Development of a P2X1-eYFP receptor knock-in mouse to track receptors in real time.

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Tytuł:: Development of a P2X1-eYFP receptor knock-in mouse to track receptors in real time.
Autorzy:: Mahaut Smith MP; Department of Molecular and Cell Biology, University of Leicester, Leicester, LE1 7RH, UK.
Evans RJ; Department of Molecular and Cell Biology, University of Leicester, Leicester, LE1 7RH, UK.
Vial C; Department of Molecular and Cell Biology, University of Leicester, Leicester, LE1 7RH, UK. .
Źródło:: Purinergic signalling [Purinergic Signal] 2019 Sep; Vol. 15 (3), pp. 397-402. Date of Electronic Publication: 2019 Jul 08.
Typ publikacji:: Journal Article; Research Support, Non-U.S. Gov't
Język:: English
Imprint Name(s):: Original Publication: Dordrecht, the Netherlands : Springer
MeSH Terms:: Gene Knock-In Techniques/*methods
Receptors, Purinergic P2X1/*analysis
Receptors, Purinergic P2X1/*metabolism
Animals ; Bacterial Proteins ; Luminescent Proteins ; Mice ; Models, Animal
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Grant Information:: United Kingdom WT_ Wellcome Trust; 080487/Z/06/Z United Kingdom WT_ Wellcome Trust
Contributed Indexing:: Keywords: ATP; Ion channels; Megakaryocytes; P2X1; P2X1-eYFP; Platelets; Smooth muscle
Substance Nomenclature:: 0 (Bacterial Proteins)
0 (Luminescent Proteins)
0 (Receptors, Purinergic P2X1)
0 (yellow fluorescent protein, Bacteria)
Entry Date(s):: Date Created: 20190710 Date Completed: 20200406 Latest Revision: 20210110
Update Code:: 20240105
PubMed Central ID:: PMC6736900
DOI:: 10.1007/s11302-019-09666-1
PMID:: 31286385
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A P2X1-eYFP knock-in mouse was generated to study receptor expression and mobility in smooth muscle and blood cells. eYFP was added to the C-terminus of the P2X1R and replaced the native P2X1R. Fluorescence corresponding to P2X1-eYFPR was detected in urinary bladder smooth muscle, platelets and megakaryocytes. ATP-evoked currents from wild type and P2X1-eYFP isolated urinary bladder smooth muscle cells had the same peak current amplitude and time-course showing that the eYFP addition had no obvious effect on properties. Fluorescence recovery after photobleaching (FRAP) in bladder smooth muscle cells demonstrated that surface P2X1Rs are mobile and their movement is reduced following cholesterol depletion. Compared to the platelet and megakaryocyte, P2X1-eYFP fluorescence was negligible in red blood cells and the majority of smaller marrow cells. The spatial pattern of P2X1-eYFP fluorescence in the megakaryocyte along with FRAP assessment of mobility suggested that P2X1Rs are expressed extensively throughout the membrane invagination system of this cell type. The current study highlights that the spatiotemporal properties of P2X1R expression can be monitored in real time in smooth muscle cells and megakaryocytes/platelets using the eYFP knock-in mouse model.

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