High-frequency HTR3B variant associated with major depression dramatically augments the signaling of the human 5-HT3AB receptor

Abstract

The 5-hydroxytryptamine-3 (5-HT3) receptor mediates the fast excitatory neurotransmission of serotonin and is known to mediate the nausea/emesis induced by radio/chemotherapy and anesthetics. A polymorphism encoding the variation Y129S in the 5-HT3B subunit exists in high frequency in the general population and has been shown to be inversely correlated to the incidence of major depression in women. We show that 5-HT3AB(Y129S) receptors exhibit a substantially increased maximal response to serotonin compared with WT receptors in two fluorescence-based cellular assays. In electrophysiological recordings, the deactivation and desensitization kinetics of the 5-HT3AB(Y129S) receptor are 20- and 10-fold slower, respectively, than those of the WT receptor. Single-channel measurements reveal a 7-fold-increased mean open time of 5-HT3AB(Y129S) receptors compared with WT receptors. The augmented signaling displayed by 5-HT3AB(Y129S) receptors may confer protection against the development of depression. The variant also may influence the development and/or treatment of nausea and other disorders involving 5-HT3 receptors. Thus, the impact of the high-frequency variant 5-HT3B(Y129S) on 5-HT3AB receptor signaling calls for a search for additional phenotypes, and the variant may thus aid in establishing the role of the 5-HT3AB receptor in pathophysiology.

Fig. 1.

The 5-HT_3B(Y129S) variant augments the serotonin-induced maximal signaling of the heteromeric 5-HT_3AB receptor. tsA cells were transiently transfected with human 5-HT_3A and human 5-HT_3B, 5-HT_3B(Y129S), or a 1:1 mixture of 5-HT_3B and 5-HT_3B(Y129S) (A/B ratio 1:4). (A–C) The concentration–response curves for serotonin in the FMP assay (A), the concentration–inhibition curves for tropisetron in the FMP assay (B), and the concentration–response curves for serotonin in the [Ca²⁺]_i assay (in the presence of 15 mM extracellular Ca²⁺) (C). ΔFU is the peak fluorescent response after application of serotonin minus baseline fluorescence. (D) Specific [³H]GR65630 binding to tsA cells transfected as described for the functional assays. Specific surface binding was determined by using intact cells, whereas specific binding to surface expressed and intracellular receptors was determined by using cells permeabilized with saponin. (E) Expression levels of myc-5-HT_3A and HA-5-HT_3B subunits in tsA cells (transfected with A/B cDNA ratios 1:4) determined in ELISA experiments (absorbance reading at 450 nm). Surface expressions were determined by using intact cells, whereas total expressions were determined in Triton X-100-permeabilized cells. All graphs display data from a representative experiment, and each data point or bar represents the mean ± SEM of duplicate or triplicate measurements.

Fig. 2.

Concentration–response curves for serotonin as determined in whole-cell recordings from HEK293 cells transfected with 5-HT_3A cDNA together with either 5-HT_3B or 5-HT_3B(Y129S) cDNA. Current amplitudes (I) are expressed as a percentage of those evoked at the respective receptors by application of 100 μM serotonin. Data points are mean ± SEM from three to six cells.

Fig. 3.

The 5-HT_3B(Y129S) variant slows deactivation and desensitization of the 5-HT_3AB receptor as determined in whole-cell recordings. (A) Examples of normalized average currents evoked by 300 μM serotonin applied for 5 ms to HEK293 cells expressing 5-HT_3AB and 5-HT_3AB(Y129S) receptors. (B) Bar graph showing the ≈20-fold-slower time constant for deactivation in receptors incorporating the 5-HT_3B(Y129S) subunit compared with WT (n = 3). (C) Normalized currents from 5-HT_3AB and 5-HT_3AB(Y129S) channels showing the different decay times in the continued presence of 300 μM serotonin. (D) Desensitization is slowed ≈10-fold in 5-HT_3AB(Y129S) receptors compared with 5-HT_3AB receptors (n = 3).

Fig. 4.

Single-channel properties of WT 5-HT_3AB and 5-HT_3AB(Y129S) channels. (A) Examples of single-channel recordings from HEK293 cells expressing either WT 5-HT_3AB (Upper) or 5-HT_3AB(Y129S) channels (Lower). Recordings made from cell-attached patches in the presence of 300 μM serotonin at a membrane potential of −60 mV. (B) Current–voltage relationship of WT 5-HT_3AB and 5-HT_3AB(Y129S) channels with slope conductance derived from the linear regression. (C) Histograms of open dwell times for open-channel events pooled from multiple patches at a membrane potential of −60 mV. The distribution was best described by the sum of four exponentials (thick dark line). Open times and their relative proportions are listed in Table 2.