AMPA receptor anchoring at CA1 synapses is determined by N-terminal domain and TARP γ8 interactions.

AMPA receptor (AMPAR) abundance and positioning at excitatory synapses regulates the strength of transmission. Changes in AMPAR localisation can enact synaptic plasticity, allowing long-term information storage, and is therefore tightly controlled. Multiple mechanisms regulating AMPAR synaptic anchoring have been described, but with limited coherence or comparison between reports, our understanding of this process is unclear. Here, combining synaptic recordings from mouse hippocampal slices and super-resolution imaging in dissociated cultures, we compare the contributions of three AMPAR interaction domains controlling transmission at hippocampal CA1 synapses. We show that the AMPAR C-termini play only a modulatory role, whereas the extracellular N-terminal domain (NTD) and PDZ interactions of the auxiliary subunit TARP γ8 are both crucial, and each is sufficient to maintain transmission. Our data support a model in which γ8 accumulates AMPARs at the postsynaptic density, where the NTD further tunes their positioning. This interplay between cytosolic (TARP γ8) and synaptic cleft (NTD) interactions provides versatility to regulate synaptic transmission and plasticity

Functional impact of human disease mutations, protein-protein and lipid-protein interactions on L-type Ca2+ [Ca hoch 2+] channels

Spannungsabhängige L-Typ-Ca2+ Kanäle (LTCK) übersetzen Membrandepolarisationen in unterschiedliche zelluläre Funktionen wie Muskelkontraktion, Genregulation, Neurotransmitterfreisetzung und Hormonausschüttung. In Rahmen meiner Doktorarbeit habe ich die Krankheitsrelevanz von menschlichen LTCK-Mutationen als auch die Modulation dieser Kanäle durch Plasmamembranlipide und Proteininteraktionen untersucht. Kürzlich wurden mehrere Cav1.3-Mutationen in Probanden mit Autismus-Spektrum-Störungen (ASD) als auch in Aldosteron produzierenden Adenomen (APA) identifiziert. Des Weiteren wurde eine zusätzliche Cav1.3 Mutation in einer Familie mit kardialen und neurologischen Dysfunktionen (KND) identifiziert. Um einen Einblick in das krankheitsfördernde Potential dieser Mutationen zu bekommen, wurden sie nach rekombinanter Expression in tsA-Zellen mittels whole-cell Patch-Clamp Experimenten auf mögliche Veränderungen ihrer biophysikalischen Eigenschaften untersucht. Dabei konnte gezeigt werden, dass die in ASD-Personen identifizieren de novo Mutationen G407R (IS6) und A749G (IIS6), zu einem komplexen Phänotyp führen, der sowohl Merkmale einer erhöhten, als auch einer verringerten Funktion aufweist. Dies deutet darauf hin, dass die Mutationen in diesen Probanden in der Tat eine wichtige Rolle in der Pathophysiologie von ASD spielen. Ähnlich zu den bereits charakterisierten Cav1.3 APA-Mutationen, resultierten auch die hier untersuchten Mutationen (F747L, IIS6; R990H, IIIS4; M1354I, IVS5), zu Veränderungen im Öffnungs- und Schließverhalten (Gating) des Kanals, welche einen erhöhten Ca2+ Einstrom in die Zelle zur Folge haben und damit zur erhöhten Aldosteronproduktion führen. Die in KND identifizierte Cav1.3 Mutation führte im Gegensatz dazu nur zu geringen Gatingveränderungen in der C-terminalen kurzen Spleißvariante des Cav1.3 Kanals. Daher ist es sehr unwahrscheinlich, dass diese Mutation eine kausale Rolle in der Entwicklung von KND spielt. Abgesehen von Mutationen, können allerdings auch andere Mechanismen das Gating von LTCKs ändern. Die I-II-Linker aller LTCKs enthalten ein positiv geladenes Motiv, bestehend aus vier Arginin Resten. Live-cell imaging Experimente zeigten, dass dieses Motiv mit negativ geladenen Phospholipiden der Plasmamembran interagiert. Elektrophysiologische Experimente zeigten zudem, dass das positiv geladene Motiv die Kanalaktivität moduliert indem es dessen Öffnungswahrscheinlichkeit reduziert und die Hemmung des Kanals durch Hydrolyse von Plasmamembranphospholipiden meditiert. Dies zeigt, dass dieses Motiv nicht nur einen zurückhaltenden Gating-Modus stabilisiert, sondern auch die Modulation der Kanalaktivität durch rezeptorvermittelten Lipidabbau mediert. Zudem hab ich im Rahmen meiner Dissertation untersucht, ob die Interaktion der präsynaptischen Proteine RIM2 und RIM-binding-protein 2 (RBP2) mit Cav1.3 für dessen langsame spannungsabhängige Inaktivierung in den inneren Haarzellen (IHZ) der Cochlea verantwortlich ist. Mittels nested PCR konnte ich die Expression von RIM2- und RBP2-Transkripten in Maus-IHZ in allen Entwicklungsstadien zeigen. Co-Lokalisationsstudien mittels Immunfluoreszenzmikroskopie nach rekombinanter Expression in tsA-Zellen als auch Co-Immunpräzipitationen von Mausgeweben, sprechen gegen eine stabile Interaktion von RBP2 mit RIM2 und Cav1.3. In elektrophysiologischen Experimenten konnte jedoch gezeigt werden, dass die Co-Expression von RBP2 und/oder RIM2 in tsA-201-Zellen, die spannungsabhängige Inaktivierung des Kanals signifikant verringert, allerdings nicht im selben Ausmaß wie in IHZ. Daher bedarf es weiterer Experimente um die Ursache für die langsame spannungsabhängige Inaktivierung in IHZ herauszufinden.Voltage gated L-type Ca2+ channels (LTCCs) transduce membrane depolarization into different cellular functions like muscle contraction, gene regulation, neurotransmitter release and hormone secretion. In my thesis, I investigated the disease relevance of human LTCC mutations and the modulation of these channels by plasma membrane lipids and protein interactions. Recently, several mutations have been identified in Cav1.3 in probands with autism spectrum disorders (ASD) as well as in aldosterone producing adenomas (APAs). An additional Cav1.3 mutation was identified in a family with cardiac and neurological disorders (CND). I investigated the biophysical alterations induced by these mutations upon heterologous expression in tsA-201 cells using whole-cell patch-clamp recordings, in order to assess their disease causing potential. I found that de novo mutations G407R (IS6) and A749G (IIS6), both identified in individuals with ASD, resulted in a complex phenotype with features of gain- as well as loss-of-function. This suggests that they play an important role in the pathophysiology of ASD in these probands. Moreover, similarly to what has already been reported for Cav1.3 mutations identified in other APAs, the mutations characterized here (F747L, IIS6; R990H, IIIS4; M1354I, IVS5) resulted in gating changes enabling enhanced Ca2+ influx, underlying the increased aldosterone production. In contrast, the Cav1.3 mutation identified in CND resulted in minor gating changes only when introduced in a C-terminal short Cav1.3 splice variant (Cav1.3S), making a causative role very unlikely. Apart from mutations, other mechanisms can also modify LTCC gating. The I-II linker of all LTCCs contain a polybasic motif comprising four arginine residues. Live-cell imaging experiments showed that this motif interacts with negatively charged phospholipids of the plasma membrane. Electrophysiological experiments revealed that the polybasic motif modulates the channel activity by reducing the open probability and by mediating current inhibition upon hydrolysis of plasma membrane phospholipids. This indicates that the polybasic motif not only stabilizes a more reluctant gating mode but also supports modulation of channel activity by receptor-mediated lipid breakdown. In my thesis I also investigated if interaction of the presynaptic proteins RIM2 and RIM-binding-protein 2 (RBP2) could account for the slow voltage dependent inactivation (VDI) observed in cochlear inner hair cells (IHCs). Nested PCR revealed expression of RIM2 and RBP2 transcripts in mouse IHCs at all developmental stages. Co-localization studies using immunofluorescence microscopy upon recombinant expression in tsA-cells as well as co-immunoprecipitations from mouse tissue, argued against a stable interaction of these proteins. However, electrophysiological experiments revealed that co-expression of RBP2 and/or RIM2 in tsA-201 cells significantly reduced the VDI of the channel, but not to the same extend as observed in IHCs. The mechanism responsible for the slow VDI in these cells therefore needs further investigation.Alexandra PinggeraInnsbruck, Univ., Diss., 2015OeBB(VLID)85631

Allosteric coupling of sub-millisecond clamshell motions in ionotropic glutamate receptor ligand-binding domains

Ionotropic glutamate receptors (iGluRs) mediate signal transmission in the brain and are important drug targets. Structural studies show snapshots of iGluRs, which provide a mechanistic understanding of gating, yet the rapid motions driving the receptor machinery are largely elusive. Here we detect kinetics of conformational change of isolated clamshell-shaped ligand-binding domains (LBDs) from the three major iGluR sub-types, which initiate gating upon binding of agonists. We design fluorescence probes to measure domain motions through nanosecond fluorescence correlation spectroscopy. We observe a broad kinetic spectrum of LBD dynamics that underlie activation of iGluRs. Microsecond clamshell motions slow upon dimerization and freeze upon binding of full and partial agonists. We uncover allosteric coupling within NMDA LBD hetero-dimers, where binding of L-glutamate to the GluN2A LBD stalls clamshell motions of the glycine-binding GluN1 LBD. Our results reveal rapid LBD dynamics across iGluRs and suggest a mechanism of negative allosteric cooperativity in NMDA receptors

New gain-of-function mutation shows CACNA1D as recurrently mutated gene in autism spectrum disorders and epilepsy

CACNA1D encodes the pore-forming alpha 1-subunit of Cav1.3, an L-type voltage-gated Ca2+-channel. Despite the recent discovery of two de novo missense gain-of-function mutations in Cav1.3 in two individuals with autism spectrum disorder (ASD) and intellectual disability CACNA1D has not been considered a prominent ASD-risk gene in large scale genetic analyses, since such studies primarily focus on likely-disruptive genetic variants. Here we report the discovery and characterization of a third de novo missense mutation in CACNA1D (V401L) in a patient with ASD and epilepsy. For the functional characterization we introduced mutation V401L into two major C-terminal long and short Cav1.3 splice variants, expressed wild-type or mutant channel complexes in tsA-201 cells and performed whole-cell patch-clamp recordings. Mutation V401L, localized within the channel's activation gate, significantly enhanced current densities, shifted voltage dependence of activation and inactivation to more negative voltages and reduced channel inactivation in both Cav1.3 splice variants. Altogether, these gating changes are expected to result in enhanced Ca2+-influx through the channel, thus representing a strong gain-of-function phenotype. Additionally, we also found that mutant channels retained full sensitivity towards the clinically available Ca2+-channel blocker isradipine. Our findings strengthen the evidence for CACNA1D as a novel candidate autism risk gene and encourage experimental therapy with available channel-blockers for this mutation. The additional presence of seizures and neurological abnormalities in our patient define a novel phenotype partially overlapping with symptoms in two individuals with PASNA (congenital primary aldosteronism, seizures and neurological abnormalities) caused by similar Cav1.3 gain-of-function mutations

Urine high-risk human papillomavirus testing as an alternative to routine cervical screening: A comparative diagnostic accuracy study of two urine collection devices using a randomised study design trial.

OBJECTIVE: To evaluate the sensitivity of human papillomavirus (HPV) tested urine to detect high-grade cervical precancer (cervical intraepithelial neoplasia grade 2+ [CIN2+]) using two urine collection devices. DESIGN: Randomised controlled trial. SETTING: St Mary's Hospital, Manchester, UK. POPULATION: Colposcopy attendees with abnormal cervical screening; a total of 480 participants were randomised. Matched urine and cervical samples were available for 235 and 230 participants using a first-void urine (FVU)-collection device and standard pot, respectively. METHODS: Urine was self-collected and mixed with preservative - randomised 1:1 to FVU-collection device (Novosanis Colli-pee® 10 mL with urine conservation medium [UCM]) or standard pot. Matched clinician-collected cervical samples were taken before colposcopy. HPV testing used Roche cobas® 8800. A questionnaire evaluated urine self-sampling acceptability. MAIN OUTCOME MEASURES: The primary outcome measured sensitivity of HPV-tested urine (FVU-collection device and standard pot) for CIN2+ detection. Secondary outcomes compared HPV-tested cervical and urine samples for CIN2+ and evaluated the acceptability of urine self-sampling. RESULTS: Urine HPV test sensitivity for CIN2+ was higher with the FVU-collection device (90.3%, 95% CI 83.7%-94.9%, 112/124) than the standard pot (73.4%, 95% CI 64.7%-80.9%, 91/124, p = 0.0005). The relative sensitivity of FVU-device-collected urine was 0.92 (95% CI 0.87-0.97, pMcN = 0.004) compared with cervical, considering that all women were referred after a positive cervical HPV test. Urine-based sampling was acceptable to colposcopy attendees. CONCLUSIONS: Testing of FVU-device-collected urine for HPV was superior to standard-pot-collected urine in colposcopy attendees and has promising sensitivity for CIN2+ detection. General population HPV testing of FVU-device-collected urine will establish its clinical performance and acceptability as an alternative to routine cervical screening

A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function.

L-type voltage-gated Ca(2+) channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The pore-forming α1-subunit comprises four repeated domains (I-IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Cav1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphosphoinositide breakdown, and was accelerated by elevated intracellular Ca(2+) levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. Neutralization of four arginine residues eliminated plasma membrane binding. Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane. Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca(2+) channel function