The long-term goal of this project is to determine the structural changes that underlie K+ channel function. These membrane-spanning proteins are critical in controlling the electrical potential difference across the membrane, which, in turn, forms the basis for solute exchange and cellular excitability. It follows that a full understanding of the relation between structure and function in K+ channels is a high priority. Achieving this goal will require knowledge of the conformational changes that mediate two key properties, ion permeation and gating. In this proposal, we will characterize at the atomic level these two properties by trapping the channel complex in different kinetic states. Subsequent structural analysis will address the following fundamental but unanswered questions: 1) what is the high-resolution structure of KcsA trapped in the open and C-type inactivated state? 2) what is the amino-acid network underlying allosteric communication between the activation gate and the selectivity filter of KcsA? and 3) which are the structural changes of the KcsA selectivity filter that lead to C-type inactivation? The answers to these questions will provide us with the structural background needed to understand ion selectivity, permeation, and gating of K+ channels in a dynamic context and eventually will assist in the rational design of drugs for the treatment of K+ channel- related diseases. We will focus our study on an archetypal prokaryotic channel, KcsA, that contains all the structural elements characteristic of K+ channel function (i.e., ion permeation, activation, and C-type inactivation gating) but possesses a structure simple enough to facilitate analysis. A dual approach combining crystallographic and electrophysiological methods will provide high-resolution functional and structural information for this model channel in various kinetic states.
K+ channels are molecules conveniently located in the plasma membrane of all living cells, from which they effectively control the flow of K+ ions coming out of the cell, which is crucial for a large number of physiological processes i.e. activation of natural killer cells in the immune system or the regulation of the blood glucose content by the pancreatic cells. Given the critical role of K+ channel in the normal functioning of the human body, it is not surprising that their dysfunction has catastrophic metabolic consequences that very often lead to death. For this reason, it is of surmount importance to determine what are the structural changes at the atomic level that a K+ channel has to endure when it is doing its biological function, which in turn it will assist us in the design of safer therapeutic drugs to treat many K+ channel related diseases.
|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