EXPRESSION CLONING OF RAT CEREBELLAR ADENOSINE-A1-RECEPTOR BY COUPLING TO KIR CHANNELS

G protein activation of inwardly rectifying K+ (Kir) channels by hepta helical receptors is an important signaling motif in slow synaptic tra nsmission in the mammalian brain. To isolate candidate receptors respo nsive to the purine nucleoside adenosine, a cerebellar cDNA library wa s constructed in the vector pSGEM and transcripts were injected into X enopus laevis oocytes coexpressing Kir3.1 and/or Kir3.2 subunits. Step wise fractionation and functional characterization of the library usin g two-electrode voltage clamp measurements resulted in the identificat ion of a single unique cDNA clone with an open reading frame of 326 am ino acids. The pharmacological properties as determined from the respo nses to cyclopentyl-adenosine (CPA, EC50 = 7 nM) and CGS21680 (EC50 = 2.6 mu M) were typical of adenosine A1 receptors. The differential rec eptor coupling to heteromeric Kir channels composed of Kir3.1-4 subuni ts provides a useful technique to isolate novel heptahelical receptors .