| Popis: |
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. |
