Differential Effects of Serotonin and Dopamine on Human 5-HT_3AReceptor Kinetics: Interpretation within an Allosteric Kinetic Model

Abstract

Serotonin type 3 (5-HT 3 ) receptors are members of the pentameric Cys-loop superfamily of receptors that modulate synaptic neurotransmission. In response to agonist binding and unbinding, members of this superfamily undergo a series of conformational transitions that define their functional properties. In this study, we report the results of electrophysiological studies using rapid solution exchange designed to characterize and compare the actions of the high-efficacy agonist serotonin and the low-efficacy agonist dopamine on human 5-HT 3A receptors expressed in human embryonic kidney HEK293 cells. In the case of serotonin, receptor activation rates varied with agonist concentration, and deactivation occurred as a single-exponential process with a rate that was similar to the maximal rate of desensitization. Receptors recovered slowly from long desensitizing pulses of serotonin with a sigmoidal time course. In the case of dopamine, receptor activation rates were independent of agonist concentration, receptor deactivation occurred as a complex process that was significantly faster than the maximal rate of desensitization, and recovery from desensitization occurred more quickly than with 5-HT and its time course was not sigmoidal. We developed an allosteric kinetic model for 5-HT 3A receptor activation, deactivation, desensitization, and resensitization. Interpretation of our results within the context of this model indicated that the distinct modulatory actions of serotonin versus dopamine are largely attributable to the vastly different rates with which these two agonists induce channel opening and dissociate from open and desensitized states.

The L293 residue in transmembrane domain 2 of the 5-HT3A receptor is a molecular determinant of allosteric modulation by 5-hydroxyindole

“…A recent study by Solt et al (2007) indicates that desensitization produced by 5-HT dominates the channel closure process even following agonist removal (i.e. our "deactivation" paradigm).…”

“…We performed kinetic simulations of receptor function designed to look at current decay in the continuous presence of 30 μM 5-HT and after agonist removal (Figure 10) to determine if our findings with 5-HI could be completely explained by reduced desensitization rate. We used the allosteric model developed by Solt et al (2007) and the kinetic parameters shown in Table 2. Indeed, we could produce reasonable simulations of the 5-HI-induced prolongation of current decay under either continuous or transient agonist application conditions by decreasing the forward desensitization rate constant (K d+ ).…”

“…In addition, our data support the existence of a second 5-HI binding site at the 5-HT 3A receptor that is not involved in the positive modulation, but likely overlaps with the orthosteric site. A Allosteric kinetic model (from Solt et al 2007) used to simulate 5-HT activation of the receptor and potential 5-HI actions. B Simulated current produced using the allosteric model assuming continuous application of 30 μM 5-HT for 10 sec with k d+ set at an estimated normal level for wild-type 5-HT 3A receptor (black trace) and reduced by ~1 order or magnitude to simulate the effect of 5-HI (Table 2).…”

“…Channel states and most rate constant estimates were as in the allosteric model developed by Solt et al (2007) except where noted. For simulation of continuous 5-HT application, 10 sec of simulated current was generated in the presence of 30 μM 5-HT with all channels initially set in the unliganded, closed state (R).…”

Number and Locations of Agonist Binding Sites Required to Activate Homomeric Cys-Loop Receptors

“…Macroscopic dose-response and single-channel kinetic analyses of homomeric Cys-loop receptors suggest that two to five agonist molecules are required for maximal activation (Amin and Weiss, 1996;Palma et al, 1996;Papke et al, 2000;Mott et al, 2001;Gentet and Clements, 2002;Beato et al, 2002Beato et al, , 2004Solt et al, 2007). However, because dose-response and single-channel measurements cannot directly reveal the number and locations of functional binding sites, this remains a crucial gap in our mechanistic understanding of homomeric Cys-loop receptors.…”