Kinetics of sodium ions release from Na2 binding site
The release of one sodium ion from the crystallographically determined sodium binding site Na2 had been identi ed as an initial step in the transport cycle which prepares the transporter for substrate translocation by stabilizing an inward-open conformation. We have constructed Markov State Models (MSMs) from extensive molecular dynamics simulations of human DAT (hDAT) to explore the mechanism of this sodium release. Our results quantify the release process triggered by hydration of the Na2 site that occurs concomitantly with a conformational transition from an outward-facing to an inward-facing state of the transporter. Our results reveal the importance of various modes of interaction of the N-terminus of hDAT in controlling the pathways of release.
The release of sodium ion (yellow sphere) from binding site after hydration of the binding site with water (red, blue, and purple spheres). Dopamine (in licorice) stays in the binding site.
Mechanism of allosteric couplings involved in the release of sodium
Allostery plays a crucial role in the mechanism of neurotransmitter-sodium symporters, such as the human dopamine transporter. Here we investigated the molecular mechanism that couples the transport-associated inward release of the sodium ion from the Na2 site to intracellular gating. We applied a combination of the thermodynamic coupling function (TCF) formalism and Markov state model analysis to a 50-µs data set of molecular dynamics trajectories of the human dopamine transporter. Our TCF approach reveals a complex landscape of thermodynamic coupling between sodium release and inward-opening, and identifies diverse, yet well-defined roles for different sodium coordinating residues.
Razavi, A. M., Khelashvili G., and Weinstein H. (2017) “A Markov State-based Quantitative Kinetic Model of Sodium Release from the Dopamine Transporter.” Scientific Reports 7, 40076. doi:10.1038/srep40076
Michael V. LeVine, Michel A. Cuendet, Asghar M. Razavi, George Khelashvili, and Harel Weinstein (2017) “Thermodynamic Coupling Function Analysis of Allosteric Mechanisms in the Human Dopamine Transporter”. Biophysical Journal, https://doi.org/10.1016/j.bpj.2017.10.030
Razavi, A. M., Khelashvili G., and Weinstein H. (2017, February) “A Markov State-based Quantitative Kinetic Model of Sodium Release from the Dopamine Transporter.” Biophysical Society Conference, New Orleans, LA