Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X ₂ -green fluorescent protein receptors

Abstract

ATP-gated P2X2 receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X 2 receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X2-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X2-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X 2-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X 2 receptor distribution on the seconds time scale.ion channel ͉ ATP ͉ filopodia C ationic P2X receptors mediate the ''fast'' milliseconds time scale actions of ATP in the nervous system (1, 2). The identity of most natively expressed P2X receptors is unclear, but many neurons express P2X 2 mRNA, P2X 2 proteins, and functional P2X 2 -like receptors (2). For example, ATP mediates synaptic transmission in a portion of CA1 neurons (3), and postnatal hippocampal neurons express P2X receptors, which include P2X 2 subunits (3-7). Moreover, cytosolic ATP concentration is 1-5 mM, and ATP released during tissue damage activates neuronal P2X receptors in the periphery (1). ATP released as a synaptic transmitter and during ischemia of brain neurons may contribute to pathophysiology, but there are no available data on the cellular consequences of P2X 2 receptor activation or on the dynamic aspects of P2X 2 receptor distribution in brain neurons.This study used P2X 2 receptors tagged with green fluorescent protein (GFP) in a quantitative method to study receptors expressed with recombinant Sindbis virus in embryonic hippocampal neurons. We report (i) the properties of functional GFP-tagged P2X 2 receptors, (ii) an optical and electrophysiological approach to measuring receptor numbers in living cells, and (iii) the cellular effects of P2X receptor activation. Materials and MethodsMolecular Biology. By PCR the P2X 2 stop codon was removed and the FLAG (f) epitope was inserted in frame with the P2X 2 cDNA cDNA (9). In the same PCR we inserted an XhoI site in the DNA. We generated GFP37 (10) with a XhoI site before the start codon and subcloned it into P2X 2 -f between the XhoI site 3Ј of the FLAG epitope and HindIII in the pcDNA3 polylinker to yield P2X 2 -GFP. The P2X 2 -f-GFP fragment was inserted into pSinRep5 between the StuI and ApaI sites, and infective Sindbis particles were generated with the use of the Sindbis Expression System (http:͞͞www.invitrogen.com͞). Site-directed mutagenesis was performed on the cDNAs with the use of synthetic oligonucleotides to generate K69A and T18A mutants (Quick Change; Stratagene).Electrophysiology and Imaging. All cell preparations, twoelectrode voltage-clamp record...

P2X7 Receptor Cell Surface Expression and Cytolytic Pore Formation Are Regulated by a Distal C-terminal Region

“…We recently examined whether the P2X7R formed clusters (9) upon agonist activation, as a prelude to density-dependent pore formation. We found that, unlike the P2X2R, which clusters upon agonist activation (1-2 m in size) (10), the P2X7R does not exist in clusters in the basal state or upon receptor activation (9). Although receptor density regulates pore formation in cells that endogenously express the P2X7R (6), a localized increase in receptor density or clusters is not associated with P2X7 pore formation.…”

Regulation of Human Recombinant P2X3Receptors by Ecto-Protein Kinase C

“…The endo-protein kinases may either phosphorylate an associated protein (Adinolfi et al, 2003;Vial et al, 2004) or the receptor channel itself (Paukert et al, 2001) and thereby regulate the ionic conductance. Phosphorylation of a large family of interacting proteins, which control P2X receptor turnover, may promote the trafficking of these receptors to the membrane and thereby cause sensitization under injurious conditions (Xu and Huang, 2004) or an increase of synaptic strength (Khakh et al, 2001;Bobanovic et al, 2002).…”

On the Role of the First Transmembrane Domain in Cation Permeability and Flux of the ATP-gated P2X2 Receptor

“…The remaining three mutants showed modest changes in calcium dynamics. Two of these occurred at sites (Gly 30 and Phe 44 ) that are unlikely to interact with permeating cations in a meaningful way. The third was a conserved tyrosine (Tyr 43 ) that may form an inter-pore binding site for calcium.…”