Decrease of current responses at human recombinant P2X3 receptors after substitution by Asp of Ser/Thr residues in protein kinase C phosphorylation sites of their ecto-domains.
The whole-cell patch-clamp technique was used to record current responses to nucleotides in HEK 293 cells transiently transfected with the human (h) P2X(3) receptor. When GDP-beta-S was included into the pipette solution, UTP at concentrations which did not alter the holding current, facilitated the alpha,beta-methylene ATP (alpha,beta-meATP)-induced current. The substitution of Ser/Thr residues situated within protein kinase C (PKC) consensus phosphorylation sites of the P2X(3) receptor ecto-domain by the neutral amino acid Ala either abolished (T134A, S178A) or did not alter (T196A, S269A) the UTP-induced potentiation of the alpha,beta-meATP current. The substitution of the same Ser/Thr residues in all four PKC sites by the negatively charged Asp prevented the potentiation by UTP. The Asp mutations abolished the first, fast offset time-constant, but did not alter, or in the case of S269D even increased, the second, slow offset time-constant; at the same time such mutations invariably increased the onset time-constant and massively depressed the peak current amplitude. None of the Ala mutations (with the exception of S269A) influenced the time-course of desensitisation or the peak current amplitude. It is concluded that constitutive activation of PKC sites at the ecto-domain of the hP2X(3) receptor both abolishes the UTP-induced potentiation of the alpha,beta-meATP current and accelerates its rate of desensitisation.
Stanchev
Doychin
D
Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Haertelstrasse 16-18, Germany.
Flehmig
Gesine
G
Gerevich
Zoltan
Z
Nörenberg
Wolfgang
W
Dihazi
Hassan
H
Fürst
Susanna
S
Eschrich
Klaus
K
Illes
Peter
P
Wirkner
Kerstin
K
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation / Q000379:methods; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D016296:Mutagenesis / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058476:Receptors, Purinergic P2X2; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014544:Uridine Triphosphate / Q000494:pharmacology
C546401:P2RX2 protein, human; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D011994:Recombinant Proteins; D013912:Threonine; D001224:Aspartic Acid; D012694:Serine; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D014544:Uridine Triphosphate