Christof T. Grewer, Ph.D

- Associate Professor, Biological and Physical Chemistry

-Member, Journal of Biological Chemistry Editorial Board (starting April 2008)

State University of New York at Binghamton
Binghamton, NY 13902

e-mail: cgrewer@binghamton.edu
Phone: 607 777 3250
fax: 607 777 4478

• Diploma in Chemistry (equivalent to M.S.), Johann Wolfgang Goethe-University, Frankurt, Germany, 1990
• Ph.D. in Physical Chemistry, Johann Wolfgang Goethe-University, Frankfurt, Germany, 1993
• Postdoctoral Fellow, Cornell University, Ithaca, NY, 1993-1996
• Senior Research Associate, Max-Planck-Institute for Biophysics, Frankurt, Germany, 1997-2003
• Assistant Professor, Dept. of Physiology and Biophysics, University of Miami School of Medicine, Miami, FL, 2003-2007
• Member, Neuroscience Program, University of Miami School of Medicine, Miami, FL, 2003-2007
• Associate Professor, Dept of Chemistry, Binghamton University, Binghamton, NY, 2008-

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. However, the actual transport mechanism(s) remain elusive. Our aim is to combine functional and structural evidence in order to obtain an understanding of how these transport proteins work.

Dynamics of the transport process

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 that combines evidence from such pre-steady-state functional data with structural information is shown in Fig. 1A 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. A typical example of transport currents generated by glutamate transporters in response to a glutamate concentration jump is shown in Fig. 1B, demonstrating the existence of two separable decay processes (assigned to the state transitions shown in Fig. 1A). 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 (Fig. 1C). 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.

Development of caged compounds

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. An example of a photolysis reaction is shown in Fig. 2 for caged GABA. When using a suitable caging group, for example the α-carboxyl-o-nitrobenzyl caging group (CNB), photolysis takes place within 100μs. 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. Fig. 2 shows also the experimental setup used for photolyzing caged compounds around voltage-clamped cells.

• Zhang, Z, Gameiro, A, and Grewer, C. "Highly-conserved asparagine 82 controls the interaction of Na+ with the sodium-coupled neutral amino acid transporter SNAT2.", J. Biol. Chem., (2008), in press.
• Erreger, K., Grewer, C., Javitch, J. A. and Galli, A., "Currents in response to rapid concentration jumps of amphetamine uncover novel aspects of human dopamine transporter function.", J. Neurosci., (2008), 28, 976-989.
• Zhang, Z., Tao, Z., Gameiro, A., Barcelona, S., Braams, S., Rauen, T. and Grewer, C., "The transport direction determines the kinetics of substrate transport by the glutamate transporter EAAC1.", Proc. Natl. Acad. Sci. USA, (2007), 104, 18025-18030.
• Mim. C., Tao, Z., and Grewer, C., "The temperature dependence of the pre-steady-state kinetics of the glutamate transporter EAAC1 reveals two conformational changes associated with glutamate translocation.", Biochemistry, (2007), 46, 9007-9018.
• Tao, Z., and Grewer, C., "Cooperation of the conserved aspartate 439 and bound glutamate is important for forming a high-affinity Na+ binding site on the glutamate transporter EAAC1.", J. Gen. Physiol., (2007), 129, 331-344.
• Zhang, Z, Papageorgiou, G, Corrie, J. E. T., and Grewer, C., "Pre-steady-state currents in neutral amino acid transporters induced by photolysis of a new caged alanine derivative." Biochemistry, (2007), 46, 3872-3880.
• Zhang, Z. and Grewer, C., "The sodium-coupled neutral amino acid transporter SNAT2 mediates an anion leak conductance that is differentially inhibited by transported substrates." Biophys. J., (2007), 92, 2621-2632.
• Maier, W., Schemm, R., Grewer, C., and Laube, B., "Disruption of interdomain interactions in the glutamate binding pocket affects differentially agonist affinity and efficacy of NMDA receptor activation." J. Biol. Chem., (2007), 282, 1863-1872.
• Tao, Z., Zhang, Z., and Grewer, C., "Neutralization of the aspartic acid residue D367, but not D454, inhibits binding of Na+ to the glutamate-free form and cycling of the glutamate transporter EAAC1." J. Biol. Chem., (2006), 281, 10263-10272.
• Mim C, Balani P, Rauen T, and Grewer C., "The Glutamate Transporter Subtypes EAAT4 and EAATs 1-3 Transport Glutamate with Dramatically Different Kinetics and Voltage Dependence but Share a Common Uptake Mechanism.", J. Gen. Physiol., (2005), 126, 571-589.
• Grewer, C. and Grabsch, A., "New inhibitors of the neutral amino acid transporter ASCT2 reveal its Na+-dependent anion leak.", (2004), J. Physiol., 557, 747-759.
• Grewer, C. Watzke, N., Rauen, T. and Bicho, A., "Is the Glutamate Residue E373 the Proton Sensor of the Glutamate Transporter EAAC1?", (2003), J. Biol. Chem., 278, 1585-2592.
• Watzke, N., Bamberg, E. and Grewer, C., "Early Intermediates in the Transport Cycle of the Neuronal Excitatory Amino Acid Carrier EAAC1.", (2001), J. Gen. Physiol., 117, 547-562. This article was featured on the cover of this JGP issue.
• Grewer, C., Watzke, N., Wiessner, M. and Rauen, T., "Glutamate translocation of the neuronal glutamate transporter EAAC1 occurs within milliseconds.", (2000), Proc. Natl. Acad. Sci. USA, 97, 9706-9711.

Curriculum Vitae