Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms

Uptake of system L amino acid substrates into isolated placental plasma membrane vesicles in the absence of opposing side amino acid (zero-trans uptake) is incompatible with the concept of obligatory exchange, where influx of amino acid is coupled to efflux. We therefore hypothesized that system L amino acid exchange transporters are not fully obligatory and/or that amino acids are initially present inside the vesicles. To address this, we combined computational modeling with vesicle transport assays and transporter localization studies to investigate the mechanism(s) mediating [14C]L-serine (a system L substrate) transport into human placental microvillous plasma membrane (MVM) vesicles. The carrier model provided a quantitative framework to test the 2 hypotheses that L-serine transport occurs by either obligate exchange or nonobligate exchange coupled with facilitated transport (mixed transport model). The computational model could only account for experimental [14C]L-serine uptake data when the transporter was not exclusively in exchange mode, best described by the mixed transport model. MVM vesicle isolates contained endogenous amino acids allowing for potential contribution to zero-trans uptake. Both L-type amino acid transporter (LAT)1 and LAT2 subtypes of system L were distributed to MVM, with L-serine transport attributed to LAT2. These findings suggest that exchange transporters do not function exclusively as obligate exchangers.—Widdows, K. L., Panitchob, N., Crocker, I. P., Please, C. P., Hanson, M. A., Sibley, C. P., Johnstone, E. D., Sengers, B. G., Lewis, R. M., Glazier, J. D. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms

Computational modelling of amino acid transfer interactions in the placenta

Placental amino acid transport is essential for fetal development during pregnancy. Impaired transport has been associated with restricted fetal growth that can potentially lead to diseases in later life. However, quantitative understanding of placenta transport remains limited and therefore requires investigation. The aim of this study was to develop a computational framework that can represent the amino acid transport system in the placenta as a whole.The transfer of amino acid across the placenta is mediated by a broad array of specific membrane transporters. Mathematical models, based on carrier-mediated transport theory, were developed to mechanistically represent these transporters. These include accumulative transporters, which use secondary active transport driven by the sodium electrochemical potential; exchangers (antiporters), which swap one substrate for another on different sides of the membrane; and facilitative transporters, which transport substrate along its concentration gradient. The transporter models were thoroughly investigated and validated with experimental data with respect to their mechanistic characteristics and parameter sensitivity. Overall, the models were demonstrated to be adequate in representing the specific transporter behaviours.There are 20 amino acids, including 9 essential ones, and over 19 different transporters, all of which act on certain overlapping subsets of these amino acids. All transporters must work interdependently for successful transfer of the required amino acids from the maternal to the fetal side; however, this complex process is not fully understood. A compartmental model of placental amino acid transport incorporating kinetic transporter models was developed and revealed to be able to sufficiently capture the integrated transport system. Modelling results clearly demonstrated how modulating specific transporter activity can increase the transport of certain classes of amino acids, but that this comes at the price of decreasing the transport of others, which could have potential implications for developing new clinical treatment options. This integrated modelling approach along with kinetic models of transporters will help in gaining an improved quantitative understanding of epithelial transport in the placenta and other systems and it is ultimately hoped that this will contribute to the development of clinical applications to intervene or prevent impaired-transport related pathologies.<br/

Computational modelling of placental amino acid transfer as an integrated system

Placental amino acid transfer is essential for fetal development and its impairment is associated with poor fetal growth. Amino acid transfer is mediated by a broad array of specific plasma membrane transporters with overlapping substrate specificity. However, it is not fully understood how these different transporters work together to mediate net flux across the placenta. Therefore the aim of this study was to develop a new computational model to describe how human placental amino acid transfer functions as an integrated system. Amino acid transfer from mother to fetus requires transport across the two plasma membranes of the placental syncytiotrophoblast, each of which contains a distinct complement of transporter proteins. A compartmental modelling approach was combined with a carrier based modelling framework to represent the kinetics of the individual accumulative, exchange and facilitative classes of transporters on each plasma membrane. The model successfully captured the principal features of transplacental transfer. Modelling results clearly demonstrate how modulating transporter activity and conditions such as phenylketonuria, can increase the transfer of certain groups of amino acids, but that this comes at the cost of decreasing the transfer of others, which has implications for developing clinical treatment options in the placenta and other transporting epithelia. The work using this model was published as: Pantichob et al, Computational modelling of placental amino acid transfer as an integrated system, BBA - Biomembranes 2016, accepted, http://dx.doi.org/10.1016/j.bbamem.2016.03.028</span

Restitution characteristics of His bundle and working myocardium in isolated rabbit hearts.

The Purkinje system (PS) and the His bundle have been recently implicated as an important driver of the rapid activation rate after 1-2 minutes of ventricular fibrillation (VF). It is unknown whether activations during VF propagate through the His-Purkinje system to other portions of the the working myocardium (WM). Little is known about restitution characteristic differences between the His bundle and working myocardium at short cycle lengths. In this study, rabbit hearts (n = 9) were isolated, Langendorff-perfused, and electromechanically uncoupled with blebbistatin (10 μM). Pacing pulses were delivered directly to the His bundle. By using standard glass microelectrodes, action potentials duration (APD) from the His bundle and WM were obtained simultaneously over a wide range of stimulation cycle lengths (CL). The global F-test indicated that the two restitution curves of the His bundle and the WM are statistically significantly different (P<0.05). Also, the APD of the His bundle was significantly shorter than that of WM throughout the whole pacing course (P<0.001). The CL at which alternans developed in the His bundle vs. the WM were shorter for the His bundle (134.2±13.1ms vs. 148.3±13.3ms, P<0.01) and 2:1 block developed at a shorter CL in the His bundle than in WM (130.0±10.0 vs. 145.6±14.2ms, P<0.01). The His bundle APD was significantly shorter than that of WM under both slow and rapid pacing rates, which suggest that there may be an excitable gap during VF and that the His bundle may conduct wavefronts from one bundle branch to the other at short cycle lengths and during VF

Boxplot of AUC.

<p>The blue boxplot is for the His bundle group, and the red one is for the WM group.</p

Scatterplot of APD versus DI with fitted lines.

<p>The blue dots and line denote the data points and the fitted line for the His bundle group, and the red dots and lines for the WM group.</p

Restitution characteristics of His bundle and working myocardium in isolated rabbit hearts - Fig 6

<p><b>Action potential duration alternans continuously recorded in the His bundle (A) and 2:1 block in the adjacent working myocardium (B) at the cycle length of 130ms pacing from the His bundle.</b> The spikes in the recordings are the pacing artifacts. In panel A, the APD₉₀ of the His bundle alternated in a long-short pattern. The long APDs ranged from 111.8ms to 113.6ms, while the short ones ranged from 97.1ms to 98.5ms. The working myocardium lost one to one capture when the His bundle had alternans.</p

Action potential durations (APD₉₀) during dynamic pacing in the His bundle (blue dots and curves) and working myocardium (red dots and curves).

<p>As cycle length progressively shortened, a transition from 1:1 capture to 2:1 block with alternation between long and short action potentials occurred.</p

Decremented-pacing protocol for measuring restitution properties.

<p>The initial interval was 300ms and was decremented to 260ms by 20-ms steps. Below 260 ms, it was reduced in 10-ms steps to the target interval, and thereafter kept at the target interval for 30 beats. Each cycle length was in ms.</p

Photograph of the experimental rabbit heart showing anatomical landmarks and impalement sites.

<p>CS, coronary sinus; AVN, atrioventricular node; His, His bundle; RA, right atrium; VS, ventricular septum. Green dot indicates the recording site of the bipolar electrode. Action potentials were recorded from the His bundle (blue dot) and endocardium (red dot), respectively.</p