TY  - JOUR
A1  - Kitzenmaier, Alexandra
A1  - Schaefer, Natascha
A1  - Kasaragod, Vikram Babu
A1  - Polster, Tilman
A1  - Hantschmann, Ralph
A1  - Schindelin, Hermann
A1  - Villmann, Carmen
T1  - The P429L loss of function mutation of the human glycine transporter 2 associated with hyperekplexia
JF  - European Journal of Neuroscience
N2  - Glycine transporter 2 (GlyT2) mutations across the entire sequence have been shown to represent the presynaptic component of the neurological disease hyperekplexia. Dominant, recessive and compound heterozygous mutations have been identified, most of them leading to impaired glycine uptake. Here, we identified a novel loss of function mutation of the GlyT2 resulting from an amino acid exchange of proline 429 to leucine in a family with both parents being heterozygous carriers. A homozygous child suffered from severe neuromotor deficits. We characterised the GlyT2P429L variant at the molecular, cellular and protein level. Functionality was determined by glycine uptake assays. Homology modelling revealed that the mutation localises to α‐helix 5, presumably disrupting the integrity of this α‐helix. GlyT2P429L shows protein trafficking through various intracellular compartments to the cellular surface. However, the protein expression at the whole cell level was significantly reduced. Although present at the cellular surface, GlyT2P429L demonstrated a loss of protein function. Coexpression of the mutant with the wild‐type protein, reflecting the situation in the parents, did not affect transporter function, thus explaining their non‐symptomatic phenotype. Nevertheless, when the mutant was expressed in excess compared with the wild‐type protein, glycine uptake was significantly reduced. Thus, these data demonstrate that the proline residue at position 429 is structurally important for the correct formation of α‐helix 5. The failure in functionality of the mutated GlyT2 is most probably due to structural changes localised in close proximity to the sodium‐binding site of the transporter.
KW  - glycine transporter 2
KW  - glyvine uptake
KW  - loss of function
KW  - presynaptic hyperekplexia
KW  - protein transport
KW  - structural disruption
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-206158
VL  - 50
IS  - 12
ER  - 
TY  - JOUR
A1  - Kasaragod, Vikram Babu
A1  - Schindelin, Hermann
T1  - Structure of Heteropentameric GABAA Receptors and Receptor-Anchoring Properties of Gephyrin
JF  - Frontiers in Molecular Neuroscience
N2  - γ-Aminobutyric acid type A receptors (GABAARs) mediate the majority of fast synaptic inhibition in the central nervous system (CNS). GABAARs belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGIC) and are assembled from 19 different subunits. As dysfunctional GABAergic neurotransmission manifests itself in neurodevelopmental disorders including epilepsy and anxiety, GABAARs are key drug targets. The majority of synaptic GABAARs are anchored at the inhibitory postsynaptic membrane by the principal scaffolding protein gephyrin, which acts as the central organizer in maintaining the architecture of the inhibitory postsynaptic density (iPSD). This interaction is mediated by the long intracellular loop located in between transmembrane helices 3 and 4 (M3–M4 loop) of the receptors and a universal receptor-binding pocket residing in the C-terminal domain of gephyrin. In 2014, the crystal structure of the β3-homopentameric GABAAR provided crucial information regarding the architecture of the receptor; however, an understanding of the structure and assembly of heteropentameric receptors at the atomic level was lacking. This review article will highlight recent advances in understanding the structure of heteropentameric synaptic GABAARs and how these structures have provided fundamental insights into the assembly of these multi-subunit receptors as well as their modulation by diverse ligands including the physiological agonist GABA. We will further discuss the role of gephyrin in the anchoring of synaptic GABAARs and glycine receptors (GlyRs), which are crucial for maintaining the architecture of the iPSD. Finally, we will also summarize how anti-malarial artemisinin drugs modulate gephyrin-mediated inhibitory neurotransmission.
KW  - GABAA
KW  - gephyrin
KW  - diazepam
KW  - GABA
KW  - PIP2
KW  - artemisinin
KW  - Cryo-EM
KW  - inhibitory neurotransmission
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-189308
SN  - 1662-5099
VL  - 12
ER  - 
TY  - JOUR
A1  - Kasaragod, Vikram Babu
A1  - Schindelin, Hermann
T1  - Structure of heteropentameric GABA\(_A\) receptors and receptor-anchoring properties of gephyrin
JF  - Frontiers in Molecular Neuroscience
N2  - γ-Aminobutyric acid type A receptors (GABA\(_A\)Rs) mediate the majority of fast synaptic inhibition in the central nervous system (CNS). GABA\(_A\)Rs belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGIC) and are assembled from 19 different subunits. As dysfunctional GABAergic neurotransmission manifests itself in neurodevelopmental disorders including epilepsy and anxiety, GABA\(_A\)Rs are key drug targets. The majority of synaptic GABA\(_A\)Rs are anchored at the inhibitory postsynaptic membrane by the principal scaffolding protein gephyrin, which acts as the central organizer in maintaining the architecture of the inhibitory postsynaptic density (iPSD). This interaction is mediated by the long intracellular loop located in between transmembrane helices 3 and 4 (M3–M4 loop) of the receptors and a universal receptor-binding pocket residing in the C-terminal domain of gephyrin. In 2014, the crystal structure of the β3-homopentameric GABA\(_A\)R provided crucial information regarding the architecture of the receptor; however, an understanding of the structure and assembly of heteropentameric receptors at the atomic level was lacking. This review article will highlight recent advances in understanding the structure of heteropentameric synaptic GABA\(_A\)Rs and how these structures have provided fundamental insights into the assembly of these multi-subunit receptors as well as their modulation by diverse ligands including the physiological agonist GABA. We will further discuss the role of gephyrin in the anchoring of synaptic GABA\(_A\)Rs and glycine receptors (GlyRs), which are crucial for maintaining the architecture of the iPSD. Finally, we will also summarize how anti-malarial artemisinin drugs modulate gephyrin-mediated inhibitory neurotransmission.
KW  - GABAA receptors
KW  - gephyrin
KW  - diazepam
KW  - GABA
KW  - PIP2
KW  - artemisinin
KW  - Cryo-EM
KW  - inhibitory neurotransmission
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201886
VL  - 12
IS  - 191
ER  - 
TY  - JOUR
A1  - Piro, Inken
A1  - Eckes, Anna-Lena
A1  - Kasaragod, Vikram Babu
A1  - Sommer, Claudia
A1  - Harvey, Robert J.
A1  - Schaefer, Natascha
A1  - Villmann, Carmen
T1  - Novel Functional Properties of Missense Mutations in the Glycine Receptor β Subunit in Startle Disease
JF  - Frontiers in Molecular Neuroscience
N2  - Startle disease is a rare disorder associated with mutations in GLRA1 and GLRB, encoding glycine receptor (GlyR) α1 and β subunits, which enable fast synaptic inhibitory transmission in the spinal cord and brainstem. The GlyR β subunit is important for synaptic localization via interactions with gephyrin and contributes to agonist binding and ion channel conductance. Here, we have studied three GLRB missense mutations, Y252S, S321F, and A455P, identified in startle disease patients. For Y252S in M1 a disrupted stacking interaction with surrounding aromatic residues in M3 and M4 is suggested which is accompanied by an increased EC\(_{50}\) value. By contrast, S321F in M3 might stabilize stacking interactions with aromatic residues in M1 and M4. No significant differences in glycine potency or efficacy were observed for S321F. The A455P variant was not predicted to impact on subunit folding but surprisingly displayed increased maximal currents which were not accompanied by enhanced surface expression, suggesting that A455P is a gain-of-function mutation. All three GlyR β variants are trafficked effectively with the α1 subunit through intracellular compartments and inserted into the cellular membrane. In vivo, the GlyR β subunit is transported together with α1 and the scaffolding protein gephyrin to synaptic sites. The interaction of these proteins was studied using eGFP-gephyrin, forming cytosolic aggregates in non-neuronal cells. eGFP-gephyrin and β subunit co-expression resulted in the recruitment of both wild-type and mutant GlyR β subunits to gephyrin aggregates. However, a significantly lower number of GlyR β aggregates was observed for Y252S, while for mutants S321F and A455P, the area and the perimeter of GlyR β subunit aggregates was increased in comparison to wild-type β. Transfection of hippocampal neurons confirmed differences in GlyR-gephyrin clustering with Y252S and A455P, leading to a significant reduction in GlyR β-positive synapses. Although none of the mutations studied is directly located within the gephyrin-binding motif in the GlyR β M3-M4 loop, we suggest that structural changes within the GlyR β subunit result in differences in GlyR β-gephyrin interactions. Hence, we conclude that loss- or gain-of-function, or alterations in synaptic GlyR clustering may underlie disease pathology in startle disease patients carrying GLRB mutations.
KW  - glycine receptor
KW  - hyperekplexia
KW  - startle disease
KW  - gephyrin
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246676
SN  - 1662-5099
VL  - 14
ER  - 
TY  - JOUR
A1  - Drehmann, Paul
A1  - Milanos, Sinem
A1  - Schaefer, Natascha
A1  - Kasaragod, Vikram Babu
A1  - Herterich, Sarah
A1  - Holzbach-Eberle, Ulrike
A1  - Harvey, Robert J.
A1  - Villmann, Carmen
T1  - Dual role of dysfunctional Asc-1 transporter in distinct human pathologies, human startle disease, and developmental delay
JF  - eNeuro
N2  - Human startle disease is associated with mutations in distinct genes encoding glycine receptors, transporters or interacting proteins at glycinergic synapses in spinal cord and brainstem. However, a significant number of diagnosed patients does not carry a mutation in the common genes GLRA1, GLRB, and SLC6A5. Recently, studies on solute carrier 7 subfamily 10 (SLC7A10; Asc-1, alanine-serine-cysteine transporter) knock-out (KO) mice displaying a startle disease-like phenotype hypothesized that this transporter might represent a novel candidate for human startle disease. Here, we screened 51 patients from our patient cohort negative for the common genes and found three exonic (one missense, two synonymous), seven intronic, and single nucleotide changes in the 5′ and 3′ untranslated regions (UTRs) in Asc-1. The identified missense mutation Asc-1\(^{G307R}\) from a patient with startle disease and developmental delay was investigated in functional studies. At the molecular level, the mutation Asc-1\(^{G307R}\) did not interfere with cell-surface expression, but disrupted glycine uptake. Substitution of glycine at position 307 to other amino acids, e.g., to alanine or tryptophan did not affect trafficking or glycine transport. By contrast, G307K disrupted glycine transport similar to the G307R mutation found in the patient. Structurally, the disrupted function in variants carrying positively charged residues can be explained by local structural rearrangements because of the large positively charged side chain. Thus, our data suggest that SLC7A10 may represent a rare but novel gene associated with human startle disease and developmental delay.
KW  - Asc-1 transporter
KW  - candidate gene
KW  - glycine receptor
KW  - glycine uptake
KW  - human startle disease
KW  - NMDAR
Y1  - 2023
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349947
VL  - 10
IS  - 11
ER  - 
TY  - THES
A1  - Kasaragod, Vikram Babu
T1  - Biochemical and Structural Basis for the Moonlighting Function of Gephyrin
T1  - Biochemische und Strukturelle Basis für die duale Funktionalität von Gephyrin
N2  - Neurons are specialized cells dedicated to transmit the nerve impulses throughout the human body across specialized structures called synapses. At the synaptic terminals, a crosstalk between multiple macromolecules regulates the structure and function of the presynaptic nerve endings and the postsynaptic recipient sites.
Gephyrin is the central organizer at inhibitory postsynaptic specializations and plays a crucial role in the organization of these structures by anchoring GABAA receptors (GABAAR) and glycine receptors (GlyR) to the postsynaptic membrane. This 93 kDa protein features an N-terminal G domain and a C-terminal E domain and the latter interacts directly with the intracellular loop between transmembrane helices 3 and 4 of certain subunits of the GlyRs and GABAARs. Biochemical and structural analyses have already provided valuable insights into the gephyrin-GlyR interaction. Interestingly, biochemical studies on the gephyrin-GABAAR interaction demonstrated that the GABAARs also depend on the same binding site as the GlyRs for the interaction with the gephyrin, but the molecular basis for this receptor specific interaction of gephyrin was still unknown. Co-crystal structures of GephE-GABAAR α3- derived peptides with supporting biochemical data presented in this study deciphered the receptor-specific interactions of gephyrin in atomic detail.
In its moonlighting function, gephyrin also catalyzes the terminal step of the evolutionarily conserved molybdenum cofactor biosynthesis. Molybdenum, an essential transition element has to be complexed with a pterin-based cofactor resulting in the formation of the molybdenum cofactor (Moco). Moco is an essential component at the active site of all molybdenum-containing enzymes with the exception of nitrogenase. Mutations in enzymes involved in this pathway lead to a rare yet severe disease called Moco deficiency, which manifest itself in severe neurodevelopmental abnormalities and early childhood death. Moco biosynthesis follows a complex multistep pathway, where in the penultimate step, the N-terminal G domain of gephyrin activates the molybdopterin to form an adenylated molybdopterin intermediate. In the terminal step, this intermediate is then transferred to the C-terminal E domain of gephyrin, which catalyzes the metal insertion and deadenylation reaction to form active Moco. Previous biochemical and structural studies provided valuable insights into the penultimate step of the Moco biosynthesis but the terminal step remained elusive. Through the course of my dissertation, I crystallized the C-terminal E domain in the apo-form as well as in complex with ADP and AMP. These structures shed lightonto the deadenylation reaction and the formation of a ternary E-domain-ADP-Mo/W complex and thus provide structural insight into the metal insertion mechanism. Moreover, the structures also provided molecular insights into a mutation leading to Moco deficiency. Finally, ternary
complexes of GephE, ADP and receptor-derived peptides provided first clues regarding the integration of gephyrin’s dual functionality.
In summary, during the course of the dissertation I was able to derive high resolution structural insights into the interactions between gephyrin and GABAARs, which explain the receptor-specific interaction of gephyrin and, furthermore, these studies can be extended in the future to understand GABAAR subunit-specific interactions of gephyrin. Finally, the understanding of Moco biosynthesis shed light on the molecular basis of the fatal Moco deficiency.
N2  - Neurone sind spezialisierte Zellen, die über die Synapsen Nervenimpulse im menschlichen Körper übertragen. An den synaptischen Enden reguliert ein Netzwerk aus einer Vielzahl von Makromolekülen die Struktur und die Funktion der präsynaptischen Nervenenden und der postsynaptischen Kontaktstellen. 
Gephyrin ist der Hauptorganisator an inhibitorischen, postsynaptischen Spezialisierungen und spielt durch die Verankerung von GABAA-Rezeptoren (GABAAR) und Glycinrezeptoren (GlyR) in der postsynaptischen Membran eine zentrale Rolle für den Aufbau dieser Strukturen. Dieses 93 kDa Protein enthält eine N-terminale G-Domäne (GephG) und eine C-terminale E-Domäne (GephE), wobei letztere direkt mit der intrazellulären unstrukturierten Region zwischen Transmembranhelices 3 und 4 bestimmter Untereinheiten der GlyR und GABAAR interagiert. Biochemische und strukturelle Analysen lieferten bereits wertvolle Erkenntnisse über die Gephyrin-GlyR Interaktion. Interessanterweise zeigten Versuche zur Gephyrin-GABAAR Interaktion, dass GABAARs die gleiche Bindungsstelle auf Gephyrin benutzen wie GlyRs, wobei die molekulare Basis für diese Interaktion nicht bekannt war. In dieser Arbeit zeige ich Co-Kristallstrukturen von GephE-GABAARα3 sowie unterstützende biochemische Daten, die die atomaren Details der rezeptorspezifischen Interaktionen von Gephyrin entschlüsseln.
Als zweite Funktion katalysiert Gephyrin den terminalen Schritt der evolutionär konservierten Molybdän Cofaktor Biosynthese. Dabei muss das essentielle Übergangselement Molybdän mit einem Pterin-basierten Cofaktor komplexiert werden, um den Molybdän Cofaktor (Moco) zu bilden. Moco ist essentieller Bestandteil im aktiven Zentrum aller Molybdän-enthaltenden Enzyme mit Ausnahme der Nitrogenase. Mutationen in Enzymen, die in die Molybdän Cofaktor Biosynthese involviert sind, verursachen eine Moco Defizienz, eine seltene, jedoch schwere Erkrankung, die sich durch schwere neurologische Entwicklungsstörungen und Tod im frühen Kindesalter äußert. Die Moco Biosynthese folgt einem komplexen mehrstufigen Ablauf. Im vorletzten Schritt adenyliert GephG das Molybdopterin und ein Zwischenprodukt entsteht. Im letzten Schritt wird dieses Zwischenprodukt auf GephE übertragen, das die Insertion des Metalls und die Deadenylierungsreaktion katalysiert, wodurch der aktive Moco entsteht. Frühere biochemische und strukturelle Studien brachten wertvolle Erkenntnisse über den vorletzten Schritt der Moco Biosynthese, aber die Kenntnisse über den letzten Schritt blieben vage. Während meiner Dissertation kristallisierte ich GephE in der apo-Form sowie im Komplex mit ADP oder AMP. Diese Strukturen gaben Aufschluss über die Deadenylierungsreaktion und die Formation eines ternären GephE-ADP-Mo/W Komplexes und gewährten so einen strukturellen Einblick in den Mechanismus der Metallinsertion. Darüber hinaus ermöglichten die Strukturen eine Mutation, die zu Moco Mangel führt, auf molekularer Ebene zu verstehen. Schließlich lieferten ternäre Komplexe aus GephE, ADP und von Rezeptoren abgeleiteten Peptiden ersten Aufschluss bezüglich der Verflechtung von Gephyrins dualer Funktion.
Zusammenfassend konnte ich während der Dissertation hochauflösende strukturelle Einblicke in den Komplex aus GephE und GABAAR α3 Untereineinheit gewinnen, die die rezeptorspezifische Interaktion von Gephyrin erklären. Weiterhin können diese Studien in der Zukunft ausgeweitet werden, um die GABAAR-untereinheitenspezifische Interaktion mit Gephyrin zu verstehen. Schließlich erlauben die Studien zur Moco Biosynthese die tödliche Moco Defizienz auf molekularer Ebene zu verstehen.
KW  - Gephyrin
KW  - Moco biosynthesis
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143077
ER  -