Ion channels and transporters are integral membrane proteins that play important roles in virtually all aspects of human physiology. Understanding the inner workings of these proteins is important, as dysfunctions of ion channels and transporters lead to human diseases. In this proposal, we use a novel approach, chemical synthesis, to investigate these proteins. Chemical synthesis is a powerful method for protein modification because it allows the incorporation of a wide variety of unnatural amino acids and protein backbone modifications for precise changes in the protein. In this application, we use the chemical synthesis of the K+ channels, KcsA and KvAP, to investigate the mechanism of slow inactivation. Slow inactivation is a conformational change at the selectivity filter of K+ channels that converts it from a conductive to a non- conductive state. Slow inactivation plays a crucial role in determining the electrical properties of an excitable cell. We will address the following unresolved issues regarding slow inactivation: i) What is the conformation of the selectivity filte in the slow inactivated state? ii) How do permeant ions modulate slow inactivation? and iii) Do similar conformational changes at the selectivity filter underlie slow inactivation in different K+ channels? We will employ a multidisciplinary approach for these investigations that combines chemical synthesis with electrophysiology and structural studies using X-ray crystallography. We also propose to extend chemical synthesis to transporters by carrying out the synthesis of GltPH, an archaeal homolog of eukaryotic glutamate transporters. Glutamate transporters mediate the concentrative uptake of glutamate by harnessing the energy from the electrochemical gradient of ions. Dysfunction of glutamate transporters has been implicated in neurological diseases such as Alzheimer's and amyotrophic lateral sclerosis (ALS). A central unanswered question in glutamate transporters is the mechanism by which the electrochemical gradients of the ions are coupled to the uptake of glutamate. Here we use chemical synthesis of GltPH to unravel the mechanism of Na+ coupled transport. The research proposed is significant because it will provide deeper mechanistic insights into the physiologically important processes of slow inactivation in K+ channels and Na+ coupled transport. Further, this research will establish the methodology of chemical synthesis for investigating integral membrane proteins.

Public Health Relevance

Integral membrane proteins such as ion channels and transporters are involved in virtually all aspects of biology. In this proposal, we use chemical synthesis of ion channels and transporters to understand how the structures of these proteins are related to their biological functions. The knowledge gained from this project is crucial as dysfunctions of ion channels and transporters are linked to human diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biophysics of Neural Systems Study Section (BPNS)
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Chin, Jean
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Oregon Health and Science University
Schools of Medicine
United States
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Matulef, Kimberly; Valiyaveetil, Francis I (2018) Patch-Clamp Recordings of the KcsA K+ Channel in Unilamellar Blisters. Methods Mol Biol 1684:181-191
Infield, Daniel T; Matulef, Kimberly; Galpin, Jason D et al. (2018) Main-chain mutagenesis reveals intrahelical coupling in an ion channel voltage-sensor. Nat Commun 9:5055
Riederer, Erika A; Focke, Paul J; Georgieva, Elka R et al. (2018) A facile approach for the in vitro assembly of multimeric membrane transport proteins. Elife 7:
Kratochvil, Huong T; Maj, Micha?; Matulef, Kimberly et al. (2017) Probing the Effects of Gating on the Ion Occupancy of the K+ Channel Selectivity Filter Using Two-Dimensional Infrared Spectroscopy. J Am Chem Soc 139:8837-8845
Valiyaveetil, Francis I (2017) A glimpse into the C-type-inactivated state for a Potassium Channel. Nat Struct Mol Biol 24:787-788
Focke, Paul J; Hein, Christopher; Hoffmann, Beate et al. (2016) Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression. Biochemistry 55:4212-9
Kratochvil, Huong T; Carr, Joshua K; Matulef, Kimberly et al. (2016) Instantaneous ion configurations in the K+ ion channel selectivity filter revealed by 2D IR spectroscopy. Science 353:1040-1044
Matulef, Kimberly; Annen, Alvin W; Nix, Jay C et al. (2016) Individual Ion Binding Sites in the K(+) Channel Play Distinct Roles in C-type Inactivation and in Recovery from Inactivation. Structure 24:750-761
Leisle, Lilia; Valiyaveetil, Francis; Mehl, Ryan A et al. (2015) Incorporation of Non-Canonical Amino Acids. Adv Exp Med Biol 869:119-51
Focke, Paul J; Annen, Alvin W; Valiyaveetil, Francis I (2015) Engineering the glutamate transporter homologue GltPh using protein semisynthesis. Biochemistry 54:1694-702

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