Tina K. Machu - US grants

The function of the 5-hydroxytryptamine3 (5-HT3) receptor is enhanced by alcohols, but the mechanism(s) of action remains unknown. In addition, regulation of the 5-HT3 receptor by post-translational modification events, such as phosphorylation, has not been well characterized. To date, one subunit of the 5-HT3 receptor has been cloned, and it possesses multiple consensus sequence sites for protein kinase dependent phosphorylaiton. Previous work with other ligand-gated ion channels, namely the GABA A and NMDA receptors, has suggested that protein kinase C may be involved in mediating these receptors' responsiveness to ethanol. Therefore, it is reasonable to speculate that kinases may modulate the responsiveness of the 5-HT 3 receptor to alcohols. In the present proposal, functional studies of the 5-HT3 receptor will be performed using Xenopus laevis oocytes as an expression system. Two-electrode voltage clamp electrophysiological recordings of expressed wild-type or mutagenized 5-HT3 receptors will be made. First, the sensitivity of the 5-HT3 receptor to alcohols will be measured. Next, the ability of serine/threonine kinases to alter 5-HT3 receptor function will be addressed. To determine which amino acid(s) are substrates for these kinases, candidate serines in consensus sequences for protein kinase dependent phosphorylation in the receptor will be mutated to nonphosphorylatable residues. Kinases which alter 5-HT3 receptor function will be examined for their ability to change the sensitivity of this receptor to alcohols. The alternative hypothesis, that tonic kinase activity underlies the 5-HT3 receptor's sensitivity to alcohols, will be tested by measuring alcohol modulation of 5-HT3 receptors with mutagenized consensus sequence sites for phosphorylation. Thus, the ultimate goal of the proposal is to determine if phosphorylation events alter the function of the 5-HT3 receptor and its modulation by alcohols. A better understanding of the regulation of the 5-HT3 receptor is important, given that this receptor may be involved in mediating the rewarding actions of drugs of abuse such as ethanol.

Ethanol and higher n-chain alcohols stimulate homomeric 5- Hyrdroxytryptamine3A receptor (5-HT3A) function. Work on this receptor, as well as studies in other laboratories on related ligand-gated ion channels, suggests that the second transmembrane domains (TM2) of these receptors play a critical role in alcohol action. However, it remains unknown whether identified residues in TMZ are part of a critical binding domain for alcohols or whether they play a key role in the transduction of n-chain alcohol action. A third possibility is that a mutation in TM2 could obscure the effects of alcohols through a kinetic mechanism. The major goal of this proposal is to delineate which of these three mechanisms is responsible for altering the alcohol sensitivity of 5-HT3A receptors mutated in the TM2 domain. The alcohol sensitivity of wild-type and mutant 5-HT3A receptors will be assessed in oocytes with the two-electrode voltage clamp technique and in HEK293 cells with patch-clamp technology and standard perfusion and ultra-fast superfusion drug application methods. Residues lining the channel pore will be identified and eliminated from study through alanine scanning mutagenesis and cysteine substitution methods. Changes in secondary structure will be assessed with tryptophan scanning mutagenesis and discrete Fourier transformation analysis. Residues facing other transmembrane domains will be mutated to amino acids differing in physicochemical properties and will be examined for differences in alcohol action. False positive receptors, whose normal gating properties are changed by mutagenesis, will be identified and eliminated if they have altered responsiveness to battery of 5-HT3A receptor drugs acting at different loci within the receptor. Candidate alcohol binding domains can be preliminarily separated from alcohol transduction sites through correlation of changes in alcohol sensitivity with physicochemical properties of substituted amino acids, changes in channel kinetics, and alteration in alcohol cut-off. Wild-type/mutant titration experiments will be used to determine the minimum number of mutant subunits required per receptor to achieve the mutant alcohol phenotype; a dominant mutant phenotype would suggest that an alcohol transduction site has been identified.

DESCRIPTION (provided by applicant): The 5-Hydroxytryptamine3 (5-HT3) receptor, which may be composed of homomers of A subunits or heteromers of A and B subunits, is the least studied member of the nicotinic acetylcholine receptor family of ligand-gated ion channels. In addition to its role in mediating synaptic transmission in the nervous system, it also regulates gastrointestinal motility and the vomiting reflex. The recently published crystal structure of the acetylcholine binding protein has identified the generalized structure of the N-terminal domains of this superfamily, but the precise three-dimensional configuration of the ligand recognition site and the residues involved in mediating ion channel activation are unknown. The goal of this proposal is to map amino acid residues that contribute to ligand recognition and model their spatial configuration. Mouse and human 5-HT3A receptors possess 84 percent identity at the amino acid level, yet have differential sensitivities to numerous drugs that bind to the ligand recognition site, such as d-tubocurarine (curare) and 3-(2-hydroxy, 4-methoxy-benzylidene)-anabaseine (2-OHMBA). The addition of the B subunit to the A subunit further alters potency of these compounds. Mouse-human and human-mouse chimeras will be constructed and expressed in Xenopus oocytes. Domains responsible for the change in drug action will be assessed with two-electrode voltage clamp electrophysiological recordings. Individual amino acids will then be identified for their roles in conferring sensitivity. Structure-activity relationships of key moieties in the substituted 3-benzylideneanabaseine analogs with identified residues will be assessed. Thermodynamic mutant cycle analysis will pinpoint the specific point of contact of the key drug moiety with the identified amino acid residue in the ligand binding domain. 5-HT3A receptors with C-terminal hexa-histidine tags will be expressed and purified. Receptor will then be photolabeled with [3H]-5-HT and [14C]-2-OHMBA, with and without azido side-chains, and radiolabeled residues will be identified through sequencing. Amino acids identified with photoaffinity labeling and electrophysiological experiments, along with the structures of 5-HT, curare, and benzylidene-anabaseine analogs, will serve as a template for molecular modeling studies of the agonist-recognition site.