The dopamine transporter counter-transports potassium to increase the uptake of dopamine

The dopamine transporter (DAT) facilitates dopamine reuptake from the extracellular space, and thereby terminates neurotransmission and refills cellular stores of dopamine. DAT belongs to the neurotransmitter:sodium symporter (NSS) family, which includes similar transporters for serotonin, norepinephrine, and GABA. A hallmark of NSS proteins is their ability to utilize the energy stored in the inward-directed Na+ gradient to drive the uphill transport of substrate. Decades ago, it was shown that the serotonin transporter also counter-transports K+, but investigations of K+-coupled transport in other NSSs have been inconclusive. Here, we show that the Drosophila dopamine transporter (dDAT) counter-transports K+. We found that ligand binding to both dDAT and human DAT is inhibited by K+ and that the conformational dynamics of dDAT in K+ is highly divergent from both the apo- and Na+-bound conformations. Furthermore, we found that K+ increased dopamine uptake by purified dDAT reconstituted in liposomes, and we visualized, in real-time, Na+ and K+ fluxes in single proteoliposomes using fluorescent ion indicators. Our results expand on the fundamentals of dopamine transport and prompt a reevaluation of the impact of K+ on other NSSs, including whether K+ counter-transport is a common mechanism for this pharmacologically important protein family.

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT

The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy of transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the neurotransmitter:sodium symporter (NSS) family, Na+stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na+transport. Substrate binding, in a step essential for coupled transport, must overcome the effect of Na+, allowing intracellular substrate and Na+release from an inward-open state. However, the specific elements of the protein that mediate this conformational response to substrate binding are unknown. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na+on conformation requires the Na2 site, where it influences conformation by fostering interaction between two domains of the protein. Here, we used cysteine accessibility to measure conformational changes of LeuT inEscherichia colimembranes. We identified a conserved tyrosine residue in the substrate binding site required for substrate to convert LeuT to inward-open states by establishing an interaction between the two transporter domains. We further identify additional required interactions between the two transporter domains in the extracellular pathway. Together with our previous work on the conformational effect of Na+, these results identify mechanistic components underlying ion–substrate coupling in NSS transporters.

The LeuT-fold neurotransmitter:sodium symporter MhsT has two substrate sites

Crystal structures of the neurotransmitter:sodium symporter MhsT revealed occluded inward-facing states with one substrate (Trp) bound in the primary substrate (S1) site and a collapsed extracellular vestibule, which in LeuT contains the second substrate (S2) site. In n-dodecyl-β-d-maltoside, the detergent used to prepare MhsT for crystallization, the substrate-to-protein binding stoichiometry was determined by using scintillation proximity to be 1 Trp:MhsT. Here, using the same experimental approach, as well as equilibrium dialysis, we report that in n-decyl-β-d-maltoside, or after reconstitution in lipid, MhsT, like LeuT, can simultaneously bind two Trp substrate molecules. Trp binding to the S2 site sterically blocks access to a substituted Cys at position 33 in the S2 site, as well as access to the deeper S1 site. Mutation of either the S1 or S2 site disrupts transport, consistent with previous studies in LeuT showing that substrate binding to the S2 site is an essential component of the transport mechanism.

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT

The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy in transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the Neurotransmitter:Sodium Symporter (NSS) family, Na+ stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na+ transport. In a step essential for coupled transport, substrate binding must overcome the effect of Na+, allowing intracellular substrate and Na+ release from an inward-open state. However, it is unclear which specific elements of the protein mediate this conformational response to substrate binding. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na+ on conformation occurs at the Na2 site, where it influences conformation by fostering interaction between two domains of the protein (JBC 291: 1456, 2016). Here, we identify a conserved tyrosine residue in the substrate binding site required for substrate to enable conversion to inward-open states by establishing an interaction between the two transporter domains. We further identify additional interactions between the two transporter domains in the extracellular pathway that are required. Together with our previous work on the conformational effect of Na+, these results identify mechanistic components underlying ion-substrate coupling in NSS transporters.

Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.