Novel channel-mediated choline transport in cholinergic neurons of the mouse retina

Abstract

Choline uptake into the presynaptic terminal of cholinergic neurons is mediated by the high-affinity choline transporter and is essential for acetylcholine synthesis. In a previous study, we reported that P2X purinoceptors are selectively expressed in OFF-cholinergic amacrine cells of the mouse retina. Under specific conditions, P2X purinoceptors acquire permeability to large cations, such as -methyl-d-glucamine, and therefore potentially could act as a noncanonical pathway for choline entry into neurons. We tested this hypothesis in OFF-cholinergic amacrine cells of the mouse retina. ATP-induced choline currents were observed in OFF-cholinergic amacrine cells, but not in ON-cholinergic amacrine cells, in mouse retinal slice preparations. High-affinity choline transporters are expressed at higher levels in ON-cholinergic amacrine cells than in OFF-cholinergic amacrine cells. In dissociated preparations of cholinergic amacrine cells, ATP-activated cation currents arose from permeation of extracellular choline. We also examined the pharmacological properties of choline currents. Pharmacologically, α,β-methylene ATP did not produce a cation current, whereas ATPγS and benzoyl-benzoyl-ATP (BzATP) activated choline currents. However, the amplitude of the choline current activated by BzATP was very small. The choline current activated by ATP was strongly inhibited by pyridoxalphosphate-6-azophenyl-2',4'-sulfonic acid. Accordingly, P2X purinoceptors expressed in HEK-293T cells were permeable to choline and similarly functioned as a choline uptake pathway. Our physiological and pharmacological findings support the hypothesis that P2 purinoceptors, including P2X purinoceptors, function as a novel choline transport pathway and may provide a new regulatory mechanism for cholinergic signaling transmission at synapses in OFF-cholinergic amacrine cells of the mouse retina. Choline transport across the membrane is exerted by both the high-affinity and low-affinity choline transporters. We found that choline can permeate P2 purinergic receptors, including P2X purinoceptors, in cholinergic neurons of the retina. Our findings show the presence of a novel choline transport pathway in cholinergic neurons. Our findings also indicate that the permeability of P2X purinergic receptors to choline observed in the heterologous expression system may have a physiological relevance in vivo.

P2X2 receptors supply extracellular choline as a substrate for acetylcholine synthesis

“…In HEK293T cells expressing P2X 2 receptors, we previously reported that the application of ATP opened P2X 2 receptors, resulting in the extracellular influx of choline into the intracellular space [ 27 ]. In the present study, to clarify whether choline that enters through the P2X 2 receptor is used for ACh synthesis, we applied ATP to ChAT‐HEK293 cells in choline‐rich solution to maximize choline influx and the opportunity for ACh synthesis (Table 1 ).…”

“…It may be explained by competition between Na + and choline + at the cation channels of P2X 2 receptors, similar to the anomalous mole fraction effects reported in Ca channels [ 32 , 33 , 34 , 35 , 36 ]. The permeability of choline + to the P2X 2 receptor is less than that of Na + [ 27 ]. We observed a significant increase in [ACh] i in choline solution because choline + freely permeates the cation channel of P2X 2 receptors without competition from Na + (Fig.…”

“…Immunocytochemistry was performed as described in our previous studies [ 27 , 30 ]. In brief, HEK293T or ChAT‐HEK293 cells were fixed with 4% paraformaldehyde (w/v) in 0.1 m phosphate buffer (PB) for 30 min, permeabilized for 15 min in 0.1 m PB containing 0.3% Triton‐X 100, and blocked with 0.1 m PB containing 1% Block Ace (Dainippon Seiyaku, Osaka, Japan) at room temperature for 60 min.…”

“…In the retina, ACh is released from cholinergic amacrine cells [ 19 , 24 ]. We recently demonstrated that P2X 2 receptors, a subtype of P2X purinoceptors that form nonselective cation channels [ 25 , 26 ], function as an alternative pathway for choline entry in the cholinergic amacrine cells of the mouse retina [ 27 ]. However, it currently remains unclear whether choline that enters through P2X 2 receptors is used to synthesize ACh because of the difficulties associated with measuring ACh concentrations in the cholinergic amacrine cells of the mouse retina.…”

Gene expression profile of the murine ischemic retina and its response to Aflibercept (VEGF-Trap)

“…Whereas this finding by itself could suggest that RGCs engage in a protective behaviour to reduce hypoxia-associated damage and death, the reduced expression levels of Glra1 , which encodes the subunit alpha-1 of the Glycine receptor (also expressed in RGCs 84 , 85 ), may instead indicate that RGC functionality and survival is compromised as a consequence of OIR and partially rescued by AFL administration. Finally, among the 10 genes associated to the chemical synaptic transmission term, P2rx2 , which encodes a subunit of a ionotropic receptor expressed in starburst amacrine cells 86 , was the most significantly down-regulated gene in OIR relative to normoxic retinas and subsequently up-regulated in response to AFL at P17. No GO terms in the biological process domain were detected as enriched in P19(AFLx2) or P19(AFLx3) retinas.…”