The type-3 serotonin (5-HT3) receptor is a target for several clinically useful compounds. Therefore, it is important to understand in detail the molecular composition and functional properties of this ligand-gated ion channel. This proposal focuses on two aspects of this receptor system. First, despite the isolation of a cDNA encoding the 5-HT3 receptor subunit four years ago, it is still unclear whether this subunit reproduces the full spectrum of microscopic properties displayed by the native receptor. This gap in our functional understanding of the 5-HT3 system reflects a trend, since the discovery process for cDNAs encoding neurotransmitter receptor subunits has outpaced our knowledge of the physiological properties of recombinant receptors. Moreover, the lack of a detailed understanding of recombinant ion channel physiology has blunted the promise that molecular cloning holds for linking structure to function. Second, dopamine has recently been found to activate recombinant 5-HT3 receptors, and kinetic and neurochemical arguments can be made that dopamine-activated 5-HT3 receptors might be utilized by neurons during normal function or pathological situations. Thus, an examination of the pharmacology and functional properties of dopamine-activated channels represents an important step towards assessing the role of dopamine as an endogenous agonist in the 5-HT3 receptor system. The experiments outlined in this proposal simultaneously address both of these issues by testing whether the pharmacological and biophysical properties of recombinant rat 5-HT3 ion channels isolated from rat dorsal root ganglia cDNA library match those of native receptors in sympathetic neurons. Proposed experiments explore potential differences between the two agonists and between recombinant versus native receptors in four sets of parameters: (1) desensitization properties (EC50, time course for onset/recovery, sensitivity to modulators), (2) Ca2+ permeability; (3) single channel properties (activation and open time, conductance, and probability of opening) and (4) Ki values for therapeutically useful serotonin and dopamine antagonists. These experiments constitute the first biophysical analysis of ion channel composed of only the recombinant 5-HT3 receptor. The results from both of these lines of experimentation hold implications for the treatment of neuropsychiatric disorders and chemotherapy-induced emesis.
