Jürgen Rettinger

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.

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).

BLUE NATIVE. PAGE AS A USEFUL METHOD FOR THE! ANALYSIS OF THE ASSEMBLY OF DISTINCT COMBINATIONS OF NICOTINIC ACETYLCHOLINE RECEPTOR SUBUNITS

Oligomerization of complete and incomplete combinations of rat muscle-type nicotinic acetylcholine receptor (nAChR) subunits in Xenopus oocytes was studied by blue native PAGE and compared with acetylcholine-activated current in these cells. The rank order of expression level judged by current was alpha 1 beta 1 gamma delta >> alpha 1 beta 1 gamma > alpha 1 beta 1 delta > alpha 1 gamma delta >> alpha 1 delta >> alpha 1 gamma. alpha 1 and alpha 1 beta 1 were not functional. Protein complexes incorporating a heptahistidyl-tagged alpha 1 subunit were chromatographically purified from digitonin extracts of oocytes and resolved by blue native PAGE. In the absence of any co-expressed nAChR subunit, the majority of alpha 1 formed aggregates. Co-expression of beta 1 had no effect on alpha 1 aggregation, whereas both gamma and delta diminished alpha 1 aggregation in favor of discrete oligomers: alpha 1 formed tetramers together with gamma and dimers, trimers, and tetramers together with delta. When alpha 1 gamma was complemented with beta 1 to form a functional alpha 1 beta 1 gamma receptor, a small amount of a pentamer was found besides a prominent alpha 1-His7 beta 1 gamma trimer. Expression of the functional alpha 1 beta 1 delta receptor yielded marked amounts of a pentamer besides dimers and trimers. These results are discussed in terms of the assembly model of Green and Claudio (Cell 74, 57-69, 1994), substantiating that blue native PAGE is suited for the investigation of ion channel assembly.

Chapter 6 Evolving view of quaternary structures of ligand-gated ion channels

Publisher Summary The scope of this chapter is to review the current view of the quaternary structure and subunit composition of the three major classes of ligandgated ion channels, with focus on P2X receptors. The chapter includes K + channels because of the emerging structural similarity of ion conduction pathways in K + channels and glutamate receptors and the initial assumption that P2X receptors too possess a K + channel-like H5-domain. Taken together, recent data suggest that each of the three major classes of ligand-gated ion channels is characterized by its own architecture, i.e., trimers (P2X receptors), tetramers (glutamate receptors), and pentamers (nicotinic acetylcholine receptor superfamily). This lack of a shared overall structural design is remarkable, because most of these ligand-gated ion channels exhibit a similar nonselective permeability for cations, which seems not to necessitate structural diversity of the array of pore-forming subunits. Functional diversity of K + channels, for instance, is brought about by a modular combination of the pore forming S5–H5–S6 module with other elements such as a voltage sensor and/or a regulatory binding domain.