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 single-channel 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.

Fig. 1.

Serotonin sensitivity of 5-HT_3A(QDA) receptors is similar to wild-type 5-HT_3A receptors. 5-HT concentration-response relationship for wild-type 5-HT_3A receptors (open circles) and 5-HT_3A(QDA) receptors (closed circles). Data fit to a Hill equation. For EC₅₀ and Hill coefficient values, see text. Data are from three to eight cells.

Fig. 2.

The macroscopic kinetics of the 5-HT_3A(QDA) receptors are similar to that wild-type 5-HT_3A receptors. A, QDA substitutions minimally affect macroscopic current recordings. Left, current traces produced by activation of wild-type 5-HT_3A receptors (top; gray trace) and by 5-HT_3A(QDA) receptors (bottom; black trace). Currents were evoked by rapidly (solution exchange time, ∼1–2 ms) applying 100 μM 5-HT to HEK293 cells expressing the indicated receptor. Right, mean activation time constants (data are mean ± S.E.M. from three to four cells). B, deactivation. A 5-ms pulse of 100 μM 5-HT was rapidly applied to HEK293 cells expressing 5-HT_3A receptors (gray trace) and 5-HT_3A(QDA) receptors (black trace). The current decay (between 90 and 10% of maximal current amplitude) immediately after the agonist pulse was fit to an exponential equation; the best fit is shown as a color-contrasted line. The two receptors had similar time constants for deactivation. Data are mean ± S.E.M. from three to four cells. C, desensitization. An exponential fit of the current decay recorded during prolonged exposure to 100 μM 5-HT rapidly applied to HEK293 cells expressing 5-HT_3A receptors (gray trace) and 5-HT_3A(QDA) receptors (black trace). The color-contrasted line during the decay phase is the exponential fit. No difference to the mean desensitization time constant was detected. Data are mean ± S.E.M. from three cells.

Fig. 3.

Alcohol enhancement of 5-HT_3A and 5-HT_3A(QDA) receptors. A, representative current recordings showing enhancement of submaximal 5-HT-evoked current amplitudes by 25 mM ethanol. 5-HT_3A and 5-HT_3A(QDA) receptors were pre-equilibrated with buffer or ethanol and then activated with an EC₁₀ response. B, mean enhancements of wild-type 5-HT_3A receptor- (open bars) and mutant 5-HT_3A(QDA) receptor (closed bars)-mediated current amplitudes by 25 mM ethanol. Data are mean ± S.E.M. from three to five cells.

Fig. 4.

Ethanol increases the number of open 5-HT_3A(QDA) channel events and increases the open probability. Left, cell-attached recordings of 5-HT_3A(QDA) receptor-mediated currents evoked by 100 μM 5-HT in the absence (A) or presence (B) of 25 mM ethanol. Twenty-second examples of single-channel activity with an expanded view of a burst. Dotted lines indicating closed and open states. Right, all-points histograms for the 20-s cell-attached recordings of 5-HT_3A(QDA) receptor-mediated currents evoked by 100 μM 5-HT shown in the left panel. Histograms were fitted by two Gaussians (gray lines). Percentages of events in the closed and open states are shown.

Fig. 5.

Ethanol induces a fourth open state distinguished by its long open time. Cell-attached recordings of 5-HT_3A(QDA) receptor-mediated currents evoked by 100 μM 5-HT (right) in the absence (A) and presence (B) of 25 mM ethanol. Pipette potential was 80 mV. Exponential fits to channel open time histograms (left), data pooled from four patches, reveal three open states in the absence and four open states in the presence of ethanol (gray lines). Resolution of fits was limited to twice the rise time of the filter (0.3 ms).