Electrophysiological consequences of ligand binding to the desensitized 5‐HT3 receptor in mammalian NG108‐15 cells.

Abstract

1. Using the whole-cell variation of the patch-clamp technique to record from mammalian NG108-15 cells, we have studied the ligand-gated ion channel current activated by a high concentration (100 /uM) of local pressure-applied 5-hydroxytryptamine (5-HT). The response was induced at intervals of at least 90-120 s, which allowed the receptor to fully recover between activations.2. The rapid inward current induced by pressure-applied 5-HT was reproducibly inhibited by the superfusion of low concentrations of 5-HT which evoked little or no detectable inward current alone (0 01-0 3 uM). This inhibitory effect was most likely to be due to a direct action on the 5-HT3 receptor as it could be recorded using intracellular solutions with or without adenosine triphosphate (ATP) and guanosine triphosphate (GTP). 3. The maximum inhibitory effect of a given concentration of 5-HT was not dependent on its superfusion time but on the number of activations of the receptor by pressure-applied 5-HT. This activation dependence was clearly evident, since the first inward current in the presence of 0 1 /M 5-HT was often unaffected in amplitude. 4. The inhibitory effect of 5-HT was evident at holding potentials of +60 and -60 mV; with the calcium chelator BAPTA in the recording pipette and with the nominal removal of extracellular calcium and magnesium ions.5. The inhibitory effect was concentration dependent, with 50% inhibition of the inward current amplitude occurring at -50 nM 5-HT. The slope factor of the inhibition curve was 1-3. The effect was mimicked by two other 5-HT3 receptor agonists, 2-methyl-5-HT and m-chlorophenylbiguanide (mCPBG) which gave 50% inhibition at -600 nm and 20 nM, respectively. These values are similar to the affinity values for these ligands determined in radioligand binding assays. 6. The 5-HT3 receptor 'antagonists' (+)-tubocurarine and quipazine (both at 3 nM) reduced the inward current amplitude by -50 %. The rate of onset of the inhibitory effect of bath-applied 5-HT was slowed in the presence of (+)-tubocurarine 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 concentrations of bath-applied 5-HT3 receptor agonists is compatible with the cyclic model of receptor activation and desensitization. We conclude that we have been studying the high-affinity binding of agonists to the desensitized form of the 5-HT3 receptor.

Conversion of the Ion Selectivity of the 5-HT3AReceptor from Cationic to Anionic Reveals a Conserved Feature of the Ligand-gated Ion Channel Superfamily

“…with WT, and the whole-cell currents recorded exhibited a marked reduction in the rate of activation and desensitization. In fact, the EC 50 for 5-HT on the 5-HT 3A -STM receptor (240 nM) is similar to the value of 230 nM reported for the EC 50 of the ␣7-2 mutant nACh receptor for acetylcholine (1) and the estimated affinity of 5-HT for the desensitized state of the 5-HT 3 receptor (50 nM) (27). The relevance of the lower Hill coefficient noted for the 5-HT 3A -STM receptor (1.45), compared with WT is not clear but may reflect an alteration to the normal gating mechanism of the receptor.…”

The Pharmacological and Functional Characteristics of the Serotonin 5-HT3A Receptor Are Specifically Modified by a 5-HT3B Receptor Subunit

“…The rank order of potency was quipazine Ͼ Ͼ 5-HT ϳ mCPBG Ͼ 2-Me-5-HT Ͼ mCPP Ͼ 1-PBG Ͼ Ͼ DA. Quipazine was also a potent agonist at murine recombinant 5-HT 3A receptors (51,52). In oocyte and mammalian studies, the EC 50 for 5-HT was about 400 nM, ϳ6 -9-fold lower than values observed for human recombinant 5-HT 3A previously reported (30,34,45,46,48).…”

Use-Dependent Inhibition of P2X3Receptors by Nanomolar Agonist

“…This creates a use dependence for P2X 3 inhibition (Fig. 6 B) similar to that seen in other channels (Bartrup and Newberry, 1996;Newell and Dunn, 2002).…”

“…Nicotinic acetylcholine receptors (nAChR), GABA A receptors, 5-hydroxytryptamine receptors, and P2X 1 receptors are desensitized by concentrations of agonist that are lower than those required for channel activation (Katz and Theseleff, 1957;Bartrup and Newberry, 1996;Newell and Dunn, 2002;Paradiso and Steinbach, 2003). We found that 60 s of 0.5 nM ATP inhibited Ͼ50% of P2X 3 current after activation by high [agonist] (Fig.…”