Do Skeletal Dynamics Mediate Sugar Uptake and Transport in Human Erythrocytes?

Abstract

We explore, herein, the hypothesis that transport of molecules or ions into erythrocytes may be affected and directly stimulated by the dynamics of the spectrin/actin skeleton. Skeleton/actin motions are driven by thermal fluctuations that may be influenced by ATP hydrolysis as well as by structural alterations of the junctional complexes that connect the skeleton to the cell's lipid membrane. Specifically, we focus on the uptake of glucose into erythrocytes via glucose transporter 1 and on the kinetics of glucose disassociation at the endofacial side of glucose transporter 1. We argue that glucose disassociation is affected by both hydrodynamic forces induced by the actin/spectrin skeleton and by probable contact of the swinging 37-nm-long F-actin protofilament with glucose, an effect we dub the "stickball effect." Our hypothesis and results are interpreted within the framework of the kinetic measurements and compartmental kinetic models of Carruthers and co-workers; these experimental results and models describe glucose disassociation as the "slow step" (i.e., rate-limiting step) in the uptake process. Our hypothesis is further supported by direct simulations of skeleton-enhanced transport using our molecular-based models for the actin/spectrin skeleton as well as by experimental measurements of glucose uptake into cells subject to shear deformations, which demonstrate the hydrodynamic effects of advection. Our simulations have, in fact, previously demonstrated enhanced skeletal dynamics in cells in shear deformations, as they occur naturally within the skeleton, which is an effect also supported by experimental observations.

Figure 1

Molecular structure of a human erythrocyte junctional complex (JC) (after Lux 2015 (6)). Note that both GLUT1 (the glucose transporter) and band 3 (the anion transporter) are associated with glycophorin C (GlyC), which binds actin. Thus, GLUT1 and band 3 are colocated at the JC. To see this figure in color, go online.

Figure 2

Schematic of the erythrocyte membrane and attached spectrin skeleton. Note that the major sites of attachment, including JCs, of the skeleton-to-membrane are the glucose transporter, GLUT1, and the anion exchanger band 3. Associated proteins are also depicted and labeled. Compartment 1, the glucose binding site, is conceptually illustrated as being either structurally or functionally associated with the endofacial side of GLUT1; it is colored in a faint yellow. To see this figure in color, go online.

Figure 3

Schematics of the three levels of our multiscale framework. The three levels (i.e., models) are connected through an information-passing algorithm: the Level I model computes the constitutive relations of the Sp (as in (8, 9)) that are then used in the Level II model. Similarly, the Level II model creates the constitutive response (e.g., areal and shear stiffness) of the cytoskeleton that is used in the Level III cell model. To see this figure in color, go online.

Figure 4

Bifurcation of the JC with shear deformations. The parameter λ (see (8, 9) for details) measures the amount of shear deformation of the skeleton, during which the skeleton is stretched by a factor of λ in one direction and compressed by a factor of $1/\lambda$ in the orthogonal direction.

Figure 5

A mode-switching event in thermal fluctuations when the JC is bistable.

Figure 6

Time histories of the penetration distance of a calcium cation into the cell in different conditions. The inset shows a cation within the field of a Gouy-Chapman electrical double layer at a bilipid membrane. To see this figure in color, go online.

Figure 7

(a) Compartment $1\to 2$ kinetic model for glucose uptake and (b) force-affected energy landscape. To see this figure in color, go online.

Figure 8

Glucose bound to a Lys residue on loop 6-7 of GLUT1 (edited from Salas-Burgos et al. (32)). Inset at upper left shows that thermal fluctuations will initially accelerate the glucose-Lys complex until the loop is sufficiently stretched. Note that when stretched, tension T develops in the loop. Note that all dimensions shown are in Å. To see this figure in color, go online.

Figure 9

Breakaway of glucose from the Lys on loop 6-7 of GLUT1. (a) A glucose bound to Lys with loop 6-7, which is initially under zero tension, is shown; the magnitude of the binding force is $f_d$. (b) Imposition of thermal fluctuation and forces f on the Lys-glucose complex are shown. This accelerates the complex and leads to stretching and a back-force on Lys, causing it to decelerate, as in (c). (c) The net force on Lys decays to zero, and the net force on glucose causes rupture on the Lys-glucose bond. Note, all dimensions shown are in Å. To see this figure in color, go online.

Figure 10

Idealization of loop 6-7; the shape of the loop as indicated is only schematic for modeling purposes. The loop is part of the GLUT1 transporter, as shown. The number of residues (abbreviated as “res”) in each section of loop 6-7 is indicated as, e.g., 18res., and the approximate length of each section is in nanometers. The “linear” dimensions of $4\times 11$nm are only meant to convey the approximate area of the loop. The Lys-glucose bond is conceived as a charge-dipole bond. F-actin protofilaments are ∼35 nm long with diameters in the range of 7–9 nm [?]. To see this figure in color, go online.

Figure 11

Idealization of the loop 6-7 binding model of Glc to Lys. The Glu-Lys complex is subjected to a combined thermal-hydrodynamic force $2f$, assumed for simplicity to be equally partitioned between Glc and Lys. As loop 6-7 is stretched, it imposes a back-force on Lys; the spring-like response of loop 6-7 is represented by $k_{\ell }$. When loop 6-7 is critically stretched, Lys is decelerated, and Glc may break away. The charge-dipole Glc-Lys bond geometry is sketched in the upper right inset. To see this figure in color, go online.

Figure 12

Idealization of the loop 6-7 binding model of Glc to Lys. The Glc-Lys complex is subjected to a combined thermal-hydrodynamic force $2f$, assumed for simplicity purposes to be equally partitioned between Glc and Lys. As loop 6-7 is stretched, it imposes a back-force on Lys; the spring-like response of loop 6-7 is represented by $k_{\ell }$. When loop 6-7 is critically stretched, Lys is decelerated, and Glc may break away. To see this figure in color, go online.

Figure 13

Thermal fluctuation of the actin protofilament illustrated by combinations of its snapshots at various instants. The small bead represents the initial location of the glucose. The pointed end of the protofilament is shown in blue. To see this figure in color, go online.

Figure 14

(Left) Time course of glucose utilization by erythrocytes suspended in plasma at different shear rates. (Middle) The erythrocyte glucose consumption is shown at different shear rates, calculated based on the slopes of the glucose concentration using least-square linear regression. (Right) The absolute values of erythrocyte glucose consumption at different shear rates are shown. To see this figure in color, go online.

Figure 15

Effects of plasma viscosity on erythrocyte glucose consumption at different shear rates. These experiments confirm that mechanical stress strongly enhances the consumption of glucose by RBCs under physiological conditions. The results show a good hydrodynamic correlation with the capillary number defined as $\eta R\dot{\gamma }/\mu$; here, η is viscosity, R is the effective cell radius, $\dot{\gamma }$ is the shearing rate, and μ is the cell stiffness.

Figure 16

(a) Glucose uptake measured at $2.3\pm {0.2}^{\circ }$C vs. time. (b) Temperature is over 250 s. (c) Uptake is shown over first the 30 s, showing linearity. To see this figure in color, go online.