Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β.

Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation

Modulation of the slow/common gating of CLC channels by intracellular cadmium.

Members of the CLC family of Cl(-) channels and transporters are homodimeric integral membrane proteins. Two gating mechanisms control the opening and closing of Cl(-) channels in this family: fast gating, which regulates opening and closing of the individual pores in each subunit, and slow (or common) gating, which simultaneously controls gating of both subunits. Here, we found that intracellularly applied Cd(2+) reduces the current of CLC-0 because of its inhibition on the slow gating. We identified CLC-0 residues C229 and H231, located at the intracellular end of the transmembrane domain near the dimer interface, as the Cd(2+)-coordinating residues. The inhibition of the current of CLC-0 by Cd(2+) was greatly enhanced by mutation of I225W and V490W at the dimer interface. Biochemical experiments revealed that formation of a disulfide bond within this Cd(2+)-binding site is also affected by mutation of I225W and V490W, indicating that these two mutations alter the structure of the Cd(2+)-binding site. Kinetic studies showed that Cd(2+) inhibition appears to be state dependent, suggesting that structural rearrangements may occur in the CLC dimer interface during Cd(2+) modulation. Mutations of I290 and I556 of CLC-1, which correspond to I225 and V490 of CLC-0, respectively, have been shown previously to cause malfunction of CLC-1 Cl(-) channel by altering the common gating. Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0. The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd(2+) likely depends on their alteration of subunit interactions

Role of physiological ClC-1 Cl- ion channel regulation for the excitability and function of working skeletal muscle.

Electrical membrane properties of skeletal muscle fibers have been thoroughly studied over the last five to six decades. This has shown that muscle fibers from a wide range of species, including fish, amphibians, reptiles, birds, and mammals, are all characterized by high resting membrane permeability for Cl(-) ions. Thus, in resting human muscle, ClC-1 Cl(-) ion channels account for ∼80% of the membrane conductance, and because active Cl(-) transport is limited in muscle fibers, the equilibrium potential for Cl(-) lies close to the resting membrane potential. These conditions-high membrane conductance and passive distribution-enable ClC-1 to conduct membrane current that inhibits muscle excitability. This depressing effect of ClC-1 current on muscle excitability has mostly been associated with skeletal muscle hyperexcitability in myotonia congenita, which arises from loss-of-function mutations in the CLCN1 gene. However, given that ClC-1 must be drastically inhibited (∼80%) before myotonia develops, more recent studies have explored whether acute and more subtle ClC-1 regulation contributes to controlling the excitability of working muscle. Methods were developed to measure ClC-1 function with subsecond temporal resolution in action potential firing muscle fibers. These and other techniques have revealed that ClC-1 function is controlled by multiple cellular signals during muscle activity. Thus, onset of muscle activity triggers ClC-1 inhibition via protein kinase C, intracellular acidosis, and lactate ions. This inhibition is important for preserving excitability of working muscle in the face of activity-induced elevation of extracellular K(+) and accumulating inactivation of voltage-gated sodium channels. Furthermore, during prolonged activity, a marked ClC-1 activation can develop that compromises muscle excitability. Data from ClC-1 expression systems suggest that this ClC-1 activation may arise from loss of regulation by adenosine nucleotides and/or oxidation. The present review summarizes the current knowledge of the physiological factors that control ClC-1 function in active muscle

Structure of the CLC-1 chloride channel from Homo sapiens.

CLC channels mediate passive Cl- conduction, while CLC transporters mediate active Cl- transport coupled to H+ transport in the opposite direction. The distinction between CLC-0/1/2 channels and CLC transporters seems undetectable by amino acid sequence. To understand why they are different functionally we determined the structure of the human CLC-1 channel. Its 'glutamate gate' residue, known to mediate proton transfer in CLC transporters, adopts a location in the structure that appears to preclude it from its transport function. Furthermore, smaller side chains produce a wider pore near the intracellular surface, potentially reducing a kinetic barrier for Cl- conduction. When the corresponding residues are mutated in a transporter, it is converted to a channel. Finally, Cl- at key sites in the pore appear to interact with reduced affinity compared to transporters. Thus, subtle differences in glutamate gate conformation, internal pore diameter and Cl- affinity distinguish CLC channels and transporters

Methods For Inhibiting Clc-2 Channel With Gatx2

Compositions and methods of using scorpion venom peptide that is a ligand for ClC channels are provided. One aspect provides a pharmaceutical composition containing an amount of GaTx2 effective to inhibit ClC activity. Methods of treating a disorder or symptom of a disorder related to aberrant ClC channel activity are also provided.Georgia Tech Research Corporatio

Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis

Epithelia frequently segregate transport processes to specific cell types, presumably for improved efficiency and control. The molecular players underlying this functional specialization are of particular interest. In Drosophila, the renal (Malpighian) tubule displays the highest per-cell transport rates known and has two main secretory cell types, principal and stellate. Electrogenic cation transport is known to reside in the principal cells, whereas stellate cells control the anion conductance, but by an as-yet-undefined route. Here, we resolve this issue by showing that a plasma membrane chloride channel, encoded by ClC-a, is exclusively expressed in the stellate cell and is required for Drosophila kinin-mediated induction of diuresis and chloride shunt conductance, evidenced by chloride ion movement through the stellate cells, leading to depolarization of the transepithelial potential. By contrast, ClC-a knockdown had no impact on resting secretion levels. Knockdown of a second CLC gene showing highly abundant expression in adult Malpighian tubules, ClC-c, did not impact depolarization of transepithelial potential after kinin stimulation. Therefore, the diuretic action of kinin in Drosophila can be explained by an increase in ClC-a–mediated chloride conductance, over and above a resting fluid transport level that relies on other (ClC-a–independent) mechanisms or routes. This key segregation of cation and anion transport could explain the extraordinary fluid transport rates displayed by some epithelia