Abstract
Adenosine triphosphate (ATP)-gated P2X7 receptors (P2X7Rs) are members of the purinergic receptor family that are expressed in several cell types including neurons. A high concentration of ATP is required for the channel opening of P2X7Rs compared to other members of this receptor family. Recent work suggests that ATP binding to members of the P2X receptor family determines the diffusion and localization of these receptors on the plasma membrane of neurons. Here, we employed single particle tracking photoactivated localization microscopy (sptPALM) to study the diffusion and ATP-dependence of rat P2X7Rs. Dendra2-tagged P2X7Rs were transfected in hippocampal neurons and imaged on proximal dendrites. Our results suggest the presence of two populations of P2X7Rs within the extra-synaptic membrane: a population composed of rapidly diffusing receptors and one stabilized within nanoclusters (~100 nm diameter). P2X7R trajectories were rarely observed at synaptic sites. P2X7R mutations in the ATP-binding site (K64A) or the conserved phosphorylation site (K17A) resulted in faster- and slower-diffusing receptors, respectively. Furthermore, ATP differentially accelerated wild type and K17A-mutant receptors but not K64A-mutant receptors. Our results indicate that receptor conformation plays a critical role in regulating ATP-mediated changes in P2X7R diffusion and micro-organization.
Highlights
Adenosine triphosphate (ATP)-gated purinergic P2X receptors (P2XRs) form homo- or hetero-trimeric receptors that can be composed of 7 different subunits, P2X1-P2X7
DENDRITIC EXPRESSION OF P2X7 RECEPTORS IN TRANSFECTED NEURONS Photo-convertible Dendra2-tagged P2X7 receptors (P2X7Rs) plasmid was transfected in hippocampal neurons at days in vitro (DIV) 12 and experiments performed at DIV 14
K64A-mutant showed a reduction in confinement as seen by an upward shift of the mean square displacement (MSD) curve. These results suggest that conformation of N-terminus phosphorylation site and ATP-binding site determines P2X7R diffusion behavior on the plasma membrane
Summary
ATP-gated purinergic P2X receptors (P2XRs) form homo- or hetero-trimeric receptors that can be composed of 7 different subunits, P2X1-P2X7. Slow-desensitizing P2X7Rs are unique members of the ATP-gated P2X receptor family with a characteristic long C-terminus (279 amino acids) and display unusually high EC50 for ATP (>100 μM) (Rassendren et al, 1997). The lack of specific antibodies and the existence of multiple splice variants of this receptor make it difficult to predict their expression and localization (Anderson and Nedergaard, 2006; Kaczmarek-Hájek et al, 2012). Recent development of P2X7-EGFP transgenic mice provides evidence of their expression in several brain regions and in both glial and neuronal cells (GENSAT, www.gensat.org). In the hippocampus of EGFPP2X7R expressing mice, expression can be seen primarily in the dentate gyrus and CA3 region (Cornu Ammonis) suggesting a cell-type specific expression (GENSAT, http://www.gensat.org/)
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