Florentina Soto

A functional P2X7 splice variant with an alternative transmembrane domain 1 escapes gene inactivation in P2X7 knock-out mice

The ATP-activated P2X7 receptor channel is involved in immune function and inflammatory pain and represents an important drug target. Here we describe a new P2X7 splice variant (P2X7(k)), containing an alternative intracellular N terminus and first transmembrane domain encoded by a novel exon 1 in the rodent P2rx7 gene. Whole cell patch clamp recordings of the rat isoform expressed in HEK293 cells revealed an 8-fold higher sensitivity to the agonist Bz-ATP and much slower deactivation kinetics when compared with the P2X7(a) receptor. Permeability measurements in Xenopus oocytes show a high permeability for N-methyl-d-glucamine immediately upon activation, suggesting that the P2X7(k) channel is constitutively dilated upon opening. The rates of agonist-induced dye uptake and membrane blebbing in HEK cells were also increased. PCR analyses and biochemical analysis by SDS-PAGE and BN-PAGE indicate that the P2X7(k) variant escapes gene deletion in one of the available P2X7−/− mice strains and is strongly expressed in the spleen. Taken together, we describe a novel P2X7 isoform with distinct functional properties that contributes to the diversity of P2X7 receptor signaling. Its presence in one of the P2X7−/− strains has important implications for our understanding of the role of this receptor in health and disease.

Molecular cloning and functional expression of a novel rat heart P2X purinoceptor.

Here we describe a novel purinergic receptor, the P2X5 receptor, cloned from rat heart. The full‐length cDNA encodes a protein 455 amino acids long which shares an overall identity of 40–47% with other members of the P2X purinergic receptor family. P2X5 mRNA transcripts are found predominantly in rat heart but are also present in brain, spinal cord and adrenal gland. Functional expression of the recombinant receptor in HEK‐293 cells shows a current that resembles mostly the P2X2 phenotype: the ATP‐activated current reveals little agonist desensitization, is not activated by α,β‐meATP and is completely blocked by suramin and PPADS.

Molecular characterization and pharmacological properties of the human P2X3 purinoceptor

Using PCR and library screening techniques, a cDNA encoding an ATP ligand-gated channel has been isolated from human heart. The full-length cDNA encodes a protein 397 amino acids long which shows a high amino-acid sequence identity with the rat P2X3 purinoceptor (93%). By fluorescence in situ hybridization, the human P2X3 gene has been mapped to region q12 of chromosome 11. Tissue distribution analysis of human P2X3 receptor mRNA shows a restricted expression pattern, i.e. transcripts are limited to the spinal cord and heart. This result contrasts with the distribution of the rat P2X3 receptor which was detected exclusively in sensory neurons of trigeminal, dorsal root and nodose ganglia. Heterologous expression of human P2X3 cRNA in Xenopus oocytes generates a fast desensitizing ATP-activated channel with pharmacological properties resembling the profile of the rat homologue receptor. Thus, the order of agonist potency is 2MeSATP > ATP > alphabeta-meATP > CTP > betagamma-meATP approximately ADP. Moreover, ATP-evoked currents on human P2X3 receptor are efficiently blocked in a reversible manner by the purinoceptor antagonists, suramin and PPADS.

Biochemical and functional evidence for heteromeric assembly of P2X1 and P2X4 subunits.

P2X receptors are ligand‐gated ion channels activated by extracellular ATP. In expression systems, P2X subunits form homo‐ and heterotrimeric receptors. Heteromerization is also likely to occur in vivo as (i) most P2X subunits show overlapping distribution in different tissues and (ii) the functional properties of many native P2X receptors differ from those of heterologously expressed homomeric receptors. Here, we used the Xenopus laevis oocyte expression system to test for heteromerization of P2X1 and P2X4 subunits. Upon co‐injection, P2X4 subunits were co‐purified with hexahistidyl‐tagged P2X1 subunits indicating heteromerization. Blue native polyacrylamide gel electrophoresis (BN‐PAGE) analysis of these P2X complexes excluded artificial aggregation and confirmed that both subunits were present in trimeric complexes of the same size. Two‐electrode voltage‐clamp experiments revealed functional P2X receptors with kinetic properties resembling homomeric P2X4 receptors and a pharmacological profile similar to homomeric P2X1 receptors. Thus, application of α,β‐methylene ATP evoked a slowly desensitizing current sensitive to the antagonists suramin and 2′,3′‐O‐(2,4,6‐trinitrophenyl)‐ATP. This study provides for the first time biochemical and functional evidence for the formation of heteromeric P2X1+4 receptors. These receptors may account for native P2X mediated responses that until now could not be correlated with previously described recombinant P2X receptors.

Cloned Ligand-gated Channels Activated by Extracellular ATP (P2X Receptors)

Adenosine 58 triphosphate (ATP) is copackaged in exocytotic granules and secreted with a number of neurotransmitters and local mediators from many cell types (Whittaker, 1982). Indeed, the exocytosis of ATP evokes fast synaptic potentials in the central and peripheral nervous system (Edwards, Gibb & Colquhoun, 1992; Evans, Derkach & Surprenant, 1992; Silinsky, Gerzanich & Vauner, 1992). ATP can also be released from the cell cytosol via nonsynaptic mechanisms, for example by diffusion after sudden rupture of intact cells by tissue injury. Moreover, cytosolic ATP can be translocated to the extracellular medium by active transporters under hypoxic conditions (Clemens & Forrester, 1981; Forrester & Williams, 1977). Extracellular ATP exerts its diverse effects by binding to membrane proteins termed P2 receptors (Dubyak & El-Moatassim, 1993). P2 receptors have been classified in two families according to amino acid sequence homology and transduction mechanisms: (i) a P2X family consisting of ligand-gated channels, of which seven subunits have been cloned (P2X1–7) (Buell, Collo & Rassendren, 1996 b) and, (ii) a P2Y family consisting of G-protein coupled receptors with eight reported members (P2Y 1–8) (Burnstock, 1996; Burnstock & King, 1996). Studies on native P2 receptors defined an additional class represented by the P2Z receptor. Its activation leads to the opening of large-conductance nonselective pores which results in cell lysis (Tatham & Landau, 1990). However, heterologously expressed P2X 7 receptor presents functional properties that strongly resembles the behavior of native P2Z receptor (Surprenant et al., 1996 a). The characterization of cloned P2X receptors constitutes a basic framework for a precise description of their physiological function. The aim of this review is to summarize recent advances in the cloning, functional characterization and tissue distribution of the P2X receptor subunits.

Genomic structure, developmental distribution and functional properties of the chicken P2X5 receptor

We report here the cloning of a chicken cDNA (402 aa) showing high sequence similarity to the previously cloned rat and human P2X5 receptors (67 and 69%, respectively). The chicken P2X5 subunit is encoded by a gene composed of 12 translated exons, which shows conserved genomic structure with mammalian P2X genes. In HEK‐293 cells heterologously expressing chicken P2X5 receptors, ATP activates a current that desensitizes in a way that is dependent on the presence of extracellular divalent cations. ATP and 2‐methylthio ATP are equipotent agonists (EC50 ∼ 2 µm) and suramin and pyridoxal 5‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid are potent antagonists. Additionally, reversal potential measurements indicate that chicken P2X5 is permeable not only to cations but also to chloride (PCs+/PCl‐ ∼ 1.9), as has been described for native P2X receptor mediated responses in embryonic chicken skeletal muscle. mRNA distribution of chicken P2X5 was determined by in situ hybridization analysis in both whole embryos and on tissue slices of heart and skeletal muscle. Our results suggest that chicken P2X5 receptors are expressed in developing muscle and might play a role in early muscle differentiation.

Spontaneous activity promotes synapse formation in a cell-type-dependent manner in the developing retina.

Spontaneous activity is thought to regulate synaptogenesis in many parts of the developing nervous system. In vivo evidence for this regulation, however, is scarce and comes almost exclusively from experiments in which normal activity was reduced or blocked completely. Thus, whether spontaneous activity itself promotes synaptogenesis or plays a purely permissive role remains uncertain. In addition, how activity influences synapse dynamics to shape connectivity and whether its effects among neurons are uniform or cell-type-dependent is unclear. In mice lacking the cone–rod homeobox gene (Crx), photoreceptors fail to establish normal connections with bipolar cells (BCs). Here, we find that retinal ganglion cells (RGCs) in Crx−/− mice become rhythmically hyperactive around the time of eye opening as a result of increased spontaneous glutamate release from BCs. This elevated neurotransmission enhances synaptogenesis between BCs and RGCs, without altering the overall circuit architecture. Using live imaging, we discover that spontaneous activity selectively regulates the rate of synapse formation, not elimination, in this circuit. Reconstructions of the connectivity patterns of three BC types with a shared RGC target further revealed that neurotransmission specifically promotes the formation of multisynaptic appositions from one BC type without affecting the maintenance or elimination of connections from the other two. Although hyperactivity in Crx−/− mice persists, synapse numbers do not increase beyond 4 weeks of age, suggesting closure of a critical period for synaptic refinement in the inner retina. Interestingly, despite their hyperactivity, RGC axons maintain normal eye-specific territories and cell-type-specific layers in the dorsal lateral geniculate nucleus.

Fluorescence measurements reveal stoichiometry of K+ channels formed by modulatory and delayed rectifier alpha-subunits.

Modulatory α-subunits, which comprise one-fourth of all voltagegated K+ channel (Kv) α-subunits, do not assemble into homomeric channels, but selectively associate with delayed rectifier Kv2 subunits to form heteromeric channels of unknown stoichiometry. Their distinct expression patterns and unique functional properties have made these channels candidate molecular correlates for a broad set of native K+ currents. Here, we combine FRET and electrophysiological measurements to determine the stoichiometry and geometry of heteromeric channels composed of the delayed rectifier Kv2.1 subunit and the modulatory Kv9.3 α-subunit. Kv channel α-subunits were fused with GFP variants, and heteromerization of different combinations of tagged and untagged α-subunits was studied. FRET, evaluated by acceptor photobleaching, was only observed upon formation of functional channels. Our results, obtained from two independent experimental paradigms, suggest the formation of heteromeric Kv2.1/Kv9.3 channels of fixed stoichiometry consisting of three Kv2.1 subunits and one Kv9.3 subunit. Strikingly, despite this uneven stoichiometry, we find that heteromeric Kv2.1/Kv9.3 channels maintain a pseudosymmetric arrangement of subunits around the central pore.

NGL-2 Regulates Pathway-Specific Neurite Growth and Lamination, Synapse Formation, and Signal Transmission in the Retina

Parallel processing is an organizing principle of many neural circuits. In the retina, parallel neuronal pathways process signals from rod and cone photoreceptors and support vision over a wide range of light levels. Toward this end, rods and cones form triad synapses with dendrites of distinct bipolar cell types, and the axons or dendrites, respectively, of horizontal cells (HCs). The molecular cues that promote the formation of specific neuronal pathways remain largely unknown. Here, we discover that developing and mature HCs express the leucine-rich repeat (LRR)-containing protein netrin-G ligand 2 (NGL-2). NGL-2 localizes selectively to the tips of HC axons, which form reciprocal connections with rods. In mice with null mutations in Ngl-2 (Ngl-2−/−), many branches of HC axons fail to stratify in the outer plexiform layer (OPL) and invade the outer nuclear layer. In addition, HC axons expand lateral territories and increase coverage of the OPL, but establish fewer synapses with rods. NGL-2 can form transsynaptic adhesion complexes with netrin-G2, which we show to be expressed by photoreceptors. In Ngl-2−/− mice, we find specific defects in the assembly of presynaptic ribbons in rods, indicating that reverse signaling of complexes involving NGL-2 regulates presynaptic maturation. The development of HC dendrites and triad synapses of cone photoreceptors proceeds normally in the absence of NGL-2 and in vivo electrophysiology reveals selective defects in rod-mediated signal transmission in Ngl-2−/− mice. Thus, our results identify NGL-2 as a central component of pathway-specific development in the outer retina.

Fe65 Interacts with P2X2 Subunits at Excitatory Synapses and Modulates Receptor Function

Ionotropic receptors in the neuronal plasma membrane are organized in macromolecular complexes, which assure their proper localization and regulate signal transduction. P2X receptors, the ionotropic receptors activated by extracellular ATP, have been shown to influence synaptic transmission. Using a yeast two-hybrid approach with the P2X2 subunit C-terminal domain as bait we isolated the β-amyloid precursor protein-binding proteins Fe65 and Fe65-like 1 as the first identified proteins interacting with neuronal P2X receptors. We confirmed the direct interaction of Fe65 and the P2X2 C-terminal domain by glutathione S-transferase pull-down experiments. No interaction was observed between Fe65 and the naturally occurring P2X2 splice variant P2X2(b), indicating that alternative splicing can regulate the receptor complex assembly. We generated two antibodies to Fe65 to determine its subcellular localization using postembedding immunogold labeling electron microscopy. We found labeling for Fe65 at the pre- and postsynaptic specialization of CA1 hippocampal pyramidal cell/Schaffer collateral synapses. By double immunogold labeling, we determined that Fe65 colocalizes with P2X2 subunits at the postsynaptic specialization of excitatory synapses. Moreover, P2X2 and Fe65 could be coimmunoprecipitated from brain membrane extracts, demonstrating that the interaction occurs in vivo. The assembly with Fe65 regulates the functional properties of P2X2 receptors. Thus, the time- and activation-dependent change in ionic selectivity of P2X2 receptors was inhibited by coexpression of Fe65, suggesting a novel role for Fe65 in regulating P2X receptor function and ATP-mediated synaptic transmission.