Luis G. Cuello, Ph.D. - US grants

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.

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.

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.