A System for Assessing Dual Action Modulators of Glycine Transporters and Glycine Receptors

Reduced inhibitory glycinergic neurotransmission is implicated in a number of neurological conditions such as neuropathic pain, schizophrenia, epilepsy and hyperekplexia. Restoring glycinergic signalling may be an effective method of treating these pathologies. Glycine transporters (GlyTs) control synaptic and extra-synaptic glycine concentrations and slowing the reuptake of glycine using specific GlyT inhibitors will increase glycine extracellular concentrations and increase glycine receptor (GlyR) activation. Glycinergic neurotransmission can also be improved through positive allosteric modulation (PAM) of GlyRs. Despite efforts to manipulate this synapse, no therapeutics currently target it. We propose that dual action modulators of both GlyTs and GlyRs may show greater therapeutic potential than those targeting individual proteins. To show this, we have characterized a co-expression system in Xenopus laevis oocytes consisting of GlyT1 or GlyT2 co-expressed with GlyRα1. We use two electrode voltage clamp recording techniques to measure the impact of GlyTs on GlyRs and the effects of modulators of these proteins. We show that increases in GlyT density in close proximity to GlyRs diminish receptor currents. Reductions in GlyR mediated currents are not observed when non-transportable GlyR agonists are applied or when Na+ is not available. GlyTs reduce glycine concentrations across different concentration ranges, corresponding with their ion-coupling stoichiometry, and full receptor currents can be restored when GlyTs are blocked with selective inhibitors. We show that partial inhibition of GlyT2 and modest GlyRα1 potentiation using a dual action compound, is as useful in restoring GlyR currents as a full and potent single target GlyT2 inhibitor or single target GlyRα1 PAM. The co-expression system developed in this study will provide a robust means for assessing the likely impact of GlyR PAMs and GlyT inhibitors on glycine neurotransmission.

A System for Assessing Dual Action Modulators of Glycine Transporters and Glycine Receptors

Reduced inhibitory glycinergic neurotransmission is implicated in a number of neurological conditions such as neuropathic pain, schizophrenia, epilepsy and hyperekplexia. Restoring glycinergic signalling may be an effective method of treating these pathologies. Glycine transporters (GlyTs) control synaptic and extra-synaptic glycine concentrations and slowing the reuptake of glycine using specific GlyT inhibitors will increase glycine extracellular concentrations and increase glycine receptor (GlyR) activation. Glycinergic neurotransmission can also be improved through positive allosteric modulation (PAM) of GlyRs. Despite efforts to manipulate this synapse, no therapeutics currently target it. We propose that dual action modulators of both GlyTs and GlyRs may show greater therapeutic potential than those targeting individual proteins. To show this, we have characterized a co-expression system in Xenopus laevis oocytes consisting of GlyT1 or GlyT2 co-expressed with GlyRα1. We use two electrode voltage clamp recording techniques to measure the impact of GlyTs on GlyRs and the effects of modulators of these proteins. We show that increases in GlyT density in close proximity to GlyRs diminish receptor currents. Reductions in GlyR mediated currents are not observed when non-transportable GlyR agonists are applied or when Na+ is not available. GlyTs reduce glycine concentrations across different concentration ranges, corresponding with their ion-coupling stoichiometry, and full receptor currents can be restored when GlyTs are blocked with selective inhibitors. We show that partial inhibition of GlyT2 and modest GlyRα1 potentiation using a dual action compound, is as useful in restoring GlyR currents as a full and potent single target GlyT2 inhibitor or single target GlyRα1 PAM.

Binding and transport of D-aspartate by the glutamate transporter homolog GltTk

Mammalian glutamate transporters are crucial players in neuronal communication as they perform neurotransmitter reuptake from the synaptic cleft. Besides L-glutamate and L-aspartate, they also recognize D-aspartate, which might participate in mammalian neurotransmission and/or neuromodulation. Much of the mechanistic insight in glutamate transport comes from studies of the archeal homologs GltPh from Pyrococcus horikoshii and GltTk from Thermococcus kodakarensis. Here, we show that GltTk transports D-aspartate with identical Na+: substrate coupling stoichiometry as L-aspartate, and that the affinities (Kd and Km) for the two substrates are similar. We determined a crystal structure of GltTk with bound D-aspartate at 2.8 Å resolution. Comparison of the L- and D-aspartate bound GltTk structures revealed that D-aspartate is accommodated with only minor rearrangements in the structure of the binding site. The structure explains how the geometrically different molecules L- and D-aspartate are recognized and transported by the protein in the same way.

A general method for determining secondary active transporter substrate stoichiometry

The number of ions required to drive substrate transport through a secondary active transporter determines the protein’s ability to create a substrate gradient, a feature essential to its physiological function, and places fundamental constraints on the transporter’s mechanism. Stoichiometry is known for a wide array of mammalian transporters, but, due to a lack of readily available tools, not for most of the prokaryotic transporters for which high-resolution structures are available. Here, we describe a general method for using radiolabeled substrate flux assays to determine coupling stoichiometries of electrogenic secondary active transporters reconstituted in proteoliposomes by measuring transporter equilibrium potentials. We demonstrate the utility of this method by determining the coupling stoichiometry of VcINDY, a bacterial Na+-coupled succinate transporter, and further validate it by confirming the coupling stoichiometry of vSGLT, a bacterial sugar transporter. This robust thermodynamic method should be especially useful in probing the mechanisms of transporters with available structures.