Extending Chemical Synthesis to Ion Channel Proteins

Summary

Principal Investigator: Francis Valiyaveetil
Abstract: DESCRIPTION (provided by applicant): The long range goal of the proposed research is to develop strategies for the chemical synthesis of ion channel proteins and to use chemical synthesis to investigate the mechanisms of ion channel function. Understanding the relationship between the atomic structure of a protein and the biological function requires the ability to perturb the protein structure in a precise manner. Chemical synthesis facilitates the incorporation of a wide variety of side chain and peptide backbone modifications that enables precise modifications of the structural and electronic properties of the protein. Similar modifications are not possible using conventional mutagenesis making chemical synthesis an important asset in investigations of protein structure and function. The size of the protein has been a major factor limiting the use of chemical synthesis to investigate proteins. In the field of membrane proteins, chemical synthesis has so far been accomplished only for relatively small (<150 amino acids) proteins. Proteins of interest such as voltage gated ion channels are much bigger and are presently not amenable to chemical synthesis. To overcome this limitation, we propose developing methods that can be used for the chemical synthesis of large (>150 amino acid) membrane proteins thereby enabling us to use chemical synthesis to investigate these proteins. We will pursue three major specific aims. Aim 1) To develop a chemical synthesis of the voltage gated K+ channel KvAP, and the non-selective channel, NaK. The chemical synthesis protocols that we develop in this aim will be useful not only in investigating these specific proteins but will also find applicability in the chemical synthesis of other important classes of membrane proteins. Aim 2) We will investigate the slow inactivation process in the KvAP channel. Understanding the process of slow inactivation is important because the rate of entry (and exit) of channels into the inactivated state can significantly alter the number of channels available and therefore the electrical properties of the cell. Aim 3) We will investigate the binding of divalent ions to the outer vestibule of the NaK channel using chemical synthesis. The NaK channel shows sequence and functional similarity to cyclic nucleotide gated ion channels. Therefore, the mechanisms that we uncover in our investigations of the NaK channel will be relevant in understanding the physiologically important interactions of divalent ions with CNG channels. PUBLIC HEALTH RELEVANCE: Ion channel function underlies many important biological processes such as the excitation of nerve and muscle cells, the secretion of hormones, and sensory transduction. The research proposed is significant because it will provide a deeper understanding of the physiologically important processes of slow inactivation and block by divalent ions. Further, the research is important as it will establish chemical synthesis as an important tool for understanding ion channel structure and function.
Funding Period: 2009-05-01 - 2015-02-28
more information: NIH RePORT

Top Publications

  1. pmc Modular strategy for the semisynthesis of a K+ channel: investigating interactions of the pore helix
    Alexander G Komarov
    Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA
    ACS Chem Biol 4:1029-38. 2009
  2. pmc Semisynthesis of NaK, a Na(+) and K(+) conducting ion channel
    Kellie M Linn
    The Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA
    Biochemistry 49:4450-6. 2010
  3. ncbi Studies of ion channels using expressed protein ligation
    Paul J Focke
    Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd, Portland, OR 97239, USA
    Curr Opin Chem Biol 14:797-802. 2010
  4. pmc In vitro folding of KvAP, a voltage-gated K+ channel
    Prasanna K Devaraneni
    The Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon 97239, United States
    Biochemistry 50:10442-50. 2011
  5. ncbi Engineering K+ channels using semisynthesis
    Alexander G Komarov
    Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR, USA
    Methods Mol Biol 995:3-17. 2013
  6. pmc Semisynthetic K+ channels show that the constricted conformation of the selectivity filter is not the C-type inactivated state
    Prasanna K Devaraneni
    Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239
    Proc Natl Acad Sci U S A 110:15698-703. 2013
  7. pmc Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K+ channels
    Kimberly Matulef
    Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239
    Proc Natl Acad Sci U S A 110:17886-91. 2013

Research Grants

  1. Voltage-dependent activation in K+ channels
    Diane M Papazian; Fiscal Year: 2013
  2. Endogenous Cannabinoids and Brain Function
    Aron H Lichtman; Fiscal Year: 2013
  3. Structural biology of neurotransmitter ion channels
    James E Gouaux; Fiscal Year: 2013
  4. Making Sense of Voltage Sensors
    Stephen H White; Fiscal Year: 2013

Detail Information

Publications7

  1. pmc Modular strategy for the semisynthesis of a K+ channel: investigating interactions of the pore helix
    Alexander G Komarov
    Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA
    ACS Chem Biol 4:1029-38. 2009
    ..These results suggest that the hydrogen bond interactions of tryptophan 68 are essential for optimizing the selectivity filter for K(+) conduction over Rb(+) conduction...
  2. pmc Semisynthesis of NaK, a Na(+) and K(+) conducting ion channel
    Kellie M Linn
    The Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA
    Biochemistry 49:4450-6. 2010
    ..Functional analysis of these mutants suggests that the presence of a negatively charged residue in the vicinity of the ion binding sites is necessary for optimal flux of ions through the NaK channel...
  3. ncbi Studies of ion channels using expressed protein ligation
    Paul J Focke
    Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd, Portland, OR 97239, USA
    Curr Opin Chem Biol 14:797-802. 2010
    ..These include studies on the KcsA K(+) channel, the non-selective cation channel NaK, and the porin OmpF. These studies are discussed in this review...
  4. pmc In vitro folding of KvAP, a voltage-gated K+ channel
    Prasanna K Devaraneni
    The Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon 97239, United States
    Biochemistry 50:10442-50. 2011
    ..Our studies suggest that the K(v)AP channel provides a good model system for investigating the folding of a multidomain integral membrane protein...
  5. ncbi Engineering K+ channels using semisynthesis
    Alexander G Komarov
    Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR, USA
    Methods Mol Biol 995:3-17. 2013
    ..We anticipate that the protocols described in this chapter will also be applicable for the semisynthesis of other integral membrane proteins of interest...
  6. pmc Semisynthetic K+ channels show that the constricted conformation of the selectivity filter is not the C-type inactivated state
    Prasanna K Devaraneni
    Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239
    Proc Natl Acad Sci U S A 110:15698-703. 2013
    ....
  7. pmc Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K+ channels
    Kimberly Matulef
    Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239
    Proc Natl Acad Sci U S A 110:17886-91. 2013
    ..Our results link C-type inactivation to ion occupancy at the S2 site. Furthermore, they suggest that the differences in inactivation of K(+) channels in K(+) compared with Rb(+) are due to different ion occupancies at the S2 site. ..

Research Grants30

  1. Voltage-dependent activation in K+ channels
    Diane M Papazian; Fiscal Year: 2013
    ..An increase in the permeability of this barrier is a newly- discovered cause of human genetic diseases;the proposed research may lead to new therapeutic approaches to seal the barrier in these diseases. ..
  2. Endogenous Cannabinoids and Brain Function
    Aron H Lichtman; Fiscal Year: 2013
    ..Ultimately, the knowledge gained from this basic research will yield novel therapeutic targets that can be exploited with the pharmacological agents developed here. PROGRAM CHARACTERISTICS ..
  3. Structural biology of neurotransmitter ion channels
    James E Gouaux; Fiscal Year: 2013
    ....
  4. Making Sense of Voltage Sensors
    Stephen H White; Fiscal Year: 2013
    ..His work focuses directly on the molecular basis of voltage sensor domains and their interactions with VSD-blocking toxins...