Günther Schmalzing

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.

Molecular characterization of human and bovine endothelin converting enzyme (ECE‐1)

A membrane‐bound protease activity that specifically converts Big endothelin‐1 has been purified from bovine endothelial cells (FBHE) The enzyme was cleaved with trypsin and the peptide sequencing analysis confirmed it to be a zinc chelating metalloprotease containing the typical HEXXH (HELTH) motif. RT‐PCR and cDNA screens were employed to isolate the complete cDNAs of the bovine and human enzymes. This human metalloprotease was expressed heterologously in cell culture oocytes. The catalytic activity of the recombinant enzyme is the same as that determined for the natural enzyme. The data suggest that the characterized enzyme represents the functional human endothelin converting enzyme ECE‐1.

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.

Trophic activity of a naturally occurring truncated isoform of the P2X7 receptor

P2X7 is the largest member of the P2X subfamily of purinergic receptors. A typical feature is the carboxyl tail, which allows formation of a large pore. Recently a naturally occurring truncated P2X7 splice variant, isoform B (P2X7B), has been identified. Here we show that P2X7B expression in HEK293 cells, a cell type lacking endogenous P2X receptors, mediated ATP‐stimulated channel activity but not plasma membrane permeabilization, raised endoplasmic reticulum Ca2+ content, activated the transcription factor NFATc1, increased the cellular ATP content, and stimulated growth. In addition, P2X7B‐transfected HEK293 cells (HEK293‐P2X7B), like most tumor cells, showed strong soft agarinfiltrating ability. When coexpressed with full‐length P2X7 (P2X7A), P2X7B coassembled with P2X7A into a heterotrimer and potentiated all known responses mediated by this latter receptor. P2X7B mRNA was found to be widely distributed in human tissues, especially in the immune and nervous systems, and to a much higher level than P2X7A. Finally, P2X7B expression was increased on mitogenic stimulation of peripheral blood lymphocyte. Altogether, these data show that P2X7B is widely expressed in several human tissues, modulates P2X7A functions, participates in the control of cell growth, and may help understand the role of the P2X7 receptor in the control of normal and cancer cell proliferation.—Adinolfi, E., Cirillo, M., Woltersdorf, R., Falzoni, S., Chiozzi, P., Pellegatti, P., Callegari, M. G., Sandonà, D., Markwardt, F., Schmalzing, G., Di Virgilio, F. Trophic activity of a naturally occurring truncated isoform of the P2X7 receptor. FASEB J. 24, 3393–3404 (2010). www.fasebj.org

Ubiquitination Precedes Internalization and Proteolytic Cleavage of Plasma Membrane-bound Glycine Receptors

The inhibitory glycine receptor (GlyR) in developing spinal neurones is internalized efficiently upon antagonist inhibition. Here we used surface labeling combined with affinity purification to show that homopentameric α1 GlyRs generated inXenopus oocytes are proteolytically nicked into fragments of 35 and 13 kDa upon prolonged incubation. Nicked GlyRs do not exist at the cell surface, indicating that proteolysis occurs exclusively in the endocytotic pathway. Consistent with this interpretation, elevation of the lysosomal pH, but not the proteasome inhibitor lactacystin, prevents GlyR cleavage. Prior to internalization, α1 GlyRs are conjugated extensively with ubiquitin in the plasma membrane. Our results are consistent with ubiquitination regulating the endocytosis and subsequent proteolysis of GlyRs residing in the plasma membrane. Ubiquitin-conjugating enzymes thus may have a crucial role in synaptic plasticity by determining postsynaptic receptor numbers.

Activation and Desensitization of the Recombinant P2X1 Receptor at Nanomolar ATP Concentrations

Activation and desensitization kinetics of the rat P2X1 receptor at nanomolar ATP concentrations were studied in Xenopus oocytes using two-electrode voltage-clamp recording. The solution exchange system used allowed complete and reproducible solution exchange in <0.5 s. Sustained exposure to 1–100 nM ATP led to a profound desensitization of P2X1 receptors. At steady-state, desensitization could be described by the Hill equation with a K1/2 value of 3.2 ± 0.1 nM. Also, the ATP dependence of peak currents could be described by a Hill equation with an EC50 value of 0.7 μM. Accordingly, ATP dose-effect relationships of activation and desensitization practically do not overlap. Recovery from desensitization could be described by a monoexponential function with the time-constant τ = 11.6 ±1.0 min. Current transients at 10–100 nM ATP, which elicited 0.1–8.5% of the maximum response, were compatible with a linear three-state model, C-O-D (closed-open-desensitized), with an ATP concentration-dependent activation rate and an ATP concentration-independent (constant) desensitization rate. In the range of 18–300 nM ATP, the total areas under the elicited current transients were equal, suggesting that P2X1 receptor desensitization occurs exclusively via the open conformation. Hence, our results are compatible with a model, according to which P2X1 receptor activation and desensitization follow the same reaction pathway, i.e., without significant C to D transition. We assume that the K1/2 of 3.2 nM for receptor desensitization reflects the nanomolar ATP affinity of the receptor found by others in agonist binding experiments. The high EC50 value of 0.7 μM for receptor activation is a consequence of fast desensitization combined with nonsteady-state conditions during recording of peak currents, which are the basis of the dose-response curve. Our results imply that nanomolar extracellular ATP concentrations can obscure P2X1 receptor responses by driving a significant fraction of the receptor pool into a long-lasting refractory closed state.

TMEM16A(a)/anoctamin-1 shares a homodimeric architecture with CLC chloride channels.

TMEM16A/anoctamin-1 has been identified as a protein with the classic properties of a Ca2+-activated chloride channel. Here, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking to assess the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac) splice variants as well as a genetically concatenated TMEM16A(a) homodimer. The constructs carried hexahistidyl (His) tags to allow for their purification using a nondenaturing metal affinity resin. Neither His-tagging nor head-to-tail concatenation of two copies of TMEM16A(a) noticeably affected Ca2+-induced measured macroscopic Cl− currents compared with the wild-type TMEM16A(a) channel. The digitonin-solubilized, nondenatured TMEM16A(a) protein migrated in the BN-PAGE gel as a homodimer, as judged by comparison with the concatenated TMEM16A(a) homodimer and channel proteins of known oligomeric structures (e.g. the voltage-gated Cl− channel CLC-1). Cross-linking with glutaraldehyde corroborated the homodimeric structure of TMEM16A(a). The TMEM16A(a) homodimer detected in Xenopus laevis oocytes and HEK 293 cells dissociated into monomers following denaturation with SDS, and reducing versus nonreducing SDS-PAGE provided no evidence for the presence of intersubunit disulfide bonds. Together, our data demonstrate that the Ca2+-activated chloride channel member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel.

Conserved Dimeric Subunit Stoichiometry of SLC26 Multifunctional Anion Exchangers

The SLC26 gene family encodes multifunctional transport proteins in numerous tissues and organs. Some paralogs function as anion exchangers, others as anion channels, and one, prestin (SLC26A5), represents a membrane-bound motor protein in outer hair cells of the inner ear. At present, little is known about the molecular basis of this functional diversity. We studied the subunit stoichiometry of one bacterial, one teleost, and two mammalian SLC26 isoforms expressed in Xenopus laevis oocytes or in mammalian cells using blue native PAGE and chemical cross-linking. All tested SLC26s are assembled as dimers composed of two identical subunits. Co-expression of two mutant prestins with distinct voltage-dependent capacitances results in motor proteins with novel electrical properties, indicating that the two subunits do not function independently. Our results indicate that an evolutionarily conserved dimeric quaternary structure represents the native and functional state of SLC26 transporters.

An intramembrane aromatic network determines pentameric assembly of Cys-loop receptors

Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that mediate fast synaptic transmission. Here functional pentameric assembly of truncated fragments comprising the ligand-binding N-terminal ectodomains and the first three transmembrane helices, M1–M3, of both the inhibitory glycine receptor (GlyR) α1 and the 5HT3A receptor subunits was found to be rescued by coexpressing the complementary fourth transmembrane helix, M4. Alanine scanning identified multiple aromatic residues in M1, M3 and M4 as key determinants of GlyR assembly. Homology modeling and molecular dynamics simulations revealed that these residues define an interhelical aromatic network, which we propose determines the geometry of M1–M4 tetrahelical packing such that nascent pLGIC subunits must adopt a closed fivefold symmetry. Because pLGIC ectodomains form random nonstoichiometric oligomers, proper pentameric assembly apparently depends on intersubunit interactions between extracellular domains and intrasubunit interactions between transmembrane segments.

Roles of Individual N-Glycans for ATP Potency and Expression of the Rat P2X1 Receptor

P2X1 receptor subunits assemble in the ER of Xenopus oocytes to homotrimers that appear as ATP-gated cation channels at the cell surface. Here we address the extent to which N-glycosylation contributes to assembly, surface appearance, and ligand recognition of P2X1receptors. SDS-polyacrylamide gel electrophoresis (PAGE) analysis of glycan minus mutants carrying Gln instead of Asn at five individual NXT/S sequons reveals that Asn284 remains unused because of a proline in the +4 position. The four other sites (Asn153, Asn184, Asn210, and Asn300) carryN-glycans, but solely Asn300 located only eight residues upstream of the predicted reentry loop of P2X1acquires complex-type carbohydrates. Like parent P2X1, glycan minus mutants migrate as homotrimers when resolved by blue native PAGE. Recording of ATP-gated currents reveals that elimination of Asn153 or Asn210 diminishes or increases functional expression levels, respectively. In addition, elimination of Asn210 causes a 3-fold reduction of the potency for ATP. If three or all four N-glycosylation sites are simultaneously eliminated, formation of P2X1 receptors is severely impaired or abolished, respectively. We conclude that at least oneN-glycan per subunit of either position is absolutely required for the formation of P2X1 receptors and that individual N-glycans possess marked positional effects on expression levels (Asn154, Asn210) and ATP potency (Asn210).