Functional and Biochemical Evidence for Heteromeric ATP-gated Channels Composed of P2X1 and P2X5Subunits

Abstract

The mammalian P2X receptor gene family encodes two-transmembrane domain nonselective cation channels gated by extracellular ATP. Anatomical localization data obtained by in situ hybridization and immunocytochemistry have shown that neuronal P2X subunits are expressed in specific but overlapping distribution patterns. Therefore, the native ionotropic ATP receptors diversity most likely arises from interactions between different P2X subunits that generate hetero-multimers phenotypically distinct from homomeric channels. Rat P2X 1 and P2X 5 mRNAs are localized within common subsets of peripheral and central sensory neurons as well as spinal motoneurons. The present study demonstrates a functional association between P2X 1 and P2X 5 subunits giving rise to hybrid ATP-gated channels endowed with the pharmacology of P2X 1 and the kinetics of P2X 5 . When expressed in Xenopus oocytes, hetero-oligomeric P2X 1؉5 ATP receptors were characterized by slowly desensitizing currents highly sensitive to the agonist ␣,␤-methylene ATP (EC 50 ‫؍‬ 1.1 M) and to the antagonist trinitrophenyl ATP (IC 50 ‫؍‬ 64 nM), observed with neither P2X 1 nor P2X 5 alone. Direct physical evidence for P2X 1؉5 co-assembly was provided by reciprocal subunitspecific co-purifications between epitope-tagged P2X 1 and P2X 5 subunits transfected in HEK-293A cells.Ionotropic ATP receptors constitute a unique class of neurotransmitter-gated ion channels generated from the assembly of P2X subunits having two transmembrane-spanning domains and a protein architecture similar to the one of the amiloridesensitive sodium channels (1, 2). Functional characterization studies of the seven mammalian cloned P2X subunits heterologously expressed as homomeric channels allowed to classify them in three groups according to their properties of desensitization and to their sensitivity to the agonist ␣,␤-methylene ATP (␣␤m-ATP) 1 : (i) rapidly desensitizing and ␣␤m-ATP-sensitive receptors including P2X 1 and P2X 3 (3-5), (ii) moderately desensitizing and ␣␤m-ATP-insensitive receptors including P2X 4 and P2X 6 (6 -12), and (iii) nondesensitizing as well as ␣␤m-ATP-insensitive receptors including P2X 2 , P2X 5 , and P2X 7 (11)(12)(13)(14). Results from Northern blots and in situ hybridization data (11) have indicated that the six neuronal P2X subunits genes are transcribed in specific but overlapping populations in the central and peripheral nervous system (1, 11). This strongly suggests that neuronal P2X subunits belonging to different functional groups might co-assemble into heteromultimeric channels.All P2X subunits have been detected in peripheral sensory ganglia, reinforcing the view that synaptically or lytically released ATP could play an important signaling role in sensory pathways (1,11,15). Rat P2X 3 subunits have been reported to be exclusively expressed in small to medium-sized isolectin B4-positive nociceptive neurons in nodose, trigeminal, and dorsal root ganglia (4, 5, 15). A significant proportion of sensory neurons are thought to express hetero-oligomeric P2X...

Ectodomain Lysines and Suramin Block of P2X1 Receptors

“…2). The pEC 50 was 6.5 Ϯ 0.12 (n ϭ 66), which is similar to values reported previously in HEK 293 cells for human (12) and rat receptors (21,22) (Fig. 2 and Table 1).…”

P2X2 and P2X5 Subunits Define a New Heteromeric Receptor with P2X7-Like Properties

“…This is in stark contrast to rodent homomeric P2X5 receptors, which are poorly functional (Collo et al, 1996;. Our functional data, together with previous studies on P2X1/5 receptors (Haines et al, 1999;Lê et al, 1999;Surprenant et al, 2000), support the idea that rodent P2X5 subunits are auxiliary subunits, associating with P2X1, P2X2, and P2X4 subunits to generate molecular diversity among P2X receptors. Rat P2X2 and P2X5 receptors, however, have similar pharmacology .…”

“…Indeed, this approach has accurately identified subunits known to interact with P2X2 or P2X5, such as P2X3 and P2X1, respectively (Lewis et al, 1995;Haines et al, 1999;Lê et al, 1999;Surprenant et al, 2000), and revealed P2X2 subunits as a new assembly partner of P2X5. In native tissue, P2X2 and P2X5 subunits were coimmunoprecipitated from brain and brainstem, and immunostaining colocalized the two proteins within specific subsets of neurons known to express P2X5 subunits (Collo et al, 1996).…”

“…The P2X2/3 receptor was initially identified in sensory neurons through its unique pharmacological and biophysical profiles, which were recapitulated in a recombinant system (Lewis et al, 1995;Radford et al, 1997). Similarly, in submucosal arterioles and cortical astrocytes, ATPinduced currents display the same biophysical and pharmacological profiles that are observed in HEK cells cotransfected with P2X1 and P2X5 subunits (Haines et al, 1999;Lê et al, 1999;Surprenant et al, 2000;Lalo et al, 2008).…”

Cross-talk and Co-trafficking between ρ1/GABA Receptors and ATP-gated Channels

“…Carboxyl-terminal epitope-tagged P2X 2 subunits with either hexahistidine (His 6 )-tagged or enhanced YFP-tagged sequences and a C-terminal His 6 -tagged 1 subunit ( 1-HIS) were obtained as described previously (28). N-terminal GFP-tagged 1 subunit ( 1-GFP) was generated by insertion of two enhanced GFPs in tandem into a unique BamHI restriction site.…”