Tsung-Yu Chen - US grants

DESCRIPTION (provided by applicant): My long-term research interest is to understand the structure and function of ion channels. These proteins catalyze the translocation of ions across cell membranes and are pivotal in controlling many fundamental physiological processes. In this application, I propose to continue our work on gating-permeation coupling mechanism of the Torpedo ClC-0 chloride (CI-) channel, which is considered as a prototype for the family of "ClC-type" channels. These CIC channels are important in their own right because they are found in many tissues such as skeletal muscle, kidney and brain, and disruptions of these genes cause myotonia, kidney stone diseases, and developmental deficits in brain structures respectively. Moreover, the operations of these channels are particularly interesting because the gating and permeation processes are tightly coupled. In particular, the voltage dependence of muscle-type ClC channels appears to come from this gating-permeation coupling, a mechanism completely different from that in the traditional "S4"-type cation channels. A mechanistic study of this coupling mechanism, therefore, is of fundamental importance to understand the malfunction of ClC channel proteins defective in human diseases. Most recently, the 3-D structures of two bacterial ClC channels were solved by the MacKinnon Lab. We will take advantage of the structures from bacterial channels to explore the structural basis of the gating mechanism of CIC-0. In particular, we will study the "fast gating" of this Torpedo channel, using heterologously expressed channels in Xenopus oocytes. We will first study an electrostatic interaction at the inner pore mouth known to be critical in controlling the fast gating of CIC-0. We will also investigate how a CI ion at the selectivity filter affects the fast gating. We will explore the functional role of a critical glutamate residue that appears to be important in interacting CI at the selectivity filter. Finally, we will study the gating motion that underlies the open-close transition of the channel. The results from this study will not only lead to a further understanding on the coupling of ion permeation to the fast gating in ClC-0 but will provide insight for understanding the gating of other CIC channels.

Ion channels are protein molecules on the cell membrane that help transport ions into and out of the cell. To accomplish the mission, ion channels consist of [unreadable]pores[unreadable] through which ions move to cross the membrane. The opening and closing of the channel pore, a process called [unreadable]gating[unreadable], determines if ions can go through the channel, and therefore is critical for controlling many physiological processes. Our long-term interest is to understand how ion channels can function as nano-machines to transport ions across cell membranes. In this application, I propose to continue our work in studying the mechanisms of gating in [unreadable]CLC channels[unreadable], which are important for Cl- transport in a variety of tissues such as skeletal muscle, kidney and brain. Disruptions of these channels, many of which cause malfunctions of channel gating, result in myotonia, kidney diseases, and developmental deficits in brain structures, to name a few. In this proposal, the molecular motions of the CLC channel proteins associated with two gating mechanisms, the [unreadable]fast-gating[unreadable] and the [unreadable]slow/common gating[unreadable] will be studied. The fast-gating controls the opening and closing of the pore with averaged transition time on the order of ~10 ms. Recent studies on the blockade of the pore of CLC-0, a prototype CLC channel, by a chemical compound, parachlorophenoxy acetate (CPA), suggested a conformational change of the pore during fast-gating. Our recent experiments found that various chemical compounds, including a series of fatty acids (FA), could also block the CLC-0 pore with a similar mechanism. We will study the blockade of CLC-0 by CPA and these various FA blockers to explore the possible gating motion associated with the fast-gating. The slow/common-gating of CLC channels operates with a rate slower than that of the fast gating. We recently found that the slow/common-gating of CLC channels may involve a movement of the C-terminal cytoplasmic region of the channel. In addition, ATP, a ubiquitous molecule in all cells, inhibits CLC-1 through modulating the common-gate, presumably by binding to an ATP-binding site in the C-terminal cytoplasmic region. Physiologically, the inhibition of CLC-1 by ATP is critical for the skeletal-muscle fiber to overcome fatigue. Thus, elucidating where the ATP-binding site is and how ATP binding exerts an inhibitory effect on CLC-1 is important for understanding the skeletal-muscle functions. To examine the gating mechanisms of CLC channels, we will employ electrophysiolgical recording and fluorescence imaging techniques. The results from this study not only will lead to a further understanding on the CLC channel functions but will provide insight for designing molecules that can modulate CLC channels, and eventually for developing drugs in treating diseases resulting from CLC channelopathy.

DESCRIPTION (provided by applicant): Homeostasis of ion distributions across cell membranes is critical for the functional operation of living cells. Ion channels help transport ions into and out of the cell, and thus play important roles in many physiological functions. To accomplish the task, ion channels consist of "pores", and the opening and closing of the pore, a process called "gating", determines if ions can go through the channel. Our long-term interest is to understand how ion channels can function as nano-machines for ion transports, and how the gating functions of these nano-machines control cellular physiology. In this application I propose to study "CLC channels", whose malfunctions result in various human diseases. Specifically, we will examine the mechanisms of the slow/common-gating of two voltage-gated CLC channels, CLC-0 and CLC-1. These two channels are highly homologous to each other-both channels are composed of two identical subunits, and each subunit forms a Cl--conducting pore. Two types of gating mechanisms are present in these channels-the fast- and the slow/common-gating. The fast-gating, a better-characterized mechanism, is known to control the opening and closing of the individual protopore. On the other hand, the mechanism of the slow/common-gating is poorly understood. Recently, we found that the slow/common-gating of CLC channels may involve a movement of the C-terminal cytoplasmic region of the channel. We also found that ATP, a physiologically critical molecule in all cells, inhibits CLC-1 by modulating the common-gating, presumably by binding to an ATP-binding site in the C- terminal cytoplasmic region. These preliminary results suggested a hypothesis that modulating the interaction between the two subunits may be the underlying mechanism of the ATP effect on the slow/common-gating. Physiologically, the inhibition of CLC-1's common-gating by ATP is critical for overcoming muscle fatigue. The slow/common-gating mechanism is also pathologically important because mutations that disrupt the common- gating of CLC-1 are known to cause the dominant form of myotonia. In this application we will combine electrophysiological and fluorescence imaging approaches to study how ATP binding exerts an inhibitory effect on CLC-1, and how myotonia mutations will disrupt the molecular function of CLC channels. These studies will unveil at the molecular level the roles of CLC-1 in muscle fatigue, and will also provide insight for the molecular mechanisms of CLC channelopathy. PUBLIC HEALTH RELEVANCE: This application will study CLC channels, which are chloride channels critical for chloride ion transports across cell membranes. In particular, the slow/common-gating mechanisms of CLC-0 and CLC-1 will be studied, and the regulation of the common-gating mechanism of CLC-1 by intracellular ATP and oxidants will be examined. Illustrating the underlying mechanisms of the interactions of the channel with these physiological ligands will further our understanding of the molecular functions of CLC-1 channels in muscle physiology and may help develop therapeutic strategies in treating diseases, such as myotonia, resulting from CLC channel defects.

DESCRIPTION (provided by applicant): Our long-term goal is to understand the detailed mechanisms of the functional role of K channels in the inner ear. The importance of K channels is underpinned by the fact that mutations of KCNQ channels results in deafness in humans as seen in Jervell Lang Nielson syndrome (JLNS), and an autosomal dominant form of nonsyndromic progressive hearing loss (PHL: DFNA2). To understand the underlying mechanisms of KCNQ channel functions, we are conducting experiments that are revealing surprising, yet insightful results that may delineate the cellular and molecular mechanisms of the channel in normal and in disease states. A diverse class of functionally distinct form of alternatively-spliced KCNQ4 channels is expressed in the cochlea with distinct tonotopic profiles. However, because the channels' form heteromultimers, a dominant-negative form of one KCNQ channel cripples the function of the entire class of K channels in the inner ear. Even more surprising is the finding that different splice variants of the channel have a paradoxical mechanism for K+ extrusion; some of the KCNQ4 isoforms are more efficient in K+ extrusion when the external K+ concentration increases, making the channel a key protein in K+ regulation in the inner ear. In Aim 1, we hypothesize although KCNQ2-5 channels are derived from different genes, the channels interact to produce diverse current phenotypes in the inner ear to confer a pivotal role a K+ homeostasis. Because of the unique interaction, a dominant negative (DN) version of one form cripples the entire class of KCNQ2-5 in the inner ear. We will clone the inner ear-specific channels, localize their differential expression and determine the molecular determinants of their functions. The mechanisms of modulation of KCNQ4 by interacting protein partners and second messengers will also be assessed. In Aim 2, we hypothesize that differential expression of different isoforms/alternative splice variants is the underlying mechanisms for the base-to-apex PHL seen in mutations of KCNQ4 channels. Finally, we will determine the functional determinants of the channel that allow it to extrude K+ in increased external K+. Thus, testing the hypothesis that crucial to the role of KCNQ4 channels in the inner ear is their paradoxical feature with some isoforms having enhanced extrusion of K+ even in the presence of increased extracellular K+. We will use molecular biological, (cloning) biochemical (yeast 2-hybrid systems and siRNA), and functional electrophysiological techniques to address the Aims of the proposal. All experiments will use mice as model. Collectively, these studies will substantially expand our understanding of the cellular mechanisms for the regulation of K+ in the inner ear. Understanding the role of KCNQ channels and their associated proteins in the inner ear is necessary to design therapies for hearing loss (e.g. PHL).7 7. Project Narrative Our study will directly examine the molecular mechanisms of the functional coupling of KCNQ channels with other binding proteins and examine differential isoform-specific subcellular localization of the channels in mice cochlea HCs and SGNs. We will also identify the novel interacting proteins and their functional significance. The study will have important implications in our understanding of how point mutations of a single animo acid (aa) in the channel can cripple an entire family of KCNQ channels and lead to PHL. Indeed, the findings may transcend inner ear-specific KCNQ channel functions. Because the channel produces characteristic muscarinic (M)-type currents in several sensory neurons, these studies will address fundamental uncertainties that surround the functions of the channels throughout the nervous system.

? DESCRIPTION (provided by applicant): Members of the CLC chloride (Cl-) channel/transporter family are ubiquitously found in various living species from bacteria to human beings. One type of CLC members, including CLC-0 in the Torpedo electric organ and CLC-1 expressed in skeletal muscle membranes, function as Cl- channels critical for controlling membrane excitability. For example, malfunction of CLC-1 often causes hyper-excitability of skeletal muscle membranes, leading to a muscle disease called myotonia. CLC-0 and CLC-1 are highly homologous to each other with similar channel-opening (also called gating) mechanisms. The gating mechanisms of CLC-0 and CLC-1 are controlled by the voltage across cell membranes as well as by the anions in the channel pore. My long-term interest is to understand how these CLC channels transport Cl- ions across cell membranes, how the gating functions of these channels control cellular physiology, and how various mutations of the channel lead to channel malfunctions (called channelopathy). In this application I propose three aims to explore two research directions. AIM 1 and AIM 2 are designed to study the slow/common gating mechanism of CLC channels, and to examine one type of CLC channel's malfunction-the inverted voltage-dependent channel activation. AIM 3 is focused on the physiological roles of CLC-1 modulations in controlling the dynamic change of the conductance of skeletal muscle membranes. In AIM 1, we hypothesize that the gating abnormality of the inverted voltage-dependent activation is due to an excessive lockdown of the channel gate by anions in the pore. We will test this hypothesis by examining the biophysical properties of the WT and mutant CLC channels in various anion and pH conditions. We will also destabilize anion binding in the pore to test if destabilizing the lockdown of the gate by anions can correct the inverted voltage activation of the channel. We also hypothesize that the lockdown of the channel gate exists in the normal slow/common gating of CLC channels though with a less strength than in the mutants with inverted voltage activation. Because slow/common gating is previously suggested to involve interaction between CLC channel's two subunits, we hypothesize in AIM 2 that the subunit interaction is altered in channel mutants with inverted voltage activation. We wil take advantage of a cadmium-binding site located at the dimer interface of the channel to examine if the mutations that reverse voltage activation alter this cadmium-binding site. In AIM 3, the roles of CLC-1 modulations in skeletal muscle fibers will be examined. We previously found that ATP inhibits expressed CLC-1 channels in acidic intracellular pH, a mechanism thought to be critical for preventing early muscle fatigue. We will translate our previous findings to muscle tissues to ask if CLC-1 modulation by ATP/H+ is indeed important in the native environment of muscle fibers. We will combine expertise from our lab and the lab of our collaborator to understand the roles of CLC-1 modulations in dynamic change of the membrane conductance of skeletal muscles.