Christof T. Grewer, Ph.D
Professor, Biological and Physical Chemistry
Member, Journal of Biological Chemistry Editorial Board
The long-term goal of our research is to understand the function and the working mechanism of membrane-bound transport proteins. In general, transporters use different types of energy sources to actively move specific substrates, such as inorganic ions or small, organic molecules across the membrane into or out of cells. Recently, significant progress has been made towards our understanding of the molecular architecture through the availability of high-resolution structures of several transporters in several different conformations. However, the structural pictures are static leaving many questions regarding the actual transport mechanism(s) unresolved. Our aim is to combine functional, structural and computational evidence in to obtain an understanding of how these transport proteins work.
Our current research focuses mainly on secondary-active Na+-coupled transporters, which are energized by coupling of substrate transport to the cotransport of sodium ions down their electrochemical potential gradient across the membrane. Neurotransmitter transporters and amino acid transporters belong to this class of transport proteins. The systems currently investigated are glutamate transporters, which contribute to the removal of the excitatory neurotransmitter glutamate from the synapse, and neutral amino acid transporters (SNATs, ASCTs), which catalyze import or export of glutamine and other important neutral amino acids into or from cells.
In many cases, membrane transport is associated with stationary or transient transport of charge. We measure this charge transport with electrophysiological techniques, such as current recording from transporter-expressing, voltage-clamped whole cells or excised inside-out patches. In order to investigate transient charge transport, we perturb a pre-existing transporter steady state by applying voltage or rapid substrate concentration jumps and subsequently measuring the kinetics of the relaxation to a new steady state with a sub-millisecond time resolution. A hypothetical transport mechanism (see Figure) combines evidence from such pre-steady-state functional data with structural information for the glutamate transporters. This mechanism predicts that two structural changes are associated with transmembrane glutamate movement: 1) The closing of an external gate after substrate binding, and 2) the subsequent opening of an internal gate, allowing dissociation of substrate to the cytoplasm.We also apply transition state theory to the pre-steady-state kinetics of the transporters. This allows us to get a better understanding of the nature of the structural changes and/or diffusional processes that are associated with transport. In addition to investigating the transport mechanism of wild-type transporters, rapid kinetic studies are extended to transporters that are fused to fluorescent proteins or site-specifically mutated by using standard molecular biological techniques. The combination of these techniques allows us to understand the relationship between the structure and the function of the transport proteins and to predict potential cation binding sites.
To apply substrate concentration jumps on a sub-millisecond time scale, amino acids or neurotransmitters are photochemically released from a photolabile, inactive caged precursor (caged amino acid) by a brief pulse of laser light. When using a suitable caging group, for example the α-carboxyl-o-nitrobenzyl caging group (CNB), photolysis takes place within 100 μs. Our lab is actively involved in developing new photolabile caging groups and caged amino acids. We have recently synthesized and applied caged alanine and proline derivatives.
We are also interested in developing pharmacological tools for the investigation of neutral amino acid transporters. For example, we have recently synthesized compounds that inhibit the neutral amino acid transporter ASCT2 with a 20μM apparent affinity. These compounds were identified using an in-silico docking approach, followed by validation of the docking results with electrophysiological analysis of inhibitor-protein interaction. We are continuing the development of those compounds with the aim to find inhibitors with nM affinities.
Investigation of secondary-active transport by using computational methods has traditionally focused on understanding the dynamics of the alternating access mechanism. However, there is a lack of computational analysis of electrical properties of electrogenic transporters. We are using numerical analysis of the Poisson-Boltzmann equation to compute valences and charge movement associated with partial reactions in the transport cycle. These partial reactions are substrate/ion binding/dissociation reactions, as well as structural changes associated with alternating access and gating. Structural information for the Poisson-Boltzmann analysis is obtained from available x-ray structures, or Molecular Dynamics (MD) simulations. An example of the glutamate transporter in an implicit membrane of dielectric constant of 2 is shown in the adjacent Figure. The results from these computations predict sign and magnitude of charge movement, which can be directly validated by experimental analysis through patch clamping.