K+ channels are key regulators of cell excitability in the nervous system, skeletal, smooth and cardiac muscle and secretory glands. Therefore, it is not surprising that dysfunction of K+ channels are the underlie cause of uncountable human pathologies, such as: neurological disorders, cardiac diseases and diabetes. For this reason, it is extremely important to understand at the atomic level the properties of K+ channels that determine cell excitability. Understanding ion selectivity, permeation and gating at atomic detail will allow us to identify highly-specific therapeutic agents that can recognize with precision a specific channel's kinetic state that need to be regulated to correct a given channelopathy. It follows that for two decades, functional, structural and computational studies, performed on the KcsA-closed structure, have improved our understanding of how the structure defines the function of K+ channels. Recently, we have made two important scientific contributions: the first atomic-resolution description of KcsA's minimal kinetic cycle and the quantification of the energetics associated with each kinetic cycle reaction. However, important unanswered questions remain, mostly due to our inability to conduct simultaneous structural and functional studies in: 1 ) the open-state of the channel 2) mutants of the highly conserved glycine residues in the selectivity filter, which are known to affect inactivation gating, ion selectivity and/or ion binding in the closed and open states of the channel, 3) tandem-tetramers to dissect cooperativity of ion channel function, and 4) mutants that dissect the non-conductive open states of KcsA by precisely uncoupling activation-gate opening from the onset of ion permeation/inactivation at the selectivity filter. Consequently, we propose the following Specific Aims: 1) To characterize the structure-function correlations between the selectivity filter, ion occupancy and conduction properties of KcsA ?trapped? with its activation gate open 2) To determine the structure-function correlations of KcsA subunit cooperativity using tandem hetero-tetramers 3) To understand the role of KcsA's allosteric coupling on the onset of ion permeation, C-type inactivation and ion selectivity and 4) To understand the structural and functional roles of the glycine residues within the K+ channel selectivity filter. The novelty of our experimental approaches, together with our vast experience working with ion channels, fully qualifies us to perform the proposed project. Finally, the completion of this project will bring us closer to a complete atomistic understanding of ion-channel function, allowing us to identify ion-channels kinetic intermediates more suitable as pharmaceutical targets for the next generation of more specific and safer therapeutic drugs.

Public Health Relevance

Because K+ selective ion channels are essential regulators of cell excitability, their dysfunction underlies a myriad of human pathological conditions that make them excellent targets for drugs to treat very diverse diseases, such as: LQT syndrome, arrhythmia, epilepsy, autoimmune diseases and diabetes. It follows that understanding the function-structure correlations of K+ channels at atomic resolution will pave the road to develop highly-specific therapeutic drugs to correct channelopathies. In this grant application, we present a systematic experimental approach that push to the limit the experimental boundaries to provide an atomic resolution understanding of K+ channel ion permeation, selectivity, subunit cooperativity and gating.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM097159-07S1
Application #
9895075
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Nie, Zhongzhen
Project Start
2012-04-01
Project End
2022-06-30
Budget Start
2019-07-01
Budget End
2020-06-30
Support Year
7
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Texas Tech University
Department
Physiology
Type
Schools of Medicine
DUNS #
609980727
City
Lubbock
State
TX
Country
United States
Zip Code
79430
Li, Jing; Ostmeyer, Jared; Cuello, Luis G et al. (2018) Rapid constriction of the selectivity filter underlies C-type inactivation in the KcsA potassium channel. J Gen Physiol 150:1408-1420
Tilegenova, Cholpon; Elberson, Benjamin W; Marien Cortes, D et al. (2018) CW-EPR Spectroscopy and Site-Directed Spin Labeling to Study the Structural Dynamics of Ion Channels. Methods Mol Biol 1684:279-288
Labro, Alain J; Cortes, D Marien; Tilegenova, Cholpon et al. (2018) Inverted allosteric coupling between activation and inactivation gates in K+ channels. Proc Natl Acad Sci U S A 115:5426-5431
Elberson, Benjamin W; Whisenant, Ty E; Cortes, D Marien et al. (2017) A cost-effective protocol for the over-expression and purification of fully-functional and more stable Erwinia chrysanthemi ligand-gated ion channel. Protein Expr Purif 133:177-186
Cuello, Luis G; Cortes, D Marien; Perozo, Eduardo (2017) The gating cycle of a K+ channel at atomic resolution. Elife 6:
Fiori, Mariana C; Krishnan, Srinivasan; Kjellgren, Abbey et al. (2017) Inhibition by Commercial Aminoglycosides of Human Connexin Hemichannels Expressed in Bacteria. Molecules 22:
Krishnan, Srinivasan; Fiori, Mariana C; Whisenant, Ty E et al. (2017) An Escherichia coli-Based Assay to Assess the Function of Recombinant Human Hemichannels. SLAS Discov 22:135-143
Rosholm, Kadla R; Baker, Matthew A B; Ridone, Pietro et al. (2017) Activation of the mechanosensitive ion channel MscL by mechanical stimulation of supported Droplet-Hydrogel bilayers. Sci Rep 7:45180
Krishnan, Srinivasan; Fiori, Mariana C; Cuello, Luis G et al. (2017) A Cell-Based Assay to Assess Hemichannel Function. Yale J Biol Med 90:87-95
Tilegenova, Cholpon; Cortes, D Marien; Cuello, Luis G (2017) Hysteresis of KcsA potassium channel's activation- deactivation gating is caused by structural changes at the channel's selectivity filter. Proc Natl Acad Sci U S A 114:3234-3239

Showing the most recent 10 out of 14 publications