Analysis of missense mutations in organic cation transporter

Monoaminergic neurotransmitters act at their cognate receptors and mediate synaptic transmission. Two types of neurotransmitter transporters actively contribute to end signalling by removing neurotransmitters from the synapse: these are the i) uptake-1 transporters (high-affinity - low capacity) and the ii) uptake-2 transporters (low-affinity - high-capacity), the polyspecific organic cation transporter 3 (OCT3). Twenty-six missense mutations have been identified in OCT3 (OCT3-MUT) from a Danish cohort of patients suffering from distinct psychiatric disorders. The aim of the current grant proposal is to establish knowledge of the structure-function relationship in OCT3 at an atomic level and to ascertain the impact of the identified mutations on OCT3 function. The proposed research strategy attempts to compare OCT3 wildtype (OCT3-WT) and OCT3-MUT on the basis of: (i) structural approaches and computational homology models to examine structural transport dynamics on validated 3D-models, (ii) in vitro activity to develop an understanding of the functional consequences of the missense mutations at the molecular level, and (iii) expression levels and oligomerization properties, to identify the interplay between mutations, oligomerisation, amount of surface expressed OCT3-WT/MUT and function. Experimental in vitro approaches will employ biochemical tracer flux experiments and several microscopy methods to assess transporter expression in the cells and oligomerization on the cell surface. We will combine several approaches in an iterative strategy: We will establish homology models of OCT3-WT and OCT3-MUT by extensive molecular dynamics simulations thereon to evaluate structural dynamics and functional consequences of the mutations. The computational methods will integrate experimental datasets to establish an experimentally verified OCT3-WT homology model and to understand disease-causing mutations at the molecular level. OCT3 is an essential, but understudied transporter, which may become an important clinical target for neurological diseases. Minimal outcome of this application is a functional characterization of OCT3-WT and OCT3-MUT, as done for uptake-1 transporters. The vision is to establish a comprehensive structure- function relationship based on knowledge of OCT3-WT transport function and oligomerization at an atomic level.

Understanding OCT3: Unraveling Its Structure and Function for Future Therapeutic Advances Our project aims to better understand a key protein in the human body called Organic Cation Transporter 3 (OCT3). This transporter plays a crucial role in moving important molecules-such as neurotransmitters dopamine and serotonin-across cell membranes. These molecules regulate mood, cognition, and various other physiological functions. A detailed understanding of how OCT3 works could have significant implications for developing new treatments for mental health conditions, cardiovascular diseases, and drug interactions. We successfully created stable cell lines containing different genetic variants (mutants) of OCT3 and tested how well they transport specific substances. Some mutant versions of the transporter showed no activity, while others were hyperactive compared to the normal (wild-type) transporter. Using advanced imaging techniques, we discovered that the hyperactive transporters were simply present in greater amounts on the cell surface, while others had defects in how they were processed within the cell. To explore whether these defects could be corrected, we plan to test a known drug, 4-phenylbutyrate, which has been successfully used for treating other diseases caused by similar protein misfolding issues. A major milestone of our project was solving the three-dimensional structure of OCT3 using an advanced imaging method called cryo-electron microscopy (cryo-EM). In collaboration with an international research team, we successfully determined the structure of OCT3 in three different states: unbound (apo), bound to the inhibitor decynium-22, and bound to the hormone corticosterone. This was a breakthrough, as it provides the first-ever high-resolution model of OCT3, laying the foundation for future studies. With this structural information, we used sophisticated computer simulations to study how OCT3 interacts with its natural substrates, dopamine and serotonin, as well as various inhibitors. By running extensive molecular dynamics simulations, we uncovered key differences in how these molecules interact with specific regions of the protein. Our findings suggest that in order for OCT3 to function properly, certain molecular interactions must take place-interactions that inhibitors appear to disrupt. We are now extending our simulations to observe how OCT3 undergoes structural changes over time and to identify new molecules that could regulate its function. These insights could lead to the development of drugs that either enhance or block OCT3 activity, which may have therapeutic benefits for neurological and cardiovascular disorders. By combining laboratory experiments, advanced imaging, and computational modeling, our project is bringing us closer to understanding OCT3 at an unprecedented level. This knowledge could pave the way for future medical breakthroughs, improving treatments for conditions where OCT3 plays a critical role.