Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Purinergic Signal
2004 Dec 01;11:75-81. doi: 10.1007/s11302-004-4744-5.
Show Gene links
Show Anatomy links
P2Y(1) receptor modulation of endogenous ion channel function in Xenopus oocytes: Involvement of transmembrane domains.
Lee SY
,
Nicholas RA
,
O'Grady SM
.
???displayArticle.abstract???
Agonist activation of the hP2Y(1) receptor expressed in Xenopus oocytes stimulated an endogenous voltage-gated ion channel, previously identified as the transient inward (T(in)) channel. When human P2Y(1) (hP2Y(1)) and skate P2Y (sP2Y) receptors were expressed in Xenopus oocytes, time-to-peak values (a measure of the response to membrane hyperpolarization) of the T(in) channel were significantly reduced compared to oocytes expressing the hB(1)-bradykinin receptor or the rat M(1)-muscarinic (rM(1)) receptor. Differences in activation were also observed in the T(in) currents elicited by various P2Y receptor subtypes. The time-to-peak values of the T(in) channel in oocytes expressing the hP2Y(4), hP2Y(11), or hB(1)-bradykinin receptors were similar, whereas the channel had significantly shorter time-to-peak values in oocytes expressing either the hP2Y(1) or sP2Y receptor. Amino acid substitutions at His-132, located in the third transmembrane domain (TM3) of the hP2Y(1) receptor, delayed the onset of channel opening, but not the kinetics of the activation process. In addition, Zn(2+) sensitivity was also dependent on the subtype of P2Y receptor expressed. Replacement of His-132 in the hP2Y(1) receptor with either Ala or Phe increased Zn(2+) sensitivity of the T(in) current. In contrast, truncation of the C-terminal region of the hP2Y(1) receptor had no affect on activation or Zn(2+) sensitivity of the T(in) channel. These results suggested that TM3 in the hP2Y(1) receptor was involved in modulating ion channel function and blocker pharmacology of the T(in) channel.
Figure 1. Effects of expressed Gq coupled receptor stimulation on hyperpolarization-induced activation of the endogenous Tin channel in Xenopus oocytes. A) Representative trace of the Tin channel current at −140 mV following stimulation of the expressed hP2Y1 receptor. Time-to-peak current measurements were used to assess hyperpolarization-induced activation of the channel and are defined as the elapsed time following the capacitance current to the peak inward current. B) Comparison of time-to-peak measurements for hB1-bradykinin (n = 13), Gq alpha subunit (n = 13), hP2Y1 receptor (n = 14), and sP2Y (n = 13). C) Comparison of time-to-peak measurements for hB1-bradykinin (n = 13), rM1-muscarinic (n = 12), hP2Y4 (n = 12), and hP2Y11 (n = 13) receptors.
Figure 2. Effect of C-terminal domain truncation on channel activation. A) The relative locations of TM3 mutations (hP2Y1-H132A, hP2Y1-H132D, and hP2Y1-H132F) and C-terminal truncation sites within the hP2Y1 receptor are indicated. B) Comparison of time-to-peak measurements for hB1-bradykinin (n = 13), hP2Y1 (n = 14), hP2Y1334tr (n = 9), hP2Y1342tr (n = 15), hP2Y1349tr (n = 11), hP2Y1360tr (n = 15), and hP2Y1369tr (n = 13) receptors as the function of voltage.
Figure 3. Effect of third transmembrane domain mutations on activation and conductance voltage relationships. A) Time-to-peak measurements for hB1-bradykinin (n = 13), wild-type hP2Y1 (n = 14), hP2Y1-H132A (n = 5), hP2Y1-H132D (n = 7), hP2Y1-H132F (n = 10), and hP2Y1-Q307K (n = 7) receptors as a function of voltage. B) Normalized conductance-voltage relationships for hB1-bradykinin (n = 13), wild-type hP2Y1 (n = 13), hP2Y1-H132A (n = 15), hP2Y1-H132D (n = 6), and hP2Y1-H132F (n = 6). The V50 values and slope factors for each conductance are listed in Table 1.
Figure 4. Effects of Zn2+ on the inward currents elicited by step hyperpolarization to −140 mV. A) Representative current traces showing Tin currents activated by hP2Y1 receptor in the presence of 20 µM 2MeS-ADP. Agonist-stimulated inward current was inhibited by ZnCl2 in a concentration-dependent manner. B) Concentration-response relationship for Gq (n = 7) and hB1-bradykinin (n = 5). C) Inhibition of hP2Y1 (n = 6), sP2Y (n = 5), and hP2Y11 (n = 5) receptor-elicited Tin currents by [Zn2+]. The IC50 values are listed in Table 2.
Figure 5. Effects of Zn2+ on the inward currents elicited after stimulation of mutant P2Y1 receptors. A) Inhibition of hP2Y1 (n = 6), hP2Y1-H132A (n = 3) and hP2Y1-H132F (n = 5) receptor-elicited Tin currents by [Zn2+]. B) Inhibition of hP2Y1 (n = 6), hP2Y1342tr (n = 9), and hP2Y1-Q307K (n = 5) receptor-elicited Tin currents by [Zn2+]. The IC50 values are listed in Table 2.
Boarder,
The regulation of vascular function by P2 receptors: multiple sites and multiple receptors.
1998, Pubmed
Boarder,
The regulation of vascular function by P2 receptors: multiple sites and multiple receptors.
1998,
Pubmed
Boarder,
G protein-coupled P2 purinoceptors: from molecular biology to functional responses.
1995,
Pubmed
Chambers,
A G protein-coupled receptor for UDP-glucose.
2000,
Pubmed
Communi,
Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase.
1997,
Pubmed
Communi,
Identification of a novel human ADP receptor coupled to G(i).
2001,
Pubmed
Communi,
Cloning and functional expression of a human uridine nucleotide receptor.
1995,
Pubmed
Dranoff,
A primitive ATP receptor from the little skate Raja erinacea.
2000,
Pubmed
,
Xenbase
Dubyak,
Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides.
1993,
Pubmed
Guttridge,
Xenopus Gq alpha subunit activates the phosphatidylinositol pathway in Xenopus oocytes but does not consistently induce oocyte maturation.
1995,
Pubmed
,
Xenbase
Hollopeter,
Identification of the platelet ADP receptor targeted by antithrombotic drugs.
2001,
Pubmed
,
Xenbase
Jiang,
A mutational analysis of residues essential for ligand recognition at the human P2Y1 receptor.
1997,
Pubmed
Kowdley,
Hyperpolarization-activated chloride currents in Xenopus oocytes.
1994,
Pubmed
,
Xenbase
Lazarowski,
Cloning and functional characterization of two murine uridine nucleotide receptors reveal a potential target for correcting ion transport deficiency in cystic fibrosis gallbladder.
2001,
Pubmed
Lee,
P2Y receptors modulate ion channel function through interactions involving the C-terminal domain.
2003,
Pubmed
,
Xenbase
Moro,
Human P2Y1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites.
1998,
Pubmed
Ni,
Efficient coupling of 5-HT1a receptors to the phospholipase C pathway in Xenopus oocytes.
1997,
Pubmed
,
Xenbase
Nicholas,
Identification of the P2Y(12) receptor: a novel member of the P2Y family of receptors activated by extracellular nucleotides.
2001,
Pubmed
O'Grady,
A guanine nucleotide-independent inwardly rectifying cation permeability is associated with P2Y1 receptor expression in Xenopus oocytes.
1996,
Pubmed
,
Xenbase
Parker,
A transient inward current elicited by hyperpolarization during serotonin activation in Xenopus oocytes.
1985,
Pubmed
,
Xenbase
Parr,
Cloning and expression of a human P2U nucleotide receptor, a target for cystic fibrosis pharmacotherapy.
1994,
Pubmed
Ralevic,
Receptors for purines and pyrimidines.
1998,
Pubmed
Sak,
A retrospective of recombinant P2Y receptor subtypes and their pharmacology.
2002,
Pubmed
Schachter,
Second messenger cascade specificity and pharmacological selectivity of the human P2Y1-purinoceptor.
1996,
Pubmed
Van Rhee,
Modelling the P2Y purinoceptor using rhodopsin as template.
1995,
Pubmed
von Kügelgen,
Molecular pharmacology of P2Y-receptors.
2000,
Pubmed