FRET-based characterization of K264A, D345A, and Y335Amutants in the human dopamine transporter

Abstract

IntroductionDopaminergic signaling has an indispensable role in neurological disorders such as schizophrenia, bipolar disorder, and attention deficit disorder. Duration of dopaminergic signaling, and therefore its regulation, is tightly coupled to the dopamine transporter (DAT), which mediates rapid uptake of dopamine (DA) from the synaptic cleft (Amara and Kuhar, 1993).The transporter belongs to the family of neurotransmitter transporters also known as sodium symporters (NSSs). These proteins form a large family encompassing other neurotransmitter transporters for norepinephrine, serotonin, glycine, and GABA. All these transporters have the common property of utilizing the energy of the Na + gradient to carry the substrate uphill (Rudnick, 1997). In addition to dopamine transport, DAT can also bind several psychostimulant drugs, including amphetamine (AMPH) and cocaine. Indeed, DAT may be the primary target for the rewarding properties of these widely abused psychostimulants.The N-terminus of transporters has indispensable roles in monoamine transporters' functions. The N-terminus is the primary target of various kinases such as protein kinase A (PKA), protein kinase C (PKC), and Ca 2+ /calmodulin-dependent kinase (CaM kinase II); however, the role of phosphorylation by these kinases is speculative. The first N-terminal 22 amino acids have previously been shown to be phosphorylated by PKC (Granas et al., 2003). When these 22 amino acids were removed from the N-terminal, DAT (del 22 DAT) phosphorylation was abolished without affecting PKC-induced internalization. In HEK-293 cells stably expressing del-22 DAT, AMPH-induced DA efflux, but not DA uptake, was substantially reduced. Phosphorylation of two serine residues at the N-terminus, Ser 7 and Ser 12, was mainly found to be essential for this response (Khoshbouei et al., 2004;Kahlig et al., 2005). Mutation of serine 7, a site for PKCmediated phosphorylation, strongly reduced cocaine analog affinity and also changed the zinc modulation of cocaine analog 2β-carbomethoxy-3β(4-fluorophenyl) tropane (CFT) binding, with opposite results in Ala and Asp mutations (Moritz et al., 2013). Other protein kinases also seem to affect nearby serine/threonine residues and phosphorylation of these presumably acts as a modulator of conformational equilibria and inhibitor binding.