P2X receptors for adenosine tri-phosphate (ATP) are a distinct family of ligand-gated cation channels with two transmembrane domains, intracellular amino and carboxy termini and a large extracellular ligand binding loop. Seven genes (P2X(1-7)) have been cloned and the channels form as either homo or heterotrimeric channels giving rise to a wide range of phenotypes. This review aims to give an account of recent work on the molecular basis of the properties of P2X receptors. In particular, to consider emerging information on the assembly of P2X receptor subunits, channel regulation and desensitisation, targeting, the molecular basis of drug action and the functional contribution of P2X receptors to physiological processes.
The cloning and characterization of a P2X receptor (schP2X) from the parasitic blood fluke Schistosoma mansoni provides the first example of a non-vertebrate ATP-gated ion channel. A number of functionally important amino acid residues conserved throughout vertebrate P2X receptors, including 10 extracellular cysteines, aromatic and positively charged residues involved in ATP recognition, and a consensus protein kinase C site in the amino-terminal tail, are also present in schP2X. Overall, the amino acid sequence identity of schP2X with human P2X 1-7 receptors ranges from 25.8 to 36.6%. ATP evoked concentration-dependent currents at schP2X channels expressed in Xenopus oocytes with an EC 50 of 22.1 M. 2,3-O-(4-Benzoylbenzoyl)adenosine 5-triphosphate (Bz-ATP) was a partial agonist (maximum response 75.4 ؎ 4.4% that of ATP) with a higher potency (EC 50 of 3.6 M) than ATP. Suramin and pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid blocked schP2X responses to 100 M ATP with IC 50 values of 9.6 and 0.5 M, respectively. Ivermectin (10 M) potentiated currents to both ATP and Bz-ATP by ϳ60% with a minimal effect on potency (EC 50 of 18.2 and 1.6 M, respectively). The relative permeability of schP2X expressed in HEK293 cells to various cations was determined under bi-ionic conditions. schP2X has a relatively high calcium permeability (P Ca /P Na ؍ 3.80 ؎ 0.29) and an estimated minimum pore diameter similar to that of vertebrate P2X receptors. SchP2X provides a useful comparative model for the better understanding of human P2X receptor function and may also provide an alternative drug target for treatment of schistosomiasis.P2X receptors comprise a family of cation-selective ion channels gated by extracellular ATP. Mammalian species possess seven distinct P2X channel subtypes (P2X 1-7 ), each encoded by a separate gene. These subunits assemble as functional homotrimeric or heterotrimeric channels and play an important role in a wide array of physiological processes including neurotransmission, smooth muscle contraction, immune cell function, and platelet aggregation (for a recent review see Ref. 1).This remarkably diverse range of physiological roles for P2X receptors has contributed to the notion that ATP is a "primitive" extracellular signaling molecule (2, 3). Functional evidence suggests that ATP-gated ion channels exist in some lower organisms including Tetrahymena thermophilia (4), leech (5), and Amoeba proteus (6), supporting the view that the development of P2X receptors for ATP occurred relatively early in the evolution of eukaryotic organisms. However, to date, definitive molecular identification of P2X channels is restricted to vertebrate species including fish (7), amphibians (8, 9), birds (10), and mammals (1).With the recent expansion in the range of species for which genomic and EST 1 sequence databases are available, it is now possible to use a bioinformatics approach to screen a wide range of lower organisms for P2X receptor-like proteins. Using such a strategy, we have identified a P2X channel from...
Researchers study how different forms of metabolic energy are coupled to drug extrusion by an ATP-binding cassette transporter.
The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell envelope. The major facilitator superfamily multidrug transporter LmrP from Lactococcus lactis catalyses drug efflux in a membrane potential and chemical proton gradient-dependent fashion. To enable the interaction with protons and cationic substrates, LmrP contains catalytic carboxyl residues on the surface of a large interior chamber that is formed by transmembrane helices. These residues colocalise together with polar and aromatic residues, and are predicted to be present in two clusters. To investigate the functional role of the catalytic carboxylates, we generated mutant proteins catalysing membrane potential-independent dye efflux by removing one of the carboxyl residues in Cluster 1. We then relocated this carboxyl residue to six positions on the surface of the interior chamber, and tested for restoration of wildtype energetics. The reinsertion at positions towards Cluster 2 reinstated the membrane potential dependence of dye efflux. Our data uncover a remarkable plasticity in proton interactions in LmrP, which is a consequence of the flexibility in the location of key residues that are responsible for proton/multidrug antiport.The ability of microbes to develop resistance to cytotoxic drugs, and to adapt rapidly to changes in the exposure to these compounds is an extremely important medical problem 1 . Drug resistance can be specific for a single drug or class of drugs, or occur simultaneously for a wide variety of toxic compounds that are structurally and functionally unrelated. The latter phenomenon is called multidrug resistance, and is recognised as an important mechanism of bacterial resistance to valuable clinical antibiotics. In many cases, the development of multidrug resistance is due to the enhanced expression levels of drug pumps in the cell 2 . These pumps mediate the extrusion of antimicrobials across the cell envelope, away from intracellular targets. Enzyme-ligand interactions are usually based on very specific interactions that allow discrimination between enantiomers of the same ligand. The ability of multidrug transporters to recognise a broad spectrum of structurally unrelated drugs is intriguing.Multidrug transporters have been reinvented on multiple occasions in the course of evolution, and are found in six different protein families: the ATP-binding cassette (ABC) superfamily, resistance-nodulation-cell division (RND) family, multiple antibiotics and toxin extrusion (MATE) family, small multidrug resistance (SMR) family, proteobacterial antimicrobial compound efflux (PACE) family, and major facilitator superfamily (MFS) 3,4 . It is generally accepted that members of the ABC family couple drug efflux to ATP binding and hydrolysis, whereas the members of the other 5 families operate by a secon...
P2X receptors are a distinct family of ATP-gated ion channels with a number of physiological roles ranging from smooth muscle contractility to the regulation of blood clotting. In this study we determined whether the UV light-reactive ATP analogues 2-azido ATP, ATP azidoanilide (ATP-AA) and 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) can be used to label the ATP binding site of P2X1 receptors. These analogues were agonists, and in patch clamp studies evoked inward currents from HEK293 cells stably expressing the P2X1 receptor. Following irradiation in the presence of these compounds subsequent responses to an EC50 concentration of ATP were reduced by >65%. These effects were partially reversed by co-application of ATP or suramin with the photo-reactive ATP analogue at the time of irradiation. In autoradiographic studies radiolabelled 2-azido [γ32P] ATP and ATP-AA-[γ32P] cross-linked to P2X1 receptors and this binding was reduced by co-incubation with ATP. These studies demonstrate that photo-reactive ATP analogues can be used to label P2X receptor and may prove useful in elucidating the ATP binding site at this novel class of ATP binding proteins.
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