Differential Regulation of Human Serotonin Receptor Type 3A by Chanoclavine and Ergonovine

Abstract

Irritable bowel syndrome (IBS) is a chronic disease that causes abdominal pain and an imbalance of defecation patterns due to gastrointestinal dysfunction. The cause of IBS remains unclear, but intestinal-brain axis problems and neurotransmitters have been suggested as factors. In this study, chanoclavine, which has a ring structure similar to 5-hydroxytryptamine (5-HT), showed an interaction with the 5-HT_3A receptor to regulate IBS. Although its derivatives are known to be involved in neurotransmitter receptors, the molecular physiological mechanism of the interaction between chanoclavine and the 5-HT_3A receptor is unknown. Electrophysiological experiments were conducted using a two-electrode voltage-clamp analysis to observe the inhibitory effects of chanoclavine on Xenopus oocytes in which the h5-HT_3A receptor was expressed. The co-application of chanoclavine and 5-HT resulted in concentration-dependent, reversible, voltage-independent, and competitive inhibition. The 5-HT_3A response induced by 5-HT was blocked by chanoclavine with half-maximal inhibitory response concentration (IC₅₀) values of 107.2 µM. Docking studies suggested that chanoclavine was positioned close F130 and N138 in the 5-HT_3A receptor-binding site. The double mutation of F130A and N138A significantly attenuated the interaction of chanoclavine compared to a single mutation or the wild type. These data suggest that F130 and N138 are important sites for ligand binding and activity. Chanoclavine and ergonovine have different effects. Asparagine, the 130th amino acid sequence of the 5-HT_3A receptor, and phenylalanine, the 138th, are important in the role of binding chanoclavine, but ergonovine has no interaction with any amino acid sequence of the 5-HT_3A receptor. The results of the electrophysiological studies and of in silico simulation showed that chanoclavine has the potential to inhibit the hypergastric stimulation of the gut by inhibiting the stimulation of signal transduction through 5-HT_3A receptor stimulation. These findings suggest chanoclavine as a potential antiemetic agent for excessive gut stimulation and offer insight into the mechanisms of 5-HT_3A receptor inhibition.

Keywords: chanoclavine; ergot alkaloids; irritable bowel syndrome; serotonin receptor.

Figure 1

Chemical structure of chanoclavine and ergonovine and the inhibitory effects on Xenopus oocytes expressing human 5-hydroxytryptamine (5-HT)_3A receptors. (A). Structure of chanoclavine (EC) and ergonovine (EG). (B). After flowing 5-HT mixed with bath solution for one minute per 2 mL, 5-HT (100 µM) induced a reversible inward current (I_5-HT). The traces indicate the inward current in the presence of 5-HT coapplied with ergonovine and chanoclavine at 100 µM. The representative traces from four different frogs elicited at the −80-mV holding potential. (C). The histogram indicates that the percentage of inhibition by chanoclavine was 42.5 ± 9.7 calculated from the mean of the peak inward current. Each value represents the mean ± S.E.M (n = 8–10 from four different frogs).

Figure 2

The concentration-dependent response of Xenopus oocytes expressing human 5-HT_3A receptors to ergot alkaloids. (A). The traces represent increases in the current inhibition with increases in the ergonovine concentration. Treatment with 5-HT (100 µM) alone induced an inward current followed by various response traces with 5-HT (100 µM) and ergonovine (10, 30, 100, and 300 µM). (B). The plot shows the ergonovine inhibition current fitted according to Hill’s equation, where the maximum inhibition (I_max) was 15 ± 5.7%. (C). The traces induced by chanoclavine with 5-HT (100 µM) appear dependent upon the concentration (10, 30, 100, and 300 µM). (D). Average percentage curves of chanoclavine fitted according to Hill’s equation based on the peak of the inward current. The I_max was 83.6 ± 12.6%, and the inhibition was 4 ± 3.8%, 7.9 ± 4.1%, 42.5 ± 5.7%, 61.6 ± 6.1%, and 75 ± 4.5% at 10, 30, 100, 200, and 300 µM, respectively. The oocytes expressing human 5-HT_3A mRNA were held at −80 mV, and each value represents the mean ± SEM (n = 8–10 from four different frogs).

Figure 3

Concentration-dependent inhibition responses of mutants to chanoclavine. (A–D). Y91A, F130A, N138A, and F130A+N138A mutant receptors are rectified at a −80 mV holding potential. The 5-HT_3A mutant receptors expressed in Xenopus oocytes elicit reversible currents using an electrode voltage clamp. All oocytes were exposed to 10, 30, 100, and 300 µM chanoclavine coapplied with 100 µM 5-HT after treatment with 5-HT alone. (E). The inhibition percentage curve according to the EC concentration of the 5-HT_3A mutant receptors. Each value represents the mean ± SEM. Additional half-maximal inhibitory response concentrations (IC₅₀), I_max, and Hill’s coefficient values of the other mutants are listed in Table 1 (n = 6–8 from four different frogs).

Figure 4

Voltage dependency and competition inhibition of chanoclavine in I_5-HT in oocytes expressing 5-HT_3A receptors. (A–D). Current–voltage relationship of I_5-HT inhibition by chanoclavine in 5-HT_3A receptors. Wild-type 5-HT_3A receptor traces showed a current–voltage relationship with EC and EG at the same concentration as 5-HT (100 µM). All control trace voltage ramps change with only the bath solution flowing without treatment. Traces were obtained using voltage changes from −80 mV to +60 mV over a duration of one sweep per two seconds. B–D Indicate F130A, N138A, and F130A+N138A, respectively. (E). Concentration-response effect of 5-HT in the absence or presence of chanoclavine on oocytes expressing human 5-HT_3A receptors. Control (○) represents various 5-HT concentration currents from 1 to 100 µM in the absence of chanoclavine, and 5-HT at 30 µM (□) and 100 µM (△) chanoclavine represents the change in the current compared to the control. Normalized currents were divided by the I_max of the control current and the I_max of 5-HT alone, with 30 µM and 100 µM EC at 99.1 ± 14.2, 94.6 ± 24.4, and 60 ± 2.2, respectively. Oocytes were exposed to 5-HT alone or with EC over a duration of 2 mL per minute and held at a −80 mV membrane holding potential. Each value represents the mean ± SEM (n = 8–10 from four different frogs).

Figure 5

Molecular docking modeling of chanoclavine to the 5-HT_3A receptor. (A–D). Side and top views of the chanoclavine docking model to the 5-HT_3A receptor. (E). Binding pocket in the 5-HT_3A receptor region of the extracellular domain membrane of the pocket side. Chanoclavine docked to the extracellular region that forms a loop complex in the 5-HT_3A receptor. (F). 2D schematic of the predicted binding mode of chanoclavine in the ligand-binding pocket. (G,H). Binding interactions of the ligand and residues in the wild and mutant types. The replaced mutants changed the interaction activity to varying degrees.