Ligand binding to 5-HT1A receptors and its regulation by Mg2+ and Mn2+

Abstract

In the first part of this thesis the binding of 5-HT1A receptor specific antagonist [3H]WAY100635 to rat brain membranes was characterized. It was found, that the binding on lower concentrations was considerably slow, and a notable amount of receptors will be inactivated before thermodynamic equilibrium can be reached, yet on higher concentrations rapid increase in reaction rate indicative of cooperative binding was detected. This means that [3H]WAY100635 is an excellent tool for determining receptor number in a given sample, but not a very good ligand in equilibrium binding studies. In the second part of this thesis the influence of Mn2+ in comparison to Mg2+ to signal transduction via 5-HT1A receptors was measured. It was shown that Mn2+ can stabilize greater number of agonist high-affinity binding sites, while the size of this effect depended on the used brain region (different in hippocampal and cortical membranes) and also on the GTPγS concentration. Since GTPγS activates G-proteins, sensitivity to its concentration refers to the involvement of G-proteins in the enhancing regulation by Mn2+. Sf9 cell lines were created for comparison expressing only 5-HT1A receptor or the receptor with Gi or Gs protein. Agonist high affinity binding was achieved only in cell line expressing the receptor with Gi protein, and the sensitivity of this cell line to Mn2+ was similar to cortical preparations but different from hippocampal membranes. To clarify the role of G-proteins on this regulation, influence of Mn2+ compared to Mg2+ to the affinity of guanosine nucleotides (GDP and GTPγS) binding to G-proteins was measured. Mn2+ lowered the affinity of both GDP and GTPγS compared to Mg2+, and the size of the effect was again dependent on used brain region, showing that there are differences in the composition of signaling complexes that are formed with 5-HT1A receptor in different brain regions. Additionally there seem to be differences in the sensitivity of these complexes to Mn2+-ions.Antud töö esimeses osas iseloomustati 5-HT1A retseptori spetsiifilise antagonisti [3H]WAY100635 sidumist roti aju rakumembraanidele ning leiti, et ligandi sidumise kiirus on madalamatel kontsentratsioonidel liiga aeglane, et saavutada termodünaamiline tasakaal enne märkimisväärse hulga retseptorite inaktiveerumist, kõrgematel kontsentratsioonidel toimus aga reaktsiooni järsk kiirenemine, mis viitab sidumise kooperatiivsusele. Seega on [3H]WAY100635 hea ligand 5-HT1A retseptorite kontsentratsiooni määramiseks, kuid selle rakendamine ligandide afiinsuse määramiseks tasakaalulises katses on vägagi piiratud. Töö teises osas uuriti Mg2+ ja Mn2+-ioonide mõju 5-HT1A retseptoriga seotud signaaliülekandele. Leiti et Mn2+ suudab stabiliseerida suurema hulga agonisti kõrge afiinsusega sidumissaite ning efekti suurus on erinevates aju osade rakumembraanides erinev (erinevus hipokampuse ja korteksi membraanide vahel) ning sõltuvuses GTPγSi kontsentratsioonist. Et GTPγS aktiveerib G-valke, viitab kontsentratsioonitundlikkus retseptoriga seotud G-valkude osalusele Mn2+ poolsel agonist kõrge afiinsusega sidumiskohtade suurendamisel. Selle hüpoteesi kinnitamiseks kasutati Sf9 rakuliine, kuhu oli ekspresseeritud kas ainult 5-HT1A retseptor, või 5-HT1A retseptor koos Gi või Gs valguga. Agonisti kõrge affinsusega sidumine saadi vaid preparaadis, kus koos retseptoriga ekspresseerus Gi valk, ning selle preparaadi tundlikkus Mn2+-le sarnanes korteksi membraanidele, kuid erines hipokampuse membraanide tundlikkusest. Et selgitada täpsemalt kuidas G-valgud osalevad Mn2+ poolses regulatsioonis, mõõdeti kuidas muutub guanosiinnukleotiidide (GDP ja GTPγS) afiinsus Mg2+ asendamisel Mn2+-ga, ja leiti et Mn2+ juuresolekul on guanosiinnukleotiidide afiinsus G-valgule oluliselt väiksem, samuti esines efekti suuruse erinevus aju osade vahel. See tulemus näitab, et ilmselt on 5-HT1A retseptoriga moodustuvad signaaliülekande kompleksid erinevates aju osades erinevad, nagu ka nende komplekside tundlikkus Mn2+-ioonile.