The 5-HT3B subunit affects high-potency inhibition of 5-HT3 receptors by morphine

Abstract

Background and purpose: Morphine is an antagonist at 5-HT(3) A receptors. 5-HT(3) and opioid receptors are expressed in many of the same neuronal pathways where they modulate gut motility, pain and reinforcement. There is increasing interest in the 5-HT3B subunit, which confers altered pharmacology to 5-HT(3) receptors. We investigated the mechanisms of inhibition by morphine of 5-HT(3) receptors and the influence of the 5-HT3B subunit.

Experimental approach: 5-HT-evoked currents were recorded from voltage-clamped HEK293 cells expressing human 5-HT3A subunits alone or in combination with 5-HT3B subunits. The affinity of morphine for the orthosteric site of 5-HT(3) A or 5-HT(3) AB receptors was assessed using radioligand binding with the antagonist [(3) H]GR65630.

Key results: When pre-applied, morphine potently inhibited 5-HT-evoked currents mediated by 5-HT(3) A receptors. The 5-HT3B subunit reduced the potency of morphine fourfold and increased the rates of inhibition and recovery. Inhibition by pre-applied morphine was insurmountable by 5-HT, was voltage-independent and occurred through a site outside the second membrane-spanning domain. When applied simultaneously with 5-HT, morphine caused a lower potency, surmountable inhibition of 5-HT(3) A and 5-HT(3) AB receptors. Morphine also fully displaced [(3) H]GR65630 from 5-HT(3) A and 5-HT(3) AB receptors with similar potency.

Conclusions and implications: These findings suggest that morphine has two sites of action, a low-affinity, competitive site and a high-affinity, non-competitive site that is not available when the channel is activated. The affinity of morphine for the latter is reduced by the 5-HT3B subunit. Our results reveal that morphine causes a high-affinity, insurmountable and subunit-dependent inhibition of human 5-HT(3) receptors.

Figure 1

High-potency insurmountable antagonism of 5-HT₃A and 5-HT₃AB receptors by morphine. (A) Concentration–response relationships of pre-applied morphine on 5-HT₃A and 5-HT₃AB receptors. Each point represents the mean ± SEM of at least four recordings from different cells. Data are normalized to the current at 30 µM 5-HT in the absence of morphine. The logistic fit to the concentration–response curve yielded an IC₅₀ of 0.33 ± 0.07 µM for 5-HT₃A receptors and 1.15 ± 0.11 µM for 5-HT₃AB receptors. Morphine was either bath applied or pre-applied for 2 s using a rapid application system (see Methods). Inset shows representative, superimposed traces of currents recorded from 5-HT₃A receptor expressing HEK293 cells in the absence or presence of 0.3, 1 and 10 µM morphine pre-applied prior to 5-HT (30 µM). (B) Graph of the 5-HT concentration–response relationship in the absence and presence of pre-applied morphine (10 µM). Current amplitudes are normalized to the amplitude of control currents activated by 100 µM 5-HT. When morphine was pre-applied, the inhibition of the 5-HT-induced current was insurmountable even by maximally efficacious concentrations of 5-HT. Inset shows superimposed traces of 5-HT (3–300 µM)-induced currents in the presence of pre-applied morphine (10 µM). The grey trace represents part of a control current induced by 5-HT (100 µM) provided for comparison.

Figure 2

Competitive and surmountable antagonism of 5-HT₃A and 5-HT₃AB receptors by morphine simultaneously applied with 5-HT. (A) Superimposed traces of 5-HT (1–300 µM)-induced currents recorded from the same HEK293 cell expressing 5-HT₃A receptors in the absence and presence of simultaneously applied morphine (10 µM). (B) Graph of the concentration–response relationship for 5-HT in the presence of increasing concentrations (3–300 µM) of co-applied morphine on 5-HT₃A receptors. Inset, Schild plot fitted with a linear regression with a slope of 0.8. (C) Superimposed traces of 5-HT (1–300 µM)-induced currents recorded from the same HEK293 cell expressing 5-HT₃AB heteromeric receptors in the absence and presence of morphine (30 µM) applied simultaneously with 5-HT. (D) Graph of the 5-HT₃AB receptor concentration–response relationship for activation by 5-HT in the absence and presence of increasing concentrations of morphine. Inset, Schild plot fitted with a linear regression with a slope of 1.2. Current amplitudes in the concentration–response relationships are expressed as percentage of the mean current induced by 5-HT (100 µM) in control conditions. Each data point represents the mean ± SEM of at least four recordings from separate cells. EC₅₀ values and Hill coefficients were determined from logistic fits to the concentration–response curves and are summarized in Table 1.

Figure 3

Morphine competes for the binding site of the competitive antagonist [³H]GR65630 on 5-HT₃ receptors. Membranes containing 5-HT₃A receptors were incubated with 150 pM [³H]GR65630. See Figure S1 for saturation binding of [³H]GR65630. The graph in this figure shows the reduction of specific [³H]GR65630 binding (expressed as percent control) with increasing concentrations of morphine. Each data point represents the mean ± SEM of at least three separate binding assays. Morphine inhibited [³H]GR65630 binding to 5-HT₃A and 5-HT₃AB receptors with IC₅₀ values of 27 and 19 µM respectively.

Figure 4

The M2 domain is not responsible for the insurmountable antagonism by pre-applied morphine. (A) Schematic diagram of the 5-HT3ABA construct, which is the 5-HT3A subunit containing the 5-HT3B M2 domain. (B) Na⁺ and Ca²⁺ permeability ratios with respect to Cs⁺ for 5-HT₃A, 5-HT₃AB and 5-HT₃A + ABA receptors. P_Ca2+/P_Cs+ in 5-HT₃AB and 5-HT₃A + ABA receptors are significantly lower than that of 5-HT₃A receptor (P < 0.05, anova, post hoc Tukey's test), thus confirming the incorporation of the ABA construct into heteromeric receptors. (C) Bar graph shows the current elicited by 5-HT (30 µM) following pre-application of morphine (1 µM) expressed as percent control current amplitude. The % control values for 5-HT₃A, 5-HT₃AB and 5-HT₃A + ABA receptors were 30 ± 6%, 52 ± 4% and 26 ± 4% respectively. The values for 5-HT₃A and 5-HT₃A +ABA receptors are significantly lower than that of 5-HT₃AB receptors (P < 0.05, one-way anova, post hoc Tukey's test), indicating that the presence of the 5-HT3B M2 domain has no effect on the inhibition by pre-applied morphine.

Figure 5

Onset and offset time course of inhibition by pre-applied morphine of 5-HT-induced currents mediated by 5-HT₃A and 5-HT₃AB receptors. (A) Typical traces of sequential 5-HT-induced currents recorded from an HEK293 cell expressing 5-HT₃A receptors. The cell was pre-exposed to morphine (10 µM) for progressively longer durations (10 ms increments) before the application of 5-HT (100 µM) plus morphine (10 µM). (B) currents recorded from a cell expressing 5-HT₃A receptors pre-exposed to morphine (10 µM) for 2 s followed by progressively increasing wash durations (100 ms increments) before the application of 5-HT (100 µM) plus morphine (10 µM). The first and last traces are shown in black, and the protocol for solution change is shown above. (C) Mean onset and (D) offset time courses for pre-applied morphine inhibition in 5-HT₃A and 5-HT₃AB receptors. Graphs show the mean peak amplitude [as % of control 5-HT (100 µM)-evoked current] with either increasing exposure time (for onset kinetics in C) or increasing wash time (for offset kinetics in D) for 5-HT₃A and 5-HT₃AB receptors. In experiments involving 5-HT₃AB receptors, a higher concentration of morphine was used (30 µM), reflecting the lower-potency inhibition of heteromeric receptors (Figure 1). Each data point represents the mean ± SEM of four to six experiments. A single exponential function was fitted to each set of data. The time constants for the onset of morphine inhibition were 35 ± 11 ms (n = 4) and 17 ± 2 ms (n = 4) for the 5-HT₃A receptor and 5-HT₃AB receptor respectively. The time constants for the offset of morphine inhibition were 0.53 ± 0.04 s (n = 6) and 0.32 ± 0.07 s (n = 5) for the 5-HT₃A receptor and 5-HT₃AB receptor respectively. There were significant differences between the onset and offset time constants (P < 0.0001 for onset, P = 0.02 for offset) for homomeric and heteromeric receptors.