OR03-06 Transmembrane Water Channels Within The Tsh Receptor

Abstract

Abstract Disclosure: R. Latif: None. T.F. Davies: Advisory Board Member; Self; Board of Kronus Inc (Starr, ID, USA);. M. Mezei: None. Using our recently reported hydrated model of the TSH receptor transmembrane (TM) region (Endocrinology 2021,162:7;1-12), we have now identified a network of putative TM water channels within this structure. By analyzing the molecular simulation trajectory for water molecules crossing the plasma membrane embedded TSHR we found evidence of putative water channels suggestive of active transport. To ascertain the biological activity of such TSHR channels we examined the movement of water down osmotic gradients using calcein aceto-oxymethyl ester (Calcein-AM) fluorescence in rat thyrocyte cells (FRTL5). Calcein-AM is a membrane permeant fluorescent dye that is quenched in a concentration-dependent manner and is, therefore, an effective probe of cell volume changes that would result from water movement across the cell membrane. Since thyroid cells are known to contain aquaporin (AQP) channels, principally AQP4, we first blocked these channels (using 3μM of TGN020) prior to examining the effects of TSHR activation using TSH (10-103 µU/ml), stimulating TSHR monoclonal antibodies (mAbs MS1 and M22 at 10μg/ml and 1μg/ml respectively) and TSHR-specific small molecule allosteric agonists (MS437 and MS438 at 10µM) and measured the changes in fluorescence quenching. D-Mannitol at 400mM was used as the hypertonic medium to induce cell shrinkage measured by continuous fluorescence measurements with read intervals of 50msec. Analyzing the normalized fluorescence of cells treated with TSH in a dose-dependent manner we observed ∼25% quenching with 103 µU/ml of TSH. On blocking the binding of TSH using a TSHR blocking mAb K1-70 (1μg/ml), we found marked de-quenching. Furthermore, using equimolar concentrations of stimulating TSHR antibodies the quenching was even more significantly enhanced to 54-60%. These data suggested that ligands that activate the TSHR by binding to the TSHR ectodomain could influence the TM water transport. However, using the allosteric activators, which act at the TM, we also showed similar changes indicating this was not ectodomain dependent. These data confirm the presence of the modeled putative TSHR water channels indicating that TSH and TSHR autoantibodies have an important role in regulating TM water passage. Presentation: Thursday, June 15, 2023