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.
This application will study physiology and pathophysiology of CLC channels, which are chloride ion channels critical for regulating skeletal muscle functions. Malfunctions of these chloride channels often lead to a congenital muscle disease called myotonia. In this application we will study the mechanisms underlying CLC channel mutations that cause myotonia. We will also study the regulation of CLC channels by ATP, pH, and oxidation in skeletal muscles. The proposed research will further our understanding of the molecular functions of CLC channels in muscle physiology and may help develop therapeutic strategies in treating diseases, such as myotonia.
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