9 research outputs found
High-Throughput Automated Patch Clamp Investigations on Ion Channels in Erythrocytes
Trotz ihrer morphologischen Einfachheit ist die Membran der roten Blutkörperchen (Erythrozyten) mit einer Reihe von Transportern und IonenkanĂ€len ausgestattet, die bisher nicht vollstĂ€ndig charakterisiert sind und deren biologische Rolle noch wenig verstanden ist. Die meisten Techniken zur Untersuchung von IonenkanĂ€len messen summierte Effekte groĂer Zellpopulationen und verbergen so jede mutmaĂliche VariabilitĂ€t von Zelle zu Zelle. Die Patch-Clamp-Technik hat sich als effektives Werkzeug zur Entdeckung und Charakterisierung von IonenkanĂ€len auf Einzelzellenebene erwiesen. Dies besonders wichtig fĂŒr Erythrozyten von SĂ€ugetieren, die eine hohe HeterogenitĂ€t der LeitfĂ€higkeit zwischen verschiedenen Spendern, und auch zwischen Zellen desselben Spenders aufweisen (Kaestner et al., 2004; Minetti et al., 2013). Die Entwicklung des automatisierten Patch-Clamps ermöglichte es, eine hohe Anzahl von Zellen gleichzeitig unter identischen experimentellen Bedingungen zu untersuchen, wodurch ZellheterogenitĂ€t erstmals umfassend bestimmt wurde.
In dieser Arbeit wurden Gårdos- und Piezo1-KanÀle als Hauptuntersuchungsziele ausgewÀhlt, da sie eine prominente Rolle in erythrozytÀren Erkrankungen, im Einzelnen Gårdos-Kanalopathie (Fermo et al., 2017) und hereditÀre Xerozytose (Zarychanski et al., 2012; Bae et al., 2013), spielen. Ziel dieser Arbeit war es, automatisierte Patch-Clamp-Assays zur Charakterisierung dieser KanÀle in Erythrozyten zu entwickeln.
Es gibt bisher nur vereinzelte Publikationen zu whole-cell Patch-Clamp-Messungen von GĂĄrdos-KanĂ€len in Erythrozyten (Grygorczyk et al., 1984; Wolff et al., 1988), wahrscheinlich aufgrund der geringen Expression des Proteins in zirkulierenden Erythrozyten. Der hochparallelisierte Ansatz der automatisierten Patch-Clamp-Technologie ermöglicht zuverlĂ€ssig die Identifizierung von GĂĄrdos-Strömen in Zelltypen mit einer oft geringen Anzahl von KanĂ€len und einer groĂen HeterogenitĂ€t der Expression, wie bei Erythrozyten.
Bisherige Piezo1-Kanaluntersuchungen zeigen, dass die Substanz Yoda1 Piezo1-Ströme bewirken kann, die empfindlich auf GdCl3 (unspezifischer Inhibitor dehnungsaktivierter KanÀle), nicht jedoch auf TRAM-34 (spezifischer Gårdos-Kanalinhibitor) reagieren. Die Anwendung dieses Assays auf Erythrozyten von Patienten mit einer neuartigen PIEZO1 R2110W-Mutation zeigte eine erhöhte Anzahl der Yoda1-empfindlichen Zellen und eine stÀrkere Antwort auf Yoda1 bei Patienten im Vergleich zu Kontroll-Erythrozyten. In Kombination mit der Untersuchung der Proteinstruktur, die den R2110W-Rests in einem gating-sensitiven Bereich des Kanals lokalisiert, deuten die Patch-Clamp-Ergebnisse darauf hin, dass die neue Piezo1-Mutation eine gain-of-function-Mutation ist (Rotordam et al., 2019).
Zusammenfassend zeigt diese Arbeit, dass die automatisierte Patch-Clamp-Methode robuste Assays zur Untersuchung von IonenkanĂ€len (GĂĄrdos und Piezo1) in PrimĂ€rzellen liefert. Die Hochdurchsatztechnologie ermöglichte die Entwicklung eines zuverlĂ€ssigen Assays fĂŒr gering exprimierte IonenkanĂ€le bei hoher HeterogenitĂ€t der Zellen. So war es möglich, eine neuartige Kanalmutation auf funktioneller Ebene direkt in Patientenzellen zu charakterisieren, ohne die Mutation in einem heterologen Expressionssystem exprimieren zu mĂŒssen. Dieser Ansatz kann zum Nachweis und zur Charakterisierung weiterer Kanalopathien verwendet werden, die nicht auf Erythrozyten beschrĂ€nkt sind, und kann generell als zur Gensequenzierung komplementĂ€rer Routine-Screening-Assay fĂŒr Krankheiten dienen, die mit Ionenkanalstörungen zusammenhĂ€ngen.Despite the morphological simplicity, the Red Blood Cell (RBC) membrane is endowed with a number of transporters and ion channels, yet not fully characterized and whose biological role is still poorly understood. Most of the techniques used to investigate ion channels are addressed to large populations of cells, thus concealing any putative cell-to-cell variability. The patch clamp technique has proven to be a valid tool for the discovery and characterization of ion channels at a single-cell level. This is of particular relevance for mammalian RBCs, which present a high heterogeneity of conductance not only between different donors but also among cells of the same donor (Kaestner et al., 2004; Minetti et al., 2013). The advent of automated patch clamp allowed to probe an increased number of cells at the same time under identical experimental conditions, thus tackling cell heterogeneity issues.
In this thesis, GĂĄrdos and Piezo1 channels were selected as main targets of investigation due to their relevance in RBC-related diseases, i.e. GĂĄrdos channelopathy (Fermo et al., 2017) and hereditary xerocytosis (Zarychanski et al., 2012; Bae et al., 2013). The aim of this work was to develop automated patch clamp assays for characterizing those channels in RBCs.
As for GĂĄrdos channels, whole cell recordings reported so far are fragmentary probably due to the low expression of the protein in circulating RBCs (Grygorczyk et al., 1984; Wolff et al., 1988). By increasing the number of cells recorded at the same time, the automated patch clamp technology allowed to identify GĂĄrdos-mediated currents in primary cells with a low-copy number of channels and a large heterogeneity of conductance as RBCs.
Piezo1 channels investigations confirmed that application of Yoda1 alone is able to elicit currents sensitive to GdCl3 (non-specific stretch-activated channels inhibitor) but not TRAM-34 (specific GĂĄrdos channel blocker). When transferred to patients carrying a novel PIEZO1 R2110W mutation, the assay revealed that the number of responders and the magnitude of the response to Yoda1 increased in patient compared to control RBCs. This result, combined with structural studies identifying the R2110W residue in a gating sensitive area of the channel, suggested that the novel Piezo1 mutation is gain-of-function (Rotordam et al., 2019).
Altogether, this work demonstrates that automated patch clamping provides robust assays to investigate ion channels (GĂĄrdos and Piezo1) in primary cells. The high-throughput technology allowed to tackle issues as response heterogeneity and low expression of the channels, and to characterize a novel channel mutation at a functional level directly from patient cells, without having to express the mutation in a heterologous expression system. This approach may be used to detect other channelopathies not limited to RBCs and may serve as routine screening assay for diseases related to ion channel dysfunctions in general, complementary to gene sequencing
A novel gain-of-function mutation of Piezo1 is functionally affirmed in red blood cells by high-throughput patch clamp
No Abstract available
Altered Ca2+ Homeostasis in Red Blood Cells of Polycythemia Vera Patients Following Disturbed Organelle Sorting during Terminal Erythropoiesis
The authors thank Thierry Peyrard, Dominique Gien, Sirandou Tounkara, and Eliane VĂ©ra at Centre National de RĂ©fĂ©rence pour les Groupes Sanguins for the management of blood samples. The authors thank Sandrine Genetet and Isabelle Mouro-Chanteloup at the Inserm UMR_S1134 unit for their assistance in experiments. The authors also thank MichaĂ«l Dussiot at the Institute Imagine for his assistance in imaging flow cytometry. We thank Johanna Bruce and Virginie Salnot at 3P5 Proteomics Platform for sample preparation and analysis, and François Guillonneau and Patrick Mayeux for their management and strategies. Funding: The work was supported by Institut National de la SantĂ© et de la Recherche MĂ©dicale (Inserm); Institut National de la Transfusion Sanguine (INTS); the University of Paris; and grants from Laboratory of Excellence (Labex) GR-Ex, reference No. ANR-11-LABX-0051. The Labex GR- Ex is funded by the IdEx program âInvestissements dâavenirâ of the French National Research Agency, reference No. ANR-11-IDEX-0005-02 and ANR-18-IDEX-0001. R.B., M.G.R., and D.M.A. were funded by the European Unionâs Horizon 2020 Research and Innovation Program under grant agreement No. 675115-RELEVANCE-H2020-MSCA-ITN-2015. R.B. also received financial support from SociĂ©tĂ© Française dâHĂ©matologie (SFH) and Club du Globule Rouge et du Fer (CGRF). R.B. is currently funded by the Innovate UK Research and Innovation Knowledge Transfer Partnership (KTP) between University of Aberdeen and Vertebrate Antibodies Ltd. (Partnership No. KTP12327). T.D. was supported by PhD grants from UniversitĂ© Paris Saclay MESR (MinistĂšre Enseignement SupĂ©rieur et de la Recherche) and then FRM (Fondation recherche mĂ©dicale). The Orbitrap Fusion mass spectrometer was acquired with funds from Fonds Europeen de Developpement Regional (FEDER) through the Operational Program for Competitiveness Factors and Employment 2007-2013 and from the Canceropole Ile de France.Peer reviewedPublisher PD
Corrigendum to âA systematic strategy for estimating hERG block potency and its implications in a new cardiac safety paradigmâ [Toxicology and Applied Pharmacology volume 394C (2020) 114961]
© 2020 The Author(s) The authors regret that one affiliation address is mistaken in the published paper. Matthew Bridgland-Taylor's affiliation was incorrectly listed as Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, Cambridge, United Kingdom. The correct affiliation is Clinical Pharmacology & Safety Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, United Kingdom. The authors would like to apologise for any inconvenience caused
A systematic strategy for estimating hERG block potency and its implications in a new cardiac safety paradigm
© 2020 Introduction: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. Methods: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. Results: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. Discussion: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment
Missense mutations in PIEZO1, which encodes the Piezo1 mechanosensor protein, define Er red blood cell antigens
Despite the identification of the high-incidence red cell antigen Era nearly 40 years ago, the molecular background of this antigen, together with the other 2 members of the Er blood group collection, has yet to be elucidated. Whole exome and Sanger sequencing of individuals with serologically defined Er alloantibodies identified several missense mutations within the PIEZO1 gene, encoding amino acid substitutions within the extracellular domain of the Piezo1 mechanosensor ion channel. Confirmation of Piezo1 as the carrier molecule for the Er blood group antigens was demonstrated using immunoprecipitation, CRISPR/Cas9-mediated gene knockout, and expression studies in an erythroblast cell line. We report the molecular bases of 5 Er blood group antigens: the recognized Era, Erb, and Er3 antigens and 2 novel high-incidence Er antigens, described here as Er4 and Er5, establishing a new blood group system. Anti-Er4 and anti-Er5 are implicated in severe hemolytic disease of the fetus and newborn. Demonstration of Piezo1, present at just a few hundred copies on the surface of the red blood cell, as the site of a new blood group system highlights the potential antigenicity of even low-abundance membrane proteins and contributes to our understanding of the in vivo characteristics of this important and widely studied protein in transfusion biology and beyond
Adverse effects of deltaâ9âtetrahydrocannabinol on sickle red blood cells
International audienc