Daniel L. Minor - US grants

DESCRIPTION (provided by applicant): The long-term goals of this project are to develop general, high-throughput methods to identify, evolve, and characterize small molecule and protein inhibitors and activators of ion channel function. Ion channels are coveted drug targets. As membrane proteins, they are readily accessible to applied extracellular compounds and their modulation brings about rapid changes in the signaling properties of excitable cells in the heart and brain. However, as membrane proteins, they also reside beyond many of the well-established approaches for inhibitor and activator development that require purified material. Consequently, many lack any significant pharmacologies. This problem leads to a large gap in our ability to connect ion channel genes with in vivo function. Unraveling the physiological and biophysical functions of ion channels demands new tools that allow the manipulation of a given type of channel's action in a variety of settings. To address this issue, we are using novel genetic selection approaches to develop activators and inhibitors of two classes of potassium channels that lack robust pharmacologies, inwardly rectifying and Two-P potassium channels. These channels are thought to play central roles in neurotransmitter regulation of neuronal and cardiac excitability but precise delineation of their functions awaits reagents that can specifically activate or block their function. We are pursuing genetic selections for both small molecule and peptides. Our approach is multidisciplinary and includes genetics, biochemistry, electrophysiology, and structural biology to dissect and characterize the modes of action of selected modulators. Because of their important roles in human physiology, ion channels are the targets for drugs to treat a wide range of diseases including epilepsy, cardiac arrhythmias, stroke, hypertension, diabetes, and memory loss. In addition to being intended drug targets, a number of ion channels, particularly cardiac ion channels, are unusually susceptible to unwanted cross-reactivity. This issue impedes the progress of many drug development trials. Thus, developing an understanding of how small molecules act on ion channel function as well as developing high-throughput methods for assaying compounds that lead to ion channel block should not only provide powerful tools for dissecting channel mechanism and function but should aid in the development of new therapeutic agents for a range of human diseases.

Abstract Not Provided.

DESCRIPTION (provided by applicant): Membrane proteins are the conduits for communication between the outside world and the interior of the cell and are central to processes that are essential for human life such as neurotransmission, cardiovascular regulation, and hormonal signaling. Despite their importance, we still know very little regarding the basic mechanisms by which they work due to a lack of three-dimensional structural information. The largest impediment to the routine biochemical and biophysical study of eukaryotic membrane proteins is the limited availability of pure protein from natural or recombinant sources. This problem is particularly acute for ion channels, which are often found in very low abundance in native tissues. Furthermore, many membrane proteins have limited stability in the detergent solutions that are required for their purification adding additional complications to their handling in biochemical and biophysical experiments. To overcome both of these limitations, we seek to develop a general strategy for the selection, overexpression, and purification of functional, stability-enhanced eukaryotic ion channels using yeast. As a model system we are investigating the prototypic inwardly rectifying potassium channel Kir2.1. Kir channels form a large family of potassium channels with central roles in the regulation of heartbeat, hormone release, sensory transduction, and cognition. Their dysfunction has been linked to a variety of human diseases including diabetes, cardiac arrhythmias, and epilepsy. Developing a means to produce large quantities of pure, functional, rare membrane proteins such as eukaryotic ion channels will have a major impact on our efforts to understand the basic biochemistry behind the membrane signaling and transport mechanisms that underlie processes like neurotransmission, cardiovascular regulation, and hormonal signaling, as well as the development of new drugs to treat dysfunctions in these systems. The methods developed here are general and, once established, should permit the overproduction of a wide range of ion channels as well as other eukaryotic membrane proteins for biochemical and structural study.

DESCRIPTION (provided by applicant): The long-term goals of this project are to develop a high-resolution understanding of ion channel function and regulation. We are investigating the KCNQ family of voltage-gated potassium channels. These channels play central roles in auditory, cardiac, and brain function. Because channel function depends on subunit composition and interactions with proteins of cellular signaling networks, we are investigating the molecular bases for both of these phenomena. Due to difficulties in studying mammalian membrane protein structure, our present efforts are focused on understanding the function cytoplasmic domains that are important for channel assembly and for the recruitment of cellular signaling factors. We are pursuing a multidisciplinary approach that includes biochemical, biophysical, X-ray crystallographic, and electrophysiological measurements to dissect KCNQ channel function. Because of their important roles in human physiology, mutations of KCNQ channels lead to a variety of hereditary diseases including congenital deafness, cardiac arrhythmias, and epilepsy. We are particularly interested in understanding how disease mutations change channel properties and interactions with other proteins. KCNQ channels are the targets for drugs directed at cardiac arrhythmias, seizures, and memory disorders. Thus, understanding their structures and mechanisms of action may lead to the development of new, valuable therapeutic agents.

The long-term goals of this project are to develop a high-resolution understanding of voltage-gated calcium channel (CaV) function and regulation. These molecular switches play pivotal roles in cardiac action potential propagation, neurotransmitter release, muscle contraction, calcium-dependent gene-transcription, and synaptic transmission. Calcium influx is a potent activator of intracellular signaling pathways but is toxic in excess. As a result, its entry into cells is tightly regulated. CaVs are major sources of activity-dependent calcium influx and possess a number of mechanisms that allow them to self-regulate. These mechanisms depend critically on interactions of the pore-forming subunit with cytoplasmic proteins that regulate channel activity. Our studies are aimed at understanding the molecular architecture that underlies CaV function and on developing novel reagents that can control channel function. We are investigating the hypothesis that two principal CaV inactivation mechanisms, calcium-dependent inactivation (CDI) and voltage-dependent inactivation (VDI) center on changes in the region of the selectivity filter. This is a paradigm-shifting view, based on our recent findings, that stands to align CaV inactivation mechanisms with a growing number of examples from other voltage-gated ion channel (VGIC) superfamily members. Due to the extraordinary challenges in studying mammalian membrane protein structure, part of our efforts focus on understanding basic structural mechanisms that are shared between CaVs and their ancestors, bacterial voltage gated sodium channels (BacNaVs). Production of multiprotein membrane proteins, such as CaVs, is a significant barrier to structural studies. To bridge this gap, we direct efforts to develop systems for production of full-length CaV complexes. In parallel, we investigate the how a novel class of reagents, anti-CaV? subunit nanobodies, interact with CaV? and modify channel function. Knowledge of such interactions will inform studies of how these novel, genetically-encodable reagents can be developed as versatile and selective agents to control CaV activity. Our studies integrate a multidisciplinary effort that includes biochemical, biophysical, X-ray crystallographic, cryo-electronmicroscopy, electrophysiological, and cell biology approaches. Because of their important role in human physiology, CaVs are the targets for drugs with great utility for the treatment of cardiac arrhythmias, hypertension, congestive heart failure, epilepsy, and chronic pain. Thus, understanding their structures and mechanisms of action in detail should greatly assist the development of valuable therapeutic agents for a wide range of human cardiac and neurological problems.

[unreadable] DESCRIPTION (provided by applicant): The long-term goals of this project are to develop a high-resolution understanding of ion channel function and regulation. We are investigating the KCNQ family of voltage-gated potassium channels. These channels play central roles in auditory, cardiac, and brain function. Because channel function depends on subunit composition and interactions with proteins of cellular signaling networks, we are investigating the molecular bases for both of these phenomena. Due to difficulties in studying mammalian membrane protein structure, our present efforts are focused on understanding the function cytoplasmic domains that are important for channel assembly and for the recruitment of cellular signaling factors. We are pursuing a multidisciplinary approach that includes biochemical, biophysical, X-ray crystallographic, and electrophysiological measurements to dissect KCNQ channel function. Because of their important roles in human physiology, mutations of KCNQ channels lead to a variety of hereditary diseases including congenital deafness, cardiac arrhythmias, and epilepsy. We are particularly interested in understanding how disease mutations change channel properties and interactions with other proteins. KCNQ channels are the targets for drugs directed at cardiac arrhythmias, seizures, and memory disorders. Thus, understanding their structures and mechanisms of action may lead to the development of new, valuable therapeutic agents. [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Electrical signaling in the brain and heart is driven by the action of ion channel proteins. These proteins from holes in the cell membrane that open and close to allow the passage of ions such as calcium and potassium. We are investigating the structures and functions of a range of ion channels, ion channel regulatory proteins, and transporters. The goals are to uncover the basic biophysical mechanisms by which these transmembrane proteins work, to understand their structures and interactions in atomic detail, and to understand how their functions are compromised in human diseases such as cardiac arrhythmias, epilepsy, deafness, and mental illness.For our research it is essential to know the molecular identity of the proteins and protein fragments that we study. Mass spectrometry provides the most accurate way to make these molecular identifications.

DESCRIPTION (provided by applicant): The voltage-gated calcium channel CaVa2d subunit is the site of action of two gabapentinoid drugs, gabapentin (Neurontin) and pregabalin (Lyrica), that are used to treat neuropathic pain, pain associated with fibromyaglia, migraine, epilepsy, and a number of mood disorders. Despite the direct connection between CaVa2d and the treatment of nervous system disorders, nothing is known about CaVa2d molecular structure at high resolution, the detailed molecular mechanisms by which CaVa2d affects voltage-gated calcium channel function, or how gabapentin and pregabalin bind to CaVa2d and modulate voltage-gated calcium channel activity. We seek to determine the high-resolution structure of CaVa2d and CaVa2d complexes with gabapentinoid drugs. Our efforts are directed at developing the appropriate expression systems to produce CaVa2d proteins for crystallographic study and to solve the high-resolution structure of CaVa2d. This work should unveil the fundamental structures that underlie CaVa2d function and reveal how gabapentinoid drugs interact with CaVa2d. This knowledge will bear directly on understanding the basic mechanisms of action of gabapentinoid drugs and should facilitate the development of better therapeutics that will improve the treatment of pain and a wide range of nervous system and mood disorders. PUBLIC HEALTH RELEVANCE: Voltage-gated calcium channels are the targets of drugs that are used to treat pain, epilepsy, migraine, fibromyalgia, and mood disorders. Our work aims to determine the molecular architecture that underlies how such drugs interact with and affect channel function. Such knowledge is indispensable for the improvement of current therapies and development of more efficacious treatments for a variety of nervous system disorders.

Membrane Proteins; Structure

DESCRIPTION (provided by applicant): The long-term goals of this project are to develop an understanding of the fundamental mechanisms that control the function of K2P (KCNK) potassium channels and to develop methods to identify and characterize small molecule, ion channel modulators for the K2P family. K2Ps are a diverse family of potassium-selective channels that are responsible for background 'leak' currents. These currents are pivotal in modulating the excitability of neurons. K2Ps respond to varied stimuli that include pH changes, temperature, and mechanical force. Although, K2Ps have well-established roles in the nervous and cardiovascular systems and are implicated in pain, anesthetic responses, thermosensation, and mood, they are the least well-understood potassium channel class. Ion channels are coveted drug targets. As membrane proteins, they are readily accessible to extracellular compounds and their modulation brings about rapid changes in the properties of excitable cells in the heart and brain. However, as membrane proteins, they also reside beyond many of the well-established approaches for modulator development that require purified material. Consequently, many channels, including those in the K2P family, lack significant pharmacologies. This problem leads to a gap in our ability to connect ion channel genes with in vivo function. We are pursuing a multidisciplinary approach that includes genetic selections, biophysical, and electrophysiological measurements to identify, dissect, and characterize the core elements that control K2P function and to define and characterize new small molecules that control K2P activity. Defining the molecular mechanisms that control K2p activity and uncovering new K2P modulators should provide the key framework and necessary tools for understanding how K2Ps function. Because of their important roles in human physiology, K2Ps are targets for drugs for the treatment of chronic pain, stroke, and depression. Thus, developing an understanding of how K2Ps function and means to find and small molecules that affect channel function should not only provide powerful tools for dissecting K2P mechanism but should aid in the development of new therapeutic agents for a range of human diseases.

DESCRIPTION (provided by applicant): The long-term goals of this project are to develop a high-resolution understanding of ion channel function and regulation. We are focused on understanding the architectural foundations that underlie the modulation and assembly of two exemplar classes of the voltage-gated ion channel (VGIC) superfamily, Kv7 and TRPM channels. Macromolecular complexes of these channels play pivotal roles in bioelectrical signaling throughout the nervous, sensory, auditory, and cardiovascular systems. Efforts are directed at understanding how intracellular modules from these channels interact with regulatory proteins and how the coiled-coil assembly domains that are a common feature of both classes direct assembly and assembly specificity determinants. We focus on two central questions: 1) What is the structural nature of the calmodulin binding apparatus in the Kv7 C-terminal tail and how do disease mutations affect calmodulin interactions with the C-terminal tail? 2) What is the structural nature of the intracellular assembly domains of TRPM and Kv7 channels and are there common themes directing heteromeric complex assembly? Elaboration of the underlying Kv7 and TRPM structural framework is essential for understanding how these and other VGICs are integrated into intracellular signaling pathways and for developing novel ways to intervene to control channel function. Our efforts encompass a multidisciplinary approach that includes biochemical, biophysical, X-ray crystallographic, and electrophysiological measurements to dissect function. Because of their important role in human physiology, VGICs are the targets for drugs with great utility for the treatment of cardiac arrhythmias, hypertension, congestive heart failure, epilepsy, and chronic pain. Thus, understanding their structures and mechanisms of action at atomic level detail should greatly assist the development of valuable therapeutic agents for a wide range of human ailments. PUBLIC HEALTH RELEVANCE: Voltage-gated ion channels (VGICs) are the targets of drugs used to treat hypertension, arrhythmia, pain, epilepsy, and mood disorders. Our work aims to understand the molecular architecture that underlies VGIC function. Such understanding has direct relevance for development of more efficacious treatments of nervous system and cardiovascular disorders.

? DESCRIPTION (provided by applicant): The long-term goals of this project are to develop an understanding of the fundamental mechanisms that control the function of K2P potassium channels and to identify, develop, and characterize small molecule, ion channel modulators for the K2P family. K2Ps are a diverse family of potassium-selective channels that are responsible for background 'leak' currents. These currents are pivotal in modulating the excitability of neurons. K2Ps respond to varied stimuli that include pH changes, temperature, and mechanical force. Although K2Ps have well-established roles in the nervous and cardiovascular systems and are implicated in pain, anesthetic responses, thermosensation, and mood, they are the least well-understood potassium channel class. Ion channels are coveted drug targets. As membrane proteins, they are readily accessible to extracellular compounds and their modulation brings about rapid changes in the properties of excitable cells in the heart and brain. However, as membrane proteins, they also reside beyond many of the well-established approaches for modulator development that require purified material. Consequently, many channels, including those in the K2P family, lack significant pharmacologies. This problem leads to a gap in our ability to connect ion channel genes with in vivo function. We are pursuing a multidisciplinary approach that includes biophysical, structural, and electrophysiological measurements and genetic selections to identify, dissect, and characterize the core elements that control K2P function and to define and characterize new small molecules that control K2P activity. Defining the molecular mechanisms that control K2p activity and uncovering new K2P modulators should provide the key framework and necessary tools for understanding how K2Ps function. Because of their important roles in human physiology, K2Ps are targets for drugs for the treatment of chronic pain, stroke, and depression. Thus, developing an understanding of how K2Ps function and means to find and small molecules that affect channel function should not only provide powerful tools for dissecting K2P mechanism but should aid in the development of new therapeutic agents for a range of human diseases.

Project Summary The long-term goals of this project are to develop new small molecules that can be used to control K2P potassium channel function and that will be able to label K2P channels so that they can be imaged in cells and in vivo. K2Ps are responsible for ?leak? potassium currents that are pivotal in modulating the excitability of neurons. Members from this diverse potassium channel family respond to varied stimuli that include pH changes, temperature, and mechanical force. K2P channels have well-established roles in the nervous and cardiovascular systems and are implicated in pain, anesthetic responses, thermosensation, and mood, but remain the least well-understood potassium channel class. Ion channels are highly desirable drug targets as they are readily accessible to extracellular compounds and their modulation brings about rapid changes in excitable cell function in the heart and brain. Nevertheless many channels, including all K2P family members, lack pharmacological agents that can selectively affect function. This lack of pharmacological control creates a serious deficiency in our ability to understand, probe, and impact K2P in vivo function. We seek to address this fundamental gap by building on recent discoveries from our laboratory that define a novel small molecule binding site in the mechano- and thermo-sensitive TREK K2P subfamily that is important for pain, analgesic responses, and mood. We will leverage a multidisciplinary approach that includes structure-guided small molecule design together with structural and electrophysiological measurements to create new, selective chemical agents that can be used to probe K2P activity. Because of their important roles in human physiology, K2Ps are targets for drugs for the treatment of chronic pain, stroke, and depression. Thus, developing new small molecules that affect K2P channel function should not only provide powerful tools for dissecting in vivo activity of K2Ps but should aid in the development of new therapeutic agents for a range of human diseases.