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Development of a Mouse Reporter Strain for the Purinergic P2X₂ Receptor.

ABSTRACT: The ATP-sensitive P2X₂ ionotropic receptor plays a critical role in a number of signal processes including taste and hearing, carotid body detection of hypoxia, the exercise pressor reflex and sensory transduction of mechanical stimuli in the airways and bladder. Elucidation of the role of P2X₂ has been hindered by the lack of selective tools. In particular, detection of P2X₂ using established pharmacological and biochemical techniques yields dramatically different expression patterns, particularly in the peripheral and central nervous systems. Here, we have developed a knock-in P2X₂-cre mouse, which we crossed with a cre-sensitive tdTomato reporter mouse to determine P2X₂ expression. P2X₂ was found in more than 80% of nodose vagal afferent neurons, but not in jugular vagal afferent neurons. Reporter expression correlated in vagal neurons with sensitivity to α,β methylene ATP (αβmATP). P2X₂ was expressed in 75% of petrosal afferents, but only 12% and 4% of dorsal root ganglia (DRG) and trigeminal afferents, respectively. P2X₂ expression was limited to very few cell types systemically. Together with the central terminals of P2X₂-expressing afferents, reporter expression in the CNS was mainly found in brainstem neurons projecting mossy fibers to the cerebellum, with little expression in the hippocampus or cortex. The structure of peripheral terminals of P2X₂-expressing afferents was demonstrated in the tongue (taste buds), carotid body, trachea and esophagus. P2X₂ was observed in hair cells and support cells in the cochlear, but not in spiral afferent neurons. This mouse strain provides a novel approach to the identification and manipulation of P2X₂-expressing cell types.

SUBMITTER: Kim SH

PROVIDER: S-EPMC7418537 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:P2X2 receptor channel, a homotrimer activated by the binding of extracellular adenosine triphosphate (ATP) to three intersubunit ATP-binding sites (each located ?50 Å from the ion permeation pore), also shows voltage-dependent activation upon hyperpolarization. Here, we used tandem trimeric constructs (TTCs) harboring critical mutations at the ATP-binding, linker, and pore regions to investigate how the ATP activation signal is transmitted within the trimer and how signals generated by ATP and hyperpolarization converge. Analysis of voltage- and [ATP]-dependent gating in these TTCs showed that: (a) Voltage- and [ATP]-dependent gating of P2X2 requires binding of at least two ATP molecules. (b) D315A mutation in the ?-14 strand of the linker region connecting the ATP-binding domains to the pore-forming helices induces two different gating modes; this requires the presence of the D315A mutation in at least two subunits. (c) The T339S mutation in the pore domains of all three subunits abolishes the voltage dependence of P2X2 gating in saturating [ATP], making P2X2 equally active at all membrane potentials. Increasing the number of T339S mutations in the TTC results in gradual changes in the voltage dependence of gating from that of the wild-type channel, suggesting equal and independent contributions of the subunits at the pore level. (d) Voltage- and [ATP]-dependent gating in TTCs differs depending on the location of one D315A relative to one K308A that blocks the ATP binding and downstream signal transmission. (e) Voltage- and [ATP]-dependent gating does not depend on where one T339S is located relative to K308A (or D315A). Our results suggest that each intersubunit ATP-binding signal is directly transmitted on the same subunit to the level of D315 via the domain that contributes K308 to the ?-14 strand. The signal subsequently spreads equally to all three subunits at the level of the pore, resulting in symmetric and independent contributions of the three subunits to pore opening.

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Development of a Mouse Reporter Strain for the Purinergic P2X₂ Receptor.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Development of a Mouse Reporter Strain for the Purinergic P2X2 Receptor.

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OmicsDI is part of the ELIXIR infrastructure

Tweets

Development of a Mouse Reporter Strain for the Purinergic P2X₂ Receptor.