Expression cloning of rat cerebellar adenosine A1 receptor by coupling to Kir channels.
G protein activation of inwardly rectifying K+ (Kir) channels by heptahelical receptors is an important signaling motif in slow synaptic transmission in the mammalian brain. To isolate candidate receptors responsive to the purine nucleoside adenosine, a cerebellar cDNA library was constructed in the vector pSGEM and transcripts were injected into Xenopus Laevis oocytes co-expressing Kir3.1 and/or Kir3.2 subunits. Stepwise fractionation and functional characterization of the library using two-electrode voltage clamp measurements resulted in the identification of a single unique cDNA clone with an open reading frame of 326 amino acids. The pharmacological properties as determined from the responses to cyclopentyl-adenosine (CPA, EC50 = 7 nM) and CGS21680 (EC50 = 2.6 microM) were typical of adenosine A1 receptors. The differential receptor coupling to heteromeric Kir channels composed of Kir3.1-4 subunits provides a useful technique to isolate novel heptahelical receptors.
Pfaff
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T
Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
Karschin
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A
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002531:Cerebellum / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005260:Female; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D015723:Gene Library; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D010627:Phenethylamines / Q000494:pharmacology; D015221:Potassium Channels / Q000096:biosynthesis / Q000502:physiology; D024661:Potassium Channels, Inwardly Rectifying; D058906:Purinergic P1 Receptor Agonists; D051381:Rats; D017208:Rats, Wistar; D018047:Receptors, Purinergic P1 / Q000096:biosynthesis* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014982:Xenopus laevis
D018076:DNA, Complementary; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D010627:Phenethylamines; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1; D011993:Recombinant Fusion Proteins; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; C048599:N(6)-cyclopentyladenosine; D000241:Adenosine