper thumbnail of Functional Characterization of the KtrAB Potassium Transport System

Biophysical Journal, 2013

Research paper thumbnail of Studies on Correlated Pairs at the NBD-NBD and NBD-TMD Interfaces in CFTR

Biophysical Journal, 2011

Research paper thumbnail of A Triad of Residues F1296-N1303-R1358 in NBD2 of CFTR is Involved in ATP-driven Gating

Biophysical Journal, 2009

Research paper thumbnail of The Structure of the KtrAB Potassium Transporter

Biophysical Journal, 2013

In bacteria, archaea, fungi and plants the Trk, Ktr and HKT ion transporters are key components o... more 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.