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

Abstract

BACKGROUND AND PURPOSEMorphine is an antagonist at 5-HT3A receptors. 5-HT3 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-HT3 receptors. We investigated the mechanisms of inhibition by morphine of 5-HT3 receptors and the influence of the 5-HT3B subunit. EXPERIMENTAL APPROACH5-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-HT3A or 5-HT3AB receptors was assessed using radioligand binding with the antagonist [ KEY RESULTSWhen pre-applied, morphine potently inhibited 5-HT-evoked currents mediated by 5-HT3A 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-HT3A and 5-HT3AB receptors. Morphine also fully displaced [ CONCLUSIONS AND IMPLICATIONSThese 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-HT3 receptors.

The Minimum M3-M4 Loop Length of Neurotransmitter-activated Pentameric Receptors Is Critical for the Structural Integrity of Cytoplasmic Portals

“…Cells were seeded at low density in 35-mm dishes for electrophysiological recording, and transfections were performed by calcium phosphate precipitation, using 1 g of total cDNA per dish, as described previously (6). cDNAs encoding wild-type and mutant 5-HT3 subunits were cloned into the pCDM8 mammalian expression vector using HindIII (5Ј) and XhoI (3Ј) sites.…”

“…A unitary rectification index was used as a marker of the successful incorporation of the 5-HT3B subunit, which causes linearization of the I-V relationship (5). Unless otherwise stated, currents were evoked by rapid application of 100 M 5-HT, using the threepipe Perfusion Fast Step system (Warner Instruments), as described previously (6). The outside-out patch configuration was used to record single channel currents from excised patches from transfected cells as detailed previously (5).…”

“…A chimeric strategy, replacing segments of 5-HT3A subunit amino acid sequences with equivalent residues in the 5-HT3B subunit, revealed that amino acid determinants of Ca 2ϩ permeability lie within the M2 domain (6). By contrast, a similar approach revealed that the molecular determinants for ␥ lie within the helical amphipathic (HA) stretch in the M3-M4loop.…”

A single channel mutation alters agonist efficacy at 5‐HT3A and 5‐HT3AB receptors

“…The 5-HT 3 A and 5-HT 3 AB receptors are the most thoroughly studied and have differing biophysical properties (Hapfelmeier et al ., 2003; Kelley et al ., 2003; Hales et al ., 2006; Hu and Peoples, 2008a). There are also differences in the potencies of non-competitive antagonists that are likely to result from different pore-lining amino-acids contributed by the different subunits (Das and Dillon, 2005; Hu and Peoples, 2008a; Thompson et al ., 2011a; 2012a; Baptista-Hon et al ., 2012). In contrast, agonists and competitive antagonists have similar affinities at 5-HT 3 A and 5-HT 3 AB receptors because both receptors contain the same binding interface, formed by adjacent 5-HT 3 A receptor subunits (Brady et al ., 2001; Lochner and Lummis, 2010; Thompson et al ., 2011b; Thompson et al ., 2012b).…”

Mutagenic Analysis of the Intracellular Portals of the Human 5-HT3A Receptor

“…Structure-function studies of Cys-loop receptors, including the 5-HT 3 A receptor, have extensively probed the M2 domain and flanking sequences for their impact upon ion conduction and selectivity (13, 35, 39–44). The amino acid sequences of the membrane-spanning regions are relatively well conserved across Cys-loop receptors, and systematic mutagenesis essentially confirms the validity of homology models of the 5-HT 3 A receptor M2 domain based on the medium resolution structure (3.6 Å) of the Torpedo nACh receptor (see Fig.…”