Jianmin Cui - US grants

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand the molecular mechanism of the voltage, Ca2+, and Mg2+ dependent activation of large-conductance K+ channels (BK channels). BK channels have road physiological functions, including the modulation of neurotransmitter release and the control of blood vessel diameters. As a consequence of these physiological functions BK channels are of significant clinical importance. For example, abnormal activity of BK channels has been associated with hypertension in animal models; their increased activity may reduce the incidence of ischemia- reperfusion-induced cardiac arrhythmia. In the activation of BK channels voltage induces movements of the voltage sensor in the channel, Ca2+ or Mg2+ binds to the channel to cause conformational changes in the channel protein to open he activation gate. Now the structure of the K+ channel pore has been solved; protein sequences underlying he activation gate, the voltage sensor, and the Ca2+ binding site have been identified. However, the manner n which voltage sensor movements, Ca2+ or Mg2+ binding are coupled to the opening of the activation gate remains unknown. Until the structural and energetic basis of these couplings is elucidated, how voltage, Ca2+ and Mg2+ sensitivities are modulated in various BK channels to subserve their physiological functions cannot be understood. Based on previous studies, we hypothesize that a structural domain of the channel protein that is physically close to the activation gate (the RCK domain for Regulating the conductance of K+ channels) is central in these couplings. Recently, the X-ray crystal structure of the RCK domain has been solved. Guided by the structural data we will perturb the channel structure using molecular biology and determine its impact on the energetic contribution of Ca2+, Mg2+, or voltage to channel opening using our recently developed electrophysiological approaches. We will also use approaches of protein biochemistry and nuclear magnetic resonance spectroscopy (NMR) to map specific intramolecular protein interactions that nay be altered during channel activation and hence control channel function. These experiments will provide a foundation for understanding how various BK channels play their role in physiological processes and define targets on BK channels for therapeutic purposes. They will also contribute to our understanding of ion channel gating in general.

DESCRIPTION (provided by applicant): The broad long-term objective of this proposal is to understand the mechanisms of ion channel gating, a key molecular event that is known to regulate a variety of physiological and pathological processes. Studying the BK-type, voltage, Ca2+ and Mg2+ dependent K+ channel as a model system, the focus of this proposal is to investigate the molecular process in which voltage sensor movements, Ca2+ or Mg2+ binding are coupled to the opening of the activation gate through intramolecular interactions. The specific aims are: I. To elucidate the role of a cytosolic domain, the AC region, in Ca2+ dependent gating. II. To investigate the interactions between the bound Mg2+ and the voltage sensor. III. To examine effects of mutations in S6 on the function of the activation gate and the sensitivity to voltage, Ca2+ and Mg2+. The role of BK channels in human health is based on their activation by voltage, Ca2+ and Mg2+, and BK channels are being pursued as a therapeutic target for various diseases. A BK channel mutation that affects voltage and Ca2+ dependent activation is linked to epilepsy and paroxysmal dyskinesia. This application seeks to dissect the molecular mechanism of BK channel gating by these stimuli, which will provide insights into BK channel related diseases and a solid basis for therapeutic developments. Several studies in recent years have provided us with X-ray crystallographic structures of potassium channels Kv1.2, KvAP, and MthK that can serve as models for BK channels. Based on these structural models and other preliminary results, a multi-disciplinary approach, including electrophysiology, mutation, chemical modification, protein biochemistry and kinetic modeling will be used to achieve the specific aims. The BK-type potassium ion channel is important for brain function and blood circulation. This research investigates the mechanism of BK channel function. The results will improve our understanding of diseases caused by the malfunction of this channel such as epilepsy and hypertension, and facilitate the development of drugs treating various diseases such as neuronal ischemia, trauma and cognitive decline.

DESCRIPTION (provided by applicant): Our long-term goal is to understand the physiological and pathophysiological role of the BK-type voltage and Ca2+-activated K+ channels. BK channels modulate physiological processes that involve Ca2+ signaling in many tissues, such as muscle contraction, renal function and neural transmission. These channels are composed of the pore-forming, voltage- and Ca2+ -sensing a subunit that is encoded by a single Slo1 gene and four types of auxiliary ¿ subunit (¿1-4). Each of these ¿ subunits modulates functional properties of the Slo1 channel with distinct characteristics and tissue-specific expression. Thus, ¿ subunits help to define the phenotypes and physiological roles of BK channels in various tissues. To achieve our long-term goal, it is necessary to study the molecular mechanism of the interaction between Slo1 and the ¿ subunits. Previous publications and our preliminary data demonstrate that, although all four types of ¿ subunit share similar structural features, the mechanisms of their modulation of Slo1 channels differ in two key aspects: 1) they target different molecular components and processes in Slo1 that are important in channel gating, and 2) they have different amino acids or motifs (active sites) that are critical for altering gating of Slo1 channels. Based on these preliminary results, we will use the methods of electrophysiology, mutagenesis, chemical modification and kinetic modeling to achieve the following specific aims: I. To identify the molecular targets of ¿ subunits modulation in Slo1 channels. II. To identify the active sites of ¿ subunits. III. To examine if ¿ subunits affect the interaction between the membrane-spanning and the cytosolic domains that is critical for BK channel gating. Aim III will establish a link between the ¿ subunit association, as studied in Aims I and II, with a structural mechanism of BK channel gating that has been revealed recently. This study will identify amino acids and structural motifs important for BK channel gating and reveal the nature of the interactions between Slo1 and ¿ subunits. It will lay the foundation for understanding the molecular basis of BK channel related pathological conditions, such as epilepsy and hypertension, and provide the target and rationale for their treatment. ¿ subunits of K+ channels with transmembrane segment, such as the KCNE family and BK ¿ subunits modulate the gating properties of their respective a subunits, which are key to the physiological roles of these channels. Our preliminary studies suggest that some of these ¿ subunits may affect channel gating through common mechanisms. Therefore, this study will provide insights to these common mechanisms of K+ channel function.

DESCRIPTION (provided by applicant): Our long-term goal is to understand the molecular mechanisms of BK channel activation. BK-type K+ channels are activated by voltage, intracellular Ca2+ and Mg2+. These channels are important in modulating muscle contraction, neural transmission and circadian pacemaker output. Recently, the voltage sensor, Ca2+ and Mg2+ binding sites in BK channels have been identified. However, the structural basis for the coupling between sensors and the activation gate, which are located in different structural domains, still remains elusive. A central question in this crucial step of BK channel gating is how these different structural domains interact with one another to mediate the coupling between the sensors and the activation gate. It has become apparent that a knowledge gap presented by this question is a critical barrier for understanding BK channel activation. Recent studies and our preliminary results lead to a general hypothesis for answering this question: interactions between the voltage sensor domain (VSD) and the cytosolic domain (CTD), and this interfacial alignment couples the ligand binding site on the CTD to the opening of the activation gate. We propose the following specific aims to examine three key aspects of this hypothesis. 1. To demonstrate that the electrostatic interactions affect the VSD-CTD alignment. 2. To demonstrate that the VSD-CTD alignment is coupled to the activation gate. 3. To show that the VSD-CTD interactions mediate coupling of Ca2+ binding to gate opening. We have developed innovative methods to measure VSD-CTD alignment and the coupling of this alignment to the activation gate. This study will identify amino acids and structural motifs important for BK channel activation and reveal the nature of the interactions among structural domains during this molecular process. A prevalent model proposed for ion channel activation by intracellular ligands is that ligand binding alters the conformation of the cytosolic domain, which pulls a peptide linker to open the activation gate. The results of our proposed study will show that in BK channels Mg2+ and Ca2+ may also activate the channel by pushing the voltage sensor via an electrostatic interaction involving the residues in different structural domains, which provides a novel mechanism of ligand dependent gating that may be shared by many other ion channels. BK channels are being pursued as therapeutic targets for neuronal ischemia, trauma and cognitive decline, and recent studies show that BK channels are associated with hypertension, schizophrenia, epilepsy and paroxysmal dyskinesia. The dissection of the molecular events during BK channel gating in this study will help identify specific targets for the development of therapeutics in addition to providing insights into the principles of ion channel gating. PUBLIC HEALTH RELEVANCE: This study will identify amino acids and structural motifs important for BK channel gating and reveal the nature of the interactions between structural domains of the channel protein. It will lay the foundation for understanding the molecular basis of BK channel related pathological conditions, such as epilepsy and hypertension, and provide the target and rationale for their treatment.

? DESCRIPTION (provided by applicant): This project is to reveal the major molecular bases for the function of KCNQ K+ channels (KCNQ1-5) that are important in the heart, brain, inner ear and epithelia. The aberrant functions of these channels are associated with cardiac arrhythmia, epilepsy, deafness, and gastric cancer. The importance of these channels is based on two prominent properties. First, all these channels, when expressed without auxiliary ß subunits, are activated by voltages at negative ranges (start activating around -60 mV). Being activated just above the resting membrane potential, the M-current through KCNQ2 and 3 in neurons reduces membrane excitability and acts as a brake to membrane discharge. Second, the association of the KCNE family K+ channel ß subunits, which radically alter gating, permeation and pharmacological properties of KCNQ1, determines the physiological role of KCNQ1. KCNQ1 associates with KCNE1 to form the IKs channel in the heart that regulates action potential duration, and with KCNE2 or KCNE3 to form the constitutively open background K+ channels in epithelia important for ion transport. What are the mechanisms underlying these properties? This is a long-standing question whose answer will provide the basis for the understanding and treatment of KCNQ associated diseases. We propose that the answer to this question lies in a novel mechanism for KCNQ1 activation. During voltage dependent activation in KCNQ1, the voltage sensor domain (VSD) moves in two steps, from the resting to intermediate and then to activated state; the channel pore opens at both intermediate and activated states of VSD, but the VSD-pore interaction differs at different states of VSD to alter channel gating, ion permeation and pharmacology. KCNE1 modulates channel function by suppressing the intermediate openings of the channel. In this project we wish to test various aspects of this hypothesis in three specific aims. 1) We wish to identify the structural motifs that are important for the VSD-pore interaction at intermediate and activated states, respectively. Mutations in KCNQ1 and KCNE1 are associated with long QT syndrome (LQT) that predispose patients to fatal cardiac arrhythmia, this study will reveal if some of the LQT mutations alter VSD-pore interaction. 2) We will examine if suppression of the intermediate and potentiation of the activated openings is the main mechanism for major functional changes upon KCNE1 association including the response to ß- adrenergic stimulation and inactivation gating. LQT patients with KCNQ1 mutations often experience symptoms of cardiac arrhythmia such as syncope and sudden death during exercise when ß-adrenergic pathway is stimulated. This study is therefore particularly significant for the understanding of molecular bases of LQT. 3) We wish to reveal if KCNE2 and 3 make the channel constitutively open by altering VSD activation, pore opening or VSD-pore interaction. We will identify if these channels are at intermediate or activated open states and their responses to drugs and cellular signaling molecules.

? DESCRIPTION (provided by applicant): This project proposes a straightforward approach to rational drug screening for ion channel-based diseases. The basic function of ion channels is to provide membrane current. In a tissue with the expression of many types of ion channels, a pathologic change in one type of channel may cause diseases. Our approach hypothesizes that the normal tissue function can be restored by compensating for the change in net current from any of the channels produced by the cell; all that is required is that a reasonable facsimile of normal net current flow be restored. We propose to apply this approach to Long Q-T Syndrome (LQTS), a condition that can cause a ventricular arrhythmia (torsades de pointe) that can lead to sudden death. The duration of the ventricular action potential (APD) depends on the balance of outward and inward currents flowing at plateau potentials. The outward currents include the delayed rectifiers IKr and IKs, while the inward currents include persistent sodium current (INaP). Specific mutations in any of these channel proteins that cause a reduction in outward current or increase in inward current are associated with congenital long QT syndrome (LQTS). There is also a much more prevalent problem called acquired LQTS (aLQTS) that is most often associated with off target effects of drugs and therefore cost the pharmaceutical industry billions of dollars and even removes from the market some compounds that could have effectively treated other diseases. To this end, we will use recent structural information concerning IKs channel activation for in silico drug screening to search for compounds with the highest probability of interacting with the IKs channel. We will apply the candidate compounds to freshly isolated guinea pig and canine cardiac ventricular myocytes to determine their effects on the ventricular action potential and the underlying ion currents in both control and LQTS conditions. The compounds that have the most favorable changes in the LQTS APD would be identified as viable candidate compounds. Our screening databases will include more than 1,500 FDA-approved small molecule drugs. If any of these FDA-approved drugs work as an IKs-enhancing compound, it should face smaller safety barriers for FDA approval. Our approach will be built on novel structural sites in the IKs channel identified by our recent work and innovative computer algorithms for molecular docking. The significance of our approach is both specific and general. Specifically if successful, candidate compounds for both congenital and acquired forms of LQTS will emerge, permitting those afflicted with the congenital form to avoid the dangers of sudden death, while allowing existing drugs or drug candidates (previous excluded for this side effect) to be made safer for clinical use. At present the various ion channels in tissues have been identified and their physiological roles defined, the structure and structure basis of function of variety of ion channels have been elucidated, and powerful computational methods have been developed. Therefore, more generally, if successful, this approach can point the way in defining how a combination of experimental studies and computer simulations can lead to rational drug development for other ion channel diseases. This new paradigm will help ion channel-targeting drug discovery be faster, cheaper, and safer and will reduce the use of animals.

Our long-term goal is to understand the molecular mechanisms of BK channel activation. BK-type K+ channels are activated by voltage and intracellular Ca2+. These channels are important in modulating muscle contraction, neural transmission and circadian pacemaker output, and have been shown to associate with hypertension, schizophrenia, epilepsy and paroxysmal dyskinesia. These channels are being pursued as therapeutic targets for neuronal ischemia, trauma and cognitive decline. The voltage sensor and Ca2+ binding sites in BK channels have been identified. However, the structural basis for the coupling between sensors and the activation gate, which are located in different structural domains, still remains elusive. A central question in this crucial step of BK channel gating is how these different structural domains interact with one another to mediate the coupling between the sensors and the activation gate. This proposal is motivated by the long- awaited and recently solved atomic structures of a whole BK channel. These structures offer new insights on fundamental mechanisms of sensor-pore coupling that may differ from the previous understanding. The structure and functional studies lead to a general hypothesis for answering this question: interactions among the voltage sensor domain (VSD), the cytosolic domain (CTD), and the pore-gate domain (PGD) all contribute to the sensor-pore coupling. We propose three specific aims to examine three key aspects of this hypothesis: 1) the VSD-PGD interactions in VSD-pore coupling, 2) the VSD-CTD interactions in both VSD-pore and Ca2+- pore couplings, and 3) the role of a peptide linker between PGD and CTD in both the VSD-pore and Ca2+-pore couplings. We will use novel structure based screening methods to identify compounds that modulate channel function. These compounds and mutations in the channel protein will serve as probes to indicate the structural motifs that are key to the sensor-pore coupling in BK channels. Threading the biophysics of mutations and modulators onto the channel structures will allow us to understand the molecular mechanisms of how physiological stimuli open BK channels. The results of these studies will identify novel binding sites, chemical cores and new mechanisms of altering channel function by drugs, which will directly help drug development targeting BK channels. Our studies on BK channels may provide insights for the understanding of other ion channels that share similar structural and functional characteristics.