ELECTROPHYSIOLOGICAL CONSEQUENCES OF LIGAND-BINDING TO THE DESENSITIZED 5-HT3 RECEPTOR IN MAMMALIAN NG108-15 CELLS

1. Using the whole-cell variation of the patch-clamp technique to reco rd from mammalian NG108-15 cells, rye have studied the ligand-gated io n channel current activated by a high concentration (100 mu M) of loca l pressure-applied 5-hydroxytryptamine (5-HT). The response was induce d at intervals of at least 90-120 s, which allowed the receptor to ful ly recover between activations. 2. The rapid inward current induced by pressure-applied 5-HT was reproducibly inhibited by the superfusion o f low concentrations of 5-HT which evoked little or no detectable inwa rd current alone (0.01-0.3 mu M). This inhibitory effect was most like ly to be due to a direct action on the 5-HT3 receptor as it could be r ecorded using intracellular solutions with Or without adenosine tripho sphate (ATP) and guanosine triphosphate (GTP). 3. The maximum inhibito ry effect of a given concentration of 5-HT was not dependent on its su perfusion time but on the number of activations of the receptor by pre ssure-applied 5-HT. This activation dependence was clearly evident, si nce the first inward current in the presence of 0.1 mu M 5-HT was ofte n unaffected in amplitude. 4. The inhibitory effect of 5-HT was eviden t at holding potentials of +60 and -60 mV; with the calcium chelator B APTA in the recording pipette and with the nominal removal of extracel lular calcium and magnesium ions. 5. The inhibitory effect was concent ration dependent, with 50% inhibition of the inward current amplitude occurring at similar to 50 nM 5-HT. The slope factor of the inhibition curve was 1.3. The effect was mimicked by two other 5-HT3 receptor ag onists, 2-methyl-5-HT and m-chlorophenylbiguanide (mCPBG) which gave 5 0% inhibition at similar to 600 nM and similar to 20 nM, respectively. These values are similar to the affinity values for these ligands det ermined in radioligand binding assays. 6. The 5-HT3 receptor 'antagoni sts' (+)-tubocurarine and quipazine (both at 3 nM) reduced the inward current amplitude by similar to 50%. The rate of onset of the inhibito ry effect of bath-applied 5-HT was slowed in the presence of (+)-tuboc urarine but not in the presence of quipazine. This difference might be explained by the agonist properties seen only with quipazine. 7. The inhibition of the 5-HT3 receptor mediated inward current by low concen trations of bath-applied 5-HT3 receptor agonists is compatible with th e cyclic model of receptor activation and desensitization. We conclude that we have been studying the high-affinity binding of agonists to t he desensitized form of the 5-HT3 receptor.