Ethanol Stabilizes the Open State of Single 5-Hydroxytryptamine_3A(QDA) Receptors

Abstract

Ethanol enhancement of 5-hydroxytryptamine (5-HT) 3A receptor-mediated responses may have important consequences in the intoxicating and addictive properties of ethanol. Although the exact mechanism is unknown, ethanol-mediated enhancement of 5-HT 3 receptor current has been proposed to occur due to stabilization of the open-channel state. It has not been possible to directly measure the open state of the channel due to the extremely low single-channel conductance of 5-HT 3A channels. Recently, three arginine residues within the large intracellular loop of the 5-HT 3A subunit were substituted by their equivalent residues (glutamine, aspartate, and alanine) of the 5-HT 3B subunit to produce a 5-HT 3A (QDA) subunit that forms functional homomeric channels exhibiting a measurable single-channel conductance. Using whole-cell rapid-agonist application techniques and the cell-attached single-channel recording configuration, we examined human 5-HT 3A (QDA) receptors expressed in human embryonic kidney 293 cells. The agonist sensitivity, macroscopic kinetics, and modulation by ethanol were similar between mutant and wild-type channels, suggesting the substitutions had not altered these channel structure-function properties. The open time histogram for single-channel events mediated by 5-HT 3A (QDA) receptors in the presence of maximal 5-HT was best fit by three exponentials, but in the presence of ethanol a fourth open state was evident. In summary, the QDA substitution greatly enhanced singlechannel conductance with little effect on 5-HT 3A channel's kinetic properties and ethanol enhances agonist action on 5-HT 3A receptors by inducing a new, long-lived open-channel state. Furthermore, the 5-HT 3A (QDA) receptor appears to be suitable for pharmacological studies of 5-HT 3A receptor modulation at a single-channel level.

Activation and modulation of recombinantly expressed serotonin receptor type 3A by terpenes and pungent substances

“…This was unexpected because the chemically close related propofol and 2-isopropylphenol were not agonists but rather inhibitors [27]. Currently, known potentiators are alcohols of low molecular size and volatile anesthetics [28].…”

Alcohol-induced serotonergic modulation: The role of histone deacetylases

“…(Engleman et al, 2008). Overall, from previous literature, it is clear that ethanol modulation of 5-HT3 receptor-mediated responses may have important consequences in the intoxicating and addictive properties of ethanol (Feinberg-Zadek and Davies, 2010). However, such conflicting results regarding the effects of alcohol on these receptors and the lack of knowledge on the mechanisms of action lead us to the search for novel research studies involving ethanol, HDACs, and serotonin modulation in CNS cells.…”

Allosteric modulation of the 5-HT3 receptor

“…Using a triple mutant receptor (5-HT 3 A(QDA)) that forms homomeric channels exhibiting a measurable single-channel conductance [11–12] the stability of the open channel by ethanol was confirmed (Figure 1). By examining the different open states evoked by saturating 5-HT in the absence and presence of ethanol the prolonged open state appeared to come at the expense of the desensitized state [13**]. This prolongation of the open state of the channel at the expense of the desensitized state is consistent with the slowed desensitization effect of ethanol and trichloroethanol on macroscopic currents [14–15].…”

“…Histograms were fitted by two Gaussians, percentages of events in the closed and open states are shown. Figure reproduced, with permission, from Feinberg-Zadek and Davies [13]. …”