P2X receptors for ATP are a family of ligand-gated cation channels. There are 11 conserved positive charges in the extracellular loop of P2X receptors. We have generated point mutants of these conserved residues (either Lys 3 Arg, Lys 3 Ala, Arg 3 Lys, or Arg 3 Ala) in the human P2X 1 receptor to determine their contribution to the binding of negatively charged ATP. ATP evoked concentration-dependent (EC 50 ϳ 0.8 M) desensitizing responses at wild-type (WT) P2X 1 receptors expressed in Xenopus oocytes. Suramin produced a parallel rightward shift in the concentration response curve with an estimated pK B of 6.7. Substitution of amino acids at positions Lys-53, Lys-190, Lys-215, Lys-325, Arg-202, Arg-305, and Arg-314 either had no effect or only a small change in ATP potency, time course, and/or suramin sensitivity. Modest changes in ATP potency were observed for mutants at K70R and R292K/A (20-and 100-fold decrease, respectively). Mutations at residues K68A and K309A reduced the potency of ATP by >1400-fold and prolonged the time course of the P2X 1 receptor current but had no effect on suramin antagonism. Lys-68, Lys-70, Arg-292, and Lys-309 are close to the predicted transmembrane domains of the receptor and suggest that the ATP binding pocket may form close to the channel vestibule.P2X receptors for ATP are ligand-gated cation channels present on many different cell types including neurons, blood cells, and smooth muscle (1). The P2X 1 receptor was originally cloned from the rat vas deferens, and its properties, in particular the rapid desensitization and sensitivity to ␣,␤-meATP, correspond closely to those of the native smooth muscle phenotype (2). This has been confirmed in recent studies on P2X 1 receptor-deficient mice that showed the P2X 1 receptor is essential for the expression of functional P2X receptors in smooth muscle (3).Seven P2X receptors (P2X 1-7 ) have been identified at the molecular level (4), and they constitute a novel family of ion channels with two transmembrane domains, intracellular amino and carboxyl termini and a large extracellular loop (5). The receptors form as either homo-or heteromultimers (6, 7) from the association of at least three P2X receptor subunits (8). The second transmembrane domain lines the ion conducting pore (9), and residues on the amino and carboxyl termini are involved in determining the time course of the response of P2X 2 receptors (10 -13). The extracellular loop is thought to be the site of ATP binding, and residues that affect antagonist action have been described (14,15). A Walker ATP binding motif (16) is not present in P2X receptors, and to date no residues associated with agonist binding have been identified.In many ATP-binding proteins, positively charged amino acids have been shown to be important in co-ordinating ATP binding. One of the key components of the Walker motif is the lysine residue, which is thought to interact directly with one of the phosphate groups of the ATP molecule (17) (for a review, see Ref. 18). Lysine residues are also important...

P2X receptors contain 10 conserved cysteines in the extracellular loop. To investigate whether these residues form disulfide bonds, we created a series of single and double cysteinealanine mutants in the human P2X 1 receptor. Mutants were expressed in Xenopus laevis oocytes and effects on ATP potency, cell-surface expression, and N-biotinoylaminoethyl methanethiosulfonate (MTSEA-Biotin) labeling of free cysteines were determined. For the majority of single mutants, only a modest decrease (2-to 5-fold) in ATP potency was recorded. For mutants C261A and C270A, the peak current amplitudes were reduced by 93.6 Ϯ 2.0 and 95.0 Ϯ 1.0%, respectively; this was a result of low cell-surface expression of these mutant receptors. Wild-type receptors showed no labeling with MT-SEA-biotin suggesting that all 10 cysteine residues in the extracellular loop are disulfide-bonded. Mutation of cysteines at positions 126, 132, 149, 159, 217, and 227 resulted in MTSEAbiotinylation of a free cysteine residue created by the disruption of a disulfide bond and provides direct biochemical evidence for at least three disulfide bonds. Based on phenotypic comparisons of single and double cysteine mutants, we propose the following disulfide bond pairs in the human P2X 1 receptor: C117-C165, C126-C149, C132-C159, C217-C227, and C261-C270. None of these bonds are individually essential for channel function. However, trafficking of the receptor to the cell membrane is severely reduced by disruption of the C261-C270 disulfide bond or disruption of C117-C165 together with another bond.

Using a method of single muscle fibre analysis, we investigated the presence of RNA transcripts for various isoforms of the myosin heavy chain (MyoHC) gene in histochemically, immunohistochemically and electrophoretically characterized individual muscle fibres (n = 65) from adult human vastus lateralis muscle. A cDNA clone isolated in this study was shown to contain the 3' end of a previously uncharacterized human MyoHC gene which is expressed specifically in human fast IIA muscle fibres and we conclude that this clone contains part of the human fast IIA MyoHC gene. In all the fibres histochemically, immunohistochemically and electrophoretically characterized as containing the previously classified IIB MyoHC (n = 23), it was shown that the human equivalent to the rat type IIX MyoHC gene is expressed. This observation was taken to suggest that the previously classified IIB muscles fibres in human muscle express a MyoHC isoform equivalent to the rat IIX, not the IIB, and would therefore be more accurately classified as IIX fibres.

Road, Leicester LE1 9HN 1 ADP, an important agonist in thrombosis and haemostasis, has been reported to activate platelets via three receptors, P2X 1 , P2Y 1 and P2T AC . Given the low potency of ADP at P2X 1 receptors and recognized contamination of commercial samples of adenosine nucleotides, we have re-examined the activation of P2X 1 receptors by ADP following HPLC and enzymatic puri®cation. 2 Native P2X 1 receptor currents in megakaryocytes were activated by a,b-meATP (10 mM) and commercial samples of ADP (10 mM), but not by puri®ed ADP (10 ± 100 mM). 3 Puri®ed ADP (up to 1 mM) was also inactive at recombinant human P2X 1 receptors expressed in Xenopus oocytes. Puri®cation did not modify the ability of ADP to activate P2Y receptors coupled to Ca 2+ mobilization in rat megakaryocytes. 4 In human platelets, P2X 1 and P2Y receptor-mediated [Ca 2+ ] i responses were distinguished by their di erent kinetics at 138C. In 1 mM Ca 2+ saline, a,b-meATP (10 mM) and commercial ADP (40 mM) activated a rapid [Ca 2+ ] i increase (lag time 40.5 s) through the activation of P2X 1 receptors. Hexokinase treatment of ADP shifted the lag time by &2 s, indicating loss of the P2X 1 receptor-mediated response. 5 A revised scheme is proposed for physiological activation of P2 receptors in human platelets. ATP stimulates P2X 1 receptors, whereas ADP is a selective agonist at metabotropic (P2Y 1 and P2T AC ) receptors.