Annette Nicke

P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels.

P2X receptors are cation channels gated by extracellular ATP. The seven known P2X isoforms possess no sequence homology with other proteins. Here we studied the quaternary structure of P2X receptors by chemical cross‐linking and blue native PAGE. P2X1 and P2X3 were N‐terminally tagged with six histidine residues to allow for non‐denaturing receptor isolation from cRNA‐injected, [35S]methionine‐labeled oocytes. The His‐tag did not change the electrophysiological properties of the P2X1 receptor. His‐P2X1 was found to carry four N‐glycans per polypeptide chain, only one of which acquired Endo H resistance en route to the plasma membrane. 3,3′‐Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and two of three bifunctional analogues of the P2X receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS) cross‐linked digitonin‐solubilized His‐P2X1 and His‐P2X3 quantitatively to homo‐trimers. Likewise, when analyzed by blue native PAGE, P2X receptors purified in digitonin or dodecyl‐β‐D‐maltoside migrated entirely as non‐covalently linked homo‐trimers, whereas the α2βγδ nicotinic acetylcholine receptor (used as a positive control) migrated as the expected pentamer. P2X monomers remained undetected soon after synthesis, indicating that trimerization occurred in the endoplasmic reticulum. The plasma membrane form of His‐P2X1 was also identified as a homo‐trimer. If n‐octylglucoside was used for P2X receptor solubilization, homo‐hexamers were observed, suggesting that trimers can aggregate to form larger complexes. We conclude that trimers represent an essential element of P2X receptor structure.

The β Subunit Determines the Ligand Binding Properties of Synaptic Glycine Receptors

Inhibitory glycine receptors (GlyRs) regulate motor coordination and sensory signal processing in spinal cord and other brain regions. GlyRs are pentameric proteins composed of membrane-spanning alpha and beta subunits. Here, site-directed mutagenesis combined with homology modeling based on the crystal structure of the acetylcholine binding protein identified key ligand binding residues of recombinant homooligomeric alpha1 and heterooligomeric alpha1beta GlyRs. This disclosed two highly conserved, oppositely charged residues located on adjacent subunit interfaces as being crucial for agonist binding. In addition, the beta subunit was found to determine the ligand binding properties of heterooligomeric GlyRs. Expression of an alpha1beta tandem construct and affinity purification of metabolically labeled GlyRs confirmed a subunit stoichiometry of 2alpha3beta. Because the beta subunit anchors GlyRs at synaptic sites, our results have important implications for the biosynthesis, clustering, and pharmacology of synaptic GlyRs.

Achbp-Targeted Alpha-Conotoxin Correlates Distinct Binding Orientations with Nachr Subtype Selectivity

Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine‐binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand‐binding domain, to discover a novel α‐conotoxin (α‐TxIA) in the venom of Conus textile. α‐TxIA bound with high affinity to AChBPs from different species and selectively targeted the α3β2 nAChR subtype. A co‐crystal structure of Ac‐AChBP with the enhanced potency analog TxIA(A10L), revealed a 20° backbone tilt compared to other AChBP–conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac‐AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co‐crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype‐selective ligands with therapeutic potential in nAChR‐related diseases.

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 determinants of glycine receptor subunit assembly.

The inhibitory glycine receptor (GlyR) is a pentameric transmembrane protein composed of homologous α and β subunits. Single expression of α subunits generates functional homo‐oligomeric GlyRs, whereas the β subunit requires a co‐expressed α subunit to assemble into hetero‐oligomeric channels of invariant stoichiometry (α3β2). Here, we identified eight amino acid residues within the N‐terminal region of the α1 subunit that are required for the formation of homo‐oligomeric GlyR channels. We show that oligomerization and N‐glycosylationq of the α1 subunit are required for transit from the endoplasmic reticulum to the Golgi apparatus and later compartments, and that addition of simple carbohydrate side chains occurs prior to GlyR subunit assembly. Our data are consistent with both intersubunit surface and conformational differences determining the different assembly behaviour of GlyR α and β subunits.

Molecular and functional properties of P2X receptors—recent progress and persisting challenges

ATP-gated P2X receptors are trimeric ion channels that assemble as homo- or heteromers from seven cloned subunits. Transcripts and/or proteins of P2X subunits have been found in most, if not all, mammalian tissues and are being discovered in an increasing number of non-vertebrates. Both the first crystal structure of a P2X receptor and the generation of knockout (KO) mice for five of the seven cloned subtypes greatly advanced our understanding of their molecular and physiological function and their validation as drug targets. This review summarizes the current understanding of the structure and function of P2X receptors and gives an update on recent developments in the search for P2X subtype-selective ligands. It also provides an overview about the current knowledge of the regulation and modulation of P2X receptors on the cellular level and finally on their physiological roles as inferred from studies on KO mice.

Isolation, Structure, and Activity of GID, a Novel alpha 4/7-Conotoxin with an Extended N-terminal Sequence

Using assay-directed fractionation ofConus geographus crude venom, we isolated α-conotoxin GID, which acts selectively at neuronal nicotinic acetylcholine receptors (nAChRs). Unlike other neuronally selective α-conotoxins, α-GID has a four amino acid N-terminal tail, γ-carboxyglutamate (Gla), and hydroxyproline (O) residues, and lacks an amidated C terminus. GID inhibits α7 and α3β2 nAChRs with IC50values of 5 and 3 nm, respectively and is at least 1000-fold less potent at the α1β1γδ, α3β4, and α4β4 combinations. GID also potently inhibits the α4β2 subtype (IC50 of 150 nm). Deletion of the N-terminal sequence (GIDΔ1–4) significantly decreased activity at the α4β2 nAChR but hardly affected potency at α3β2 and α7 nAChRs, despite enhancing the off-rates at these receptors. In contrast, Arg12 contributed to α4β2 and α7 activity but not to α3β2 activity. The three-dimensional structure of GID is well defined over residues 4–19 with a similar motif to other α-conotoxins. However, despite its influence on activity, the tail appears to be disordered in solution. Comparison of GID with other α4/7-conotoxins which possess an NN(P/O) motif in loop II, revealed a correlation between increasing length of the aliphatic side-chain in position 10 (equivalent to 13 in GID) and greater α7 versus α3β2 selectivity.

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.

Identification of an Intersubunit Cross-Link between Substituted Cysteine Residues Located in the Putative ATP Binding Site of the P2X1 Receptor

P2X receptors are ATP-gated nonselective cation channels. Functional receptors are assembled as homotrimers or heterotrimers of seven cloned subunits. Each subunit contains two transmembrane domains linked by a large extracellular loop that is required for agonist binding. So far, there is no direct evidence indicating whether the agonist binding site is formed within one subunit or at the interface of two neighboring subunits. Here we used a disulfide cross-linking approach to identify pairs of residues that are in close proximity within the ATP binding site of the P2X1 homotrimer. Eight amino acid residues that have previously been shown to be essential for high ATP potency (K68, K70, F185, K190, F291, R292, R305, and K309) were substituted by cysteine residues, and the respective mutant subunits were pairwise expressed in Xenopus laevis oocytes. Nonreducing SDS-PAGE analysis of the purified receptors revealed a spontaneous and specific dimer formation between the K68C and F291C mutants. An almost complete cross-link into trimers was achieved with the K68C/F291C double mutant, consistent with the formation of intersubunit disulfide bridges. In support of this interpretation, two-electrode voltage-clamp analysis of the K68C/F291C mutations introduced into a nondesensitizing P2X2–1 chimera showed only small ATP-activated currents that, however, increased ∼60-fold after extracellular application of the reducing agent dithiothreitol. In addition, we show that a K68C/K309C double mutant is nonfunctional and can be functionally rescued by coexpression with nonmutated subunits. Our data are consistent with loops from neighboring P2X subunits forming the ATP-binding site in P2X receptors.

Homotrimeric complexes are the dominant assembly state of native P2X7 subunits

P2X receptors (P2XRs) are trimeric ATP-gated cation channels. Seven subunits have been cloned. P2X4 and P2X7 subunits show overlapping expression and both subunits are involved in pathophysiological processes such as inflammatory and neuropathic pain. A recent study provides evidence for heteromeric P2X4/7Rs. In this study, subtype-specific antibodies in combination with BN-PAGE are used to directly visualize P2XR complexes solubilized from membrane extracts of native tissues. The results show specific P2X7R and P2X4R staining in many tissues. The P2X7 complex has a clearly different size than the P2X4 complex but is likewise composed of three subunits. No complexes corresponding to more than three subunits could be detected. Also, no complexes of intermediate size or reactive to both antibodies were detected. These data suggest that either heteromerization between P2X4 and P2X7 subunits results not in stable heteromeric complexes or P2X4/7 heteromers do not represent a dominant subtype in the tissues investigated.