Transport dynamics in a glutamate transporter homologue

Abstract

Glutamate transporters are integral membrane proteins that catalyse neurotransmitter uptake from the synaptic cleft into the cytoplasm of glial cells and neurons. Their mechanism of action involves transitions between extracellular (outward)-facing and intracellular (inward)-facing conformations, whereby substrate binding sites become accessible to either side of the membrane. This process has been proposed to entail transmembrane movements of three discrete transport domains within a trimeric scaffold. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging, we have directly observed large-scale transport domain movements in a bacterial homologue of glutamate transporters. We find that individual transport domains alternate between periods of quiescence and periods of rapid transitions, reminiscent of bursting patterns first recorded in single ion channels using patch-clamp methods. We propose that the switch to the dynamic mode in glutamate transporters is due to separation of the transport domain from the trimeric scaffold, which precedes domain movements across the bilayer. This spontaneous dislodging of the substrate-loaded transport domain is approximately 100-fold slower than subsequent transmembrane movements and may be rate determining in the transport cycle.

Figure 1. FRET efficiency changes reflect relative orientations of the transport domains

a, Glt_Ph protomer pairs in symmetrical outward- and inward-facing states viewed within the membrane plane. Trimerization and transport domains are colored wheat and blue, respectively. Bound Asp and Na⁺ ions are emphasized as spheres and colored by atom type. Introduced cysteines are highlighted in cyan with inter-protomer distances above the dotted lines. Magenta arrows mark sites of mutations altering state distributions. b, Labeling and surface-immobilization strategies. c-g, FRET efficiency population histograms for Asp/Na⁺-bound transporters. Introduced mutations are indicated above the panels. The number of molecules analyzed (N) is shown. Population contour plots (left) are color-coded from tan (lowest) to red (highest population) with the color scale shown next to the graphs. In the cumulative population histogram (right), the solid black lines are fits to the sums of individual Gaussian functions (red lines).

Figure 2. Dynamics in the apo and substrate bound transporter

a-b, Shown are smFRET trajectories (blue), acquired for apo (a) and Na⁺/Asp-bound (b) Glt_Ph-N378C. Overlaid are idealizations generated in QuB (red). Arrows mark population averages for the low- (L), intermediate- (I) and high- (H) FRET efficiencies. Data in panel b was collected using 400 ms integration time. c-d, Transition density plots for the apo (c) and Na⁺/Asp-bound (d) transporters show that transitions occur between three distinct FRET states (L, I and H) with an average frequency of ∼0.5 s⁻¹ and ∼0.02 s⁻¹, respectively. Initial and final FRET values for each transition are accumulated into two-dimensional histograms. Color scale is from tan (lowest frequency) to red (highest frequency).

Figure 3. Na⁺ ions and Asp favor the outward facing state

Populations of low- (top, green), intermediate- (center, red) and high- (bottom, blue) FRET states as a function of a, Na⁺ ions added in the presence of 10 μM Asp and b, Asp added in the presence of 2 mM Na⁺. The titrations yielded dissociation constants (K_D s) of 1.6± 0.3 mM and 6.5± 2.5 μM and Hill coefficients of 1.3± 0.3 and 0.9 ± 0.3, respectively. Shown are averages and standard deviations from at least three independent datasets (each containing at least 250 molecules). Solid lines through the data points are the results of global fitting of the data to Hill equation.

Figure 4. Modulation of dynamics by substrate and inhibitor binding

a, SmFRET traces obtained in the presence of 2 mM Na⁺ and increasing Asp concentrations. b, The apparent durations of the dynamic (black) and quiescent (red) periods as a function of Asp concentration. Shown are averages and standard deviations for 3 independent datasets containing at least 250 molecules each. c, A smFRET trace obtained in the presence of 200 mM Na⁺ and 100 μM Asp. Expanded views of the flicker events (shaded in pink) are shown below the trace. d, Survival plot of the observed flickers. Solid line is a fit to a single exponential decay. e, Transition density plots for flicker events in saturating Asp (left) or TBOA (right). Average transition frequencies are ∼ 0.02 s⁻¹ and < 0.005 s⁻¹, respectively. Data in panels d and e were collected with 400 ms integration time.