Spontaneous Inward Opening of the Dopamine Transporter is Triggered by PIP2-Regulated Dynamics of the N-Terminus

Abstract

Valvular interstitial cells (VICs) play a critical role in the pathophysiology of heart valves. The objective of this study was to investigate the roles of different subcellular structures of the VICs, especially a-SMA stress fibers, to the VIC mechanical responses under different mechanical loading conditions and activation states. We modeled the VIC as a continuum with two distinct domains: the cell nucleus and cytoplasm. The nucleus was modeled as an incompressible neo-Hookean material while the cytoplasm was modeled as a mixture of two solid phases: the basal, isotropic cytoskeleton phase and a-SMA stress fiber phase, which exhibit some orientations at each point described by an orientation density function. The a-SMA stress fibers also exhibit passive elastic and active contractile responses in the direction of the orientations. We developed VIC mechanical model, which integrated the data from two experiments: micropipette aspiration (MA) and atomic force microscopy (AFM) of the aortic VIC (AVIC) and pulmonary VIC (PVIC) that each exhibits different expression levels of the a-SMA and contraction strength. In the MA experiment, VICs are in inactivated states while in AFM experiment, VICs are in activated states, exhibiting higher level of a-SMA expression and contraction. Thus, using our model in conjunction with the experimental data, we investigated how the expression level and active contraction of the a-SMA fibers affect the mechanical responses of the VICs. We implemented our model on the finite element method. We determined a~10-fold difference in the contractions strength between the AVICs and PVICs, implying that not only the expression level of the a-SMA fibers but also the contraction level increases from PVICs compared to AVICs. The higher expression of the a-SMA fibers may facilitate more efficient contraction and increased ability to synthesize collagen and other ECM components.

Combined in vitro and in silico approaches to the assessment of stimulant properties of novel psychoactive substances – The case of the benzofuran 5-MAPB

“…We present here, on the basis of the crystal structure from Drosophila melanogaster dopamine transporter (dDAT) (PDB ID: 4XP1) (Wang et al, 2015), a molecular model of 5-MAPB bound to rat DAT (rDAT), in comparison with that of dopamine, amphetamine, 5-APB, MDMA, cocaine and RTI-121 also bound to rDAT. Our studies compare the structural changes produced by various ligands binding to rDAT to the already ongoing work in hDAT models (Hamilton et al, 2013;Hansen et al, 2014;Khelashvili et al, 2015aKhelashvili et al, , 2015b since it is the primary animal model used in functional studies and pre-clinical drug abuse testing.…”

“…The construction of the homology model of rDAT (rDAT dDAT ) in complex with dopamine uses established protocols previously described for the construction of a human DAT (hDAT) model (Hamilton et al, 2013;Hansen et al, 2014;Khelashvili et al, 2015aKhelashvili et al, , 2015b. Briefly, as the template, the homology model uses the recent crystal structure of the neurotransmitter sodium symporter (NSS) DAT from Drosophila melanogaster (dDAT) bound to dopamine (PDB ID: 4XP1) (Wang et al, 2015).…”

“…', n.d.). Dopamine was then re-docked into this prepared model of rDAT dDAT as described earlier (Khelashvili et al, 2015a(Khelashvili et al, , 2015b.…”

“…cocaine, amphetamine and RTI-121 were prepared using established protocols (Khelashvili et al, 2015a(Khelashvili et al, , 2015b.…”

“…The previously well-equilibrated structure of the occluded hDAT dDAT model (homology model of the full-length hDAT based on dDAT X-ray structure 4M48 with added N-and C-terminal domains) in complex with dopamine and in a corresponding membrane environment (Khelashvili et al, 2015b) . After a short (0.5ns) equilibration, this new system was subjected to unbiased MD simulations totalling ~250ns.…”