Structure and Function of Calcium-Activated Chloride Channels and Phospholipid Scramblases in the TMEM16 Family.

The transmembrane protein 16 (TMEM16) family consists of Ca2+-activated chloride channels and phospholipid scramblases. Ten mammalian TMEM16 proteins, TMEM16A-K (with no TMEM16I), and several non-mammalian TMEM16 proteins, such as afTMEM16 and nhTMEM16, have been discovered. All known TMEM16 proteins are homodimeric proteins containing two subunits. Each subunit consists of ten transmembrane helices with Ca2+-binding sites and a single ion-permeation/phospholipid transport pathway. The ion-permeation pathway and the phospholipid transport pathway of TMEM16 proteins have a wide intracellular vestibule, a narrow neck, and a smaller extracellular vestibule. Interestingly, the lining wall of the ion-permeation/phospholipid transport pathway may be formed, at least partially, by membrane phospholipids, though the degree of pore-wall forming by phospholipids likely varies among TMEM16 proteins. Thus, the biophysical properties and activation mechanisms of TMEM16 proteins could differ from each other accordingly. Here we review the current understanding of the structure and function of TMEM16 molecules.

Biophysical and Pharmacological Insights to CLC Chloride Channels.

The CLC family encompasses two functional categories of transmembrane proteins: chloride conducting channels and proton-chloride antiporters. All members in this chloride channel/transporter family consist of two identical protein subunits, and each subunit forms an independent ion-transport pathway, a structural architecture known as "double barrel." These CLC proteins serve biological functions ranging from membrane excitability and cell volume regulation to acidification of endosomes. Despite their ubiquitous expression, physiological significance, and resolved molecular structures of some of the family members, the mechanisms governing these molecules' biophysical functions are still not completely settled. However, a series of functional and structural studies have brought insights into interesting questions related to these proteins. This chapter explores the functional peculiarities underlying CLC channels aided by information observed from the chloride-proton antiporters in the CLC family. The overall structural features of these CLC proteins will be presented, and the biophysical functions will be addressed. Finally, the mechanism of pharmacological agents that interact with CLC channels will also be discussed.

Divalent Cation Modulation of Ion Permeation in TMEM16 Proteins.

Intracellular divalent cations control the molecular function of transmembrane protein 16 (TMEM16) family members. Both anion channels (such as TMEM16A) and phospholipid scramblases (such as TMEM16F) in this family are activated by intracellular Ca2+ in the low µM range. In addition, intracellular Ca2+ or Co2+ at mM concentrations have been shown to further potentiate the saturated Ca2+-activated current of TMEM16A. In this study, we found that all alkaline earth divalent cations in mM concentrations can generate similar potentiation effects in TMEM16A when applied intracellularly, and that manipulations thought to deplete membrane phospholipids weaken the effect. In comparison, mM concentrations of divalent cations minimally potentiate the current of TMEM16F but significantly change its cation/anion selectivity. We suggest that divalent cations may increase local concentrations of permeant ions via a change in pore electrostatic potential, possibly acting through phospholipid head groups in or near the pore. Monovalent cations appear to exert a similar effect, although with a much lower affinity. Our findings resolve controversies regarding the ion selectivity of TMEM16 proteins. The physiological role of this mechanism, however, remains elusive because of the nearly constant high cation concentrations in cytosols.

Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect.

The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2-deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90ß (Hsp90ß) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90ß-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.

FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane.

Mutations in the skeletal muscle-specific CLC-1 chloride channel are associated with the human hereditary disease myotonia congenita. The molecular pathophysiology underlying some of the disease-causing mutations can be ascribed to defective human CLC-1 protein biosynthesis. CLC-1 protein folding is assisted by several molecular chaperones and co-chaperones, including FK506-binding protein 8 (FKBP8). FKBP8 is generally considered an endoplasmic reticulum- and mitochondrion-resident membrane protein, but is not thought to contribute to protein quality control at the cell surface. Herein, we aim to test the hypothesis that FKBP8 may regulate CLC-1 protein at the plasma membrane. Surface biotinylation and subcellular fractionation analyses reveal that a portion of FKBP8 is present at the plasma membrane, and that co-expression with CLC-1 enhances surface localization of FKBP8. Immunoblotting analyses of plasma membrane proteins purified from skeletal muscle further confirm surface localization of FKBP8. Importantly, FKBP8 promotes CLC-1 protein stability at the plasma membrane. Together, our data underscore the importance of FKBP8 in the peripheral quality control of CLC-1 channel.

Protein kinase C-dependent regulation of ClC-1 channels in active human muscle and its effect on fast and slow gating.

KEY POINTS: Regulation of ion channel function during repeated firing of action potentials is commonly observed in excitable cells. Recently it was shown that muscle activity is associated with rapid, protein kinase C (PKC)-dependent ClC-1 Cl(-) channel inhibition in rodent muscle. While this PKC-dependent ClC-1 inhibition during muscle activity was shown to be important for the maintenance of contractile endurance in rat muscle it is unknown whether a similar regulation exists in human muscle. Also, the molecular mechanisms underlying the observed PKC-dependent ClC-1 inhibition are unclear. Here we present the first demonstration of ClC-1 inhibition in active human muscle fibres, and we determine the changes in ClC-1 gating that underlie the PKC-dependent ClC-1 inhibition in active muscle using human ClC-1 expressed in Xenopus oocytes. This activity-induced ClC-1 inhibition is suggested to represent a mechanism by which human muscle fibres maintain their excitability during sustained activity. ABSTRACT: Repeated firing of action potentials (APs) is known to trigger rapid, protein kinase C (PKC)-dependent inhibition of ClC-1 Cl(-) ion channels in rodent muscle and this inhibition is important for contractile endurance. It is currently unknown whether similar regulation exists in human muscle, and the molecular mechanisms underlying PKC-dependent ClC-1 inhibition are unclear. This study first determined whether PKC-dependent ClC-1 inhibition exists in active human muscle, and second, it clarified how PKC alters the gating of human ClC-1 expressed in Xenopus oocytes. In human abdominal and intercostal muscles, repeated AP firing was associated with 30-60% reduction of ClC-1 function, which could be completely prevented by PKC inhibition (1 µm GF109203X). The role of the PKC-dependent ClC-1 inhibition was evaluated from rheobase currents before and after firing 1000 APs: while rheobase current was well maintained after activity under control conditions it rose dramatically if PKC-dependent ClC-1 inhibition had been prevented with the inhibitor. This demonstrates that the ClC-1 inhibition is important for maintenance of excitability in active human muscle fibres. Oocyte experiments showed that PKC activation lowered the overall open probability of ClC-1 in the voltage range relevant for AP initiation in muscle fibres. More detailed analysis of this reduction showed that PKC mostly affected the slow gate of ClC-1. Indeed, there was no effect of PKC activation in C277S mutated ClC-1 in which the slow gate is effectively locked open. It is concluded that regulation of excitability of active human muscle fibres relies on PKC-dependent ClC-1 inhibition via a gating mechanism.

Intracellular ß-nicotinamide adenine dinucleotide inhibits the skeletal muscle ClC-1 chloride channel.

ClC-1 is the dominant sarcolemmal chloride channel and plays an important role in regulating membrane excitability that is underscored by ClC-1 mutations in congenital myotonia. Here we show that the coenzyme ß-nicotinamide adenine dinucleotide (NAD), an important metabolic regulator, robustly inhibits ClC-1 when included in the pipette solution in whole cell patch clamp experiments and when transiently applied to inside-out patches. The oxidized (NAD(+)) form of the coenzyme was more efficacious than the reduced (NADH) form, and inhibition by both was greatly enhanced by acidification. Molecular modeling, based on the structural coordinates of the homologous ClC-5 and CmClC proteins and in silico docking, suggest that NAD(+) binds with the adenine base deep in a cleft formed by ClC-1 intracellular cystathionine ß-synthase domains, and the nicotinamide base interacts with the membrane-embedded channel domain. Consistent with predictions from the models, mutation of residues in cystathionine ß-synthase and channel domains either attenuated (G200R, T636A, H847A) or abrogated (L848A) the effect of NAD(+). In addition, the myotonic mutations G200R and Y261C abolished potentiation of NAD(+) inhibition at low pH. Our results identify a new biological role for NAD and suggest that the main physiological relevance may be the exquisite sensitivity to intracellular pH that NAD(+) inhibition imparts to ClC-1 gating. These findings are consistent with the reduction of sarcolemmal chloride conductance that occurs upon acidification of skeletal muscle and suggest a previously unexplored mechanism in the pathophysiology of myotonia.

Full-field microimaging with 8 keV X-rays achieves a spatial resolutions better than 20 nm.

Fresnel zone plates (450 nm thick Au, 25 nm outermost zone width) used as objective lenses in a full field transmission reached a spatial resolution better than 20 nm and 1.5% efficiency with 8 keV photons. Zernike phase contrast was also realized without compromising the resolution. These are very significant achievements in the rapid progress of high-aspect-ratio zone plate fabrication by combined electron beam lithography and electrodeposition.

Hard x-ray Zernike microscopy reaches 30 nm resolution.

Since its invention in 1930, Zernike phase contrast has been a pillar in optical microscopy and more recently in x-ray microscopy, in particular for low-absorption-contrast biological specimens. We experimentally demonstrate that hard-x-ray Zernike microscopy now reaches a lateral resolution below 30 nm while strongly enhancing the contrast, thus opening many new research opportunities in biomedicine and materials science.

Physiology and pathophysiology of CLC-1: mechanisms of a chloride channel disease, myotonia.

The CLC-1 chloride channel, a member of the CLC-channel/transporter family, plays important roles for the physiological functions of skeletal muscles. The opening of this chloride channel is voltage dependent and is also regulated by protons and chloride ions. Mutations of the gene encoding CLC-1 result in a genetic disease, myotonia congenita, which can be inherited as an autosmal dominant (Thomsen type) or an autosomal recessive (Becker type) pattern. These mutations are scattered throughout the entire protein sequence, and no clear relationship exists between the inheritance pattern of the mutation and the location of the mutation in the channel protein. The inheritance pattern of some but not all myotonia mutants can be explained by a working hypothesis that these mutations may exert a "dominant negative" effect on the gating function of the channel. However, other mutations may be due to different pathophysiological mechanisms, such as the defect of protein trafficking to membranes. Thus, the underlying mechanisms of myotonia are likely to be quite diverse, and elucidating the pathophysiology of myotonia mutations will require the understanding of multiple molecular/cellular mechanisms of CLC-1 channels in skeletal muscles, including molecular operation, protein synthesis, and membrane trafficking mechanisms.

Large movement in the C terminus of CLC-0 chloride channel during slow gating.

Chloride channels and transporters of the CLC gene family are expressed in virtually all cell types and are crucial in the regulation of membrane potential, chloride homeostasis and intravesicular pH. There are two gating processes that open CLC channels-fast and slow. The fast gating process in CLC channels has recently been linked to a small movement of a glutamate side chain. However, the molecular mechanism underlying the slow gating process is still elusive. Using spectroscopic microscopy, we observed a large backbone movement in the C terminus of the CLC-0 chloride channel that was functionally linked to slow gating. We further showed that the C-terminal movement had a time course similar to slow gating. In addition, a mutation known to lock the slow gate open prevented movement of the C terminus. When combined with recent structural information on the CLC C terminus, our findings provide a structural model for understanding the conformational changes linked to slow gating in CLC transport proteins.

The link between abnormalities of calcium handling proteins and catecholaminergic polymorphic ventricular tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT), a rare autosomal dominant or recessive disease, usually results in syncope or sudden cardiac death. Most CPVT patients do not show abnormal cardiac structure and electrocardiogram features and symptoms, usually onset during adrenergically mediated physiological conditions. CPVT tends to occur at a younger age and is not easy to be diagnosed and managed. The main cause of CPVT is associated with mishandling Ca2+ in cardiomyocytes. Intracellular Ca2+ is strictly controlled by a protein located in the sarcoplasm reticulum (SR), such as ryanodine receptor, histidine-rich Ca2+-binding protein, triadin, and junctin. Mutation in these proteins results in misfolding or malfunction of these proteins, thereby affecting their Ca2+-binding affinity, and subsequently disturbs Ca2+ homeostasis during excitation-contraction coupling (E-C coupling). Furthermore, transient disturbance of Ca2+ homeostasis increases membrane potential and causes Ca2+ store overload-induced Ca2+ release, which in turn leads to delayed after depolarization and arrhythmia. Previous studies have focused on the interaction between ryanodine receptors and protein kinase or phosphatase in the cytosol. However, recent studies showed the regulation signaling for ryanodine receptor not only from the cytosol but also within the SR. The changing of Ca2+ concentration is critical for protein interaction inside the SR which changes protein conformation to regulate the open probability of ryanodine receptors. Thus, it influences the threshold of Ca2+ released from the SR, making it easier to release Ca2+ during E-C coupling. In this review, we briefly discuss how Ca2+ handling protein variations affect the Ca2+ handling in CPVT.

Accessibility of the CLC-0 pore to charged methanethiosulfonate reagents.

Using the substituted-cysteine-accessibility method, we previously showed that a cysteine residue introduced to the Y512 position of CLC-0 was more rapidly modified by a negatively charged methanethiosulfonate (MTS) reagent, 2-sulfonatoethyl MTS (MTSES), than by the positively charged 2-(trimethylammonium)ethyl MTS (MTSET). This result suggests that a positive intrinsic pore potential attracts the negatively charged MTS molecule. In this study, we further test this hypothesis of a positive pore potential in CLC-0 and find that the preference for the negatively charged MTS is diminished significantly in modifying the substituted cysteine at a deeper pore position, E166. To examine this conundrum, we study the rates of MTS inhibitions of the E166C current and those of the control mutant current from E166A. The results suggest that the inhibition of E166C by intracellularly applied MTS reagents is tainted by the modification of an endogenous cysteine, C229, located at the channel's dimer interface. After this endogenous cysteine is mutated, CLC-0 resumes its preference for selecting MTSES in modifying E166C, reconfirming the idea that the pore of CLC-0 is indeed built with a positive intrinsic potential. These experiments also reveal that MTS modification of C229 can inhibit the current of CLC-0 depending on the amino acid placed at position 166.

A three-state multi-ion kinetic model for conduction properties of ClC-0 chloride channel.

A three-state, multiion kinetic model was proposed to enable the conduction properties of the mammalian channel ClC-0 to be well characterized. Using this rate-theory based model, the current-voltage and conductance-concentration relations were obtained. The five parameters needed were determined by fitting the data of conduction experiments of the wild-type ClC-0 and its K519C mutant. The model was then tested against available calculation and simulation data, and the energy differences between distinct chloride-occupancy states computed agreed with an independent calculation on the binding free energies solved by using the Poisson-Boltzmann equation. The average ion number of conduction and the ion passing duration calculated closely resembled the values obtained from Brownian dynamics simulations. According to the model, the decrease of conductance caused by mutating residue K519 to C519 can be attributed to the effect of K519C mutation on translocation rate constants. Our study sets up a theoretical model for ion permeation and conductance in ClC-0. It provides a starting point for experimentalists to test the three-state model, and would help in understanding the conduction mechanism of ClC-0.

Chloride channels and transporters in human corneal epithelium.

Transport of water and electrolytes is critical for corneal clarity. Recent studies indicate another important function of transport of ions and electrolytes - establishing wound electric fields that guide cell migration. We found chloride (Cl(-)) flux is a major component of the corneal wound electric current. In order to elucidate the mechanisms of Cl(-) transport, we studied Cl(-) channels and transporters in human corneal epithelial (HCE) cells. We tested a transformed human corneal epithelial cell line (tHCE), primary cultures of human corneal epithelial cells (pHCE), and human donor corneas. We first used RT-PCR to determine expression levels of mRNA of CLC (Cl(-) channels/transporters of CLC gene family) family members and CFTR (cystic fibrosis transmembrane conductance regulator) in HCE cells. We then confirmed protein expression and distribution of selected CLC family members and CFTR with Western blot and immunofluorescence confocal microscopy. Finally, Cl(-) currents were recorded with electrophysiological techniques. The mRNAs of CLC-2, CLC-3, CLC-4, CLC-5, CLC-6, and CFTR were detected in the HCE cell line. CLC-1 and CLC-7 were not detectable. Western blot and immunostaining confirmed protein expression and distribution of CLC-2, CLC-3, CLC-4, CLC-6 and CFTR in human corneal epithelium. CLC-2 preferentially labeled the apical and basal layers, while CLC-3 and CLC-4 labeled only the superficial layer. CLC-6 and CFTR labeling showed a unique gradient with strong staining in apical layers which gradually decreased towards the basal layers. Corneal endothelium was positive for CLC-2, CLC-3, CLC-4, CLC-6 and possibly CFTR. Human corneal epithelial cells demonstrated voltage dependent Cl(-) currents. HCE cells express functional Cl(-) channels and transporters. CLC-2, CLC-3, CLC-4, CLC-6, and CFTR had distinct expression patterns in human corneal epithelium. Those molecules and their distribution may play important roles in maintaining resting Cl(-) fluxes and in regulating Cl(-) flux at corneal wounds, which may be a major contributor to wound electrical signaling.

Proton-dependent inhibition, inverted voltage activation, and slow gating of CLC-0 Chloride Channel.

CLC-0, a prototype Cl- channel in the CLC family, employs two gating mechanisms that control its ion-permeation pore: fast gating and slow gating. The negatively-charged sidechain of a pore glutamate residue, E166, is known to be the fast gate, and the swinging of this sidechain opens or closes the pore of CLC-0 on the millisecond time scale. The other gating mechanism, slow gating, operates with much slower kinetics in the range of seconds to tens or even hundreds of seconds, and it is thought to involve still-unknown conformational rearrangements. Here, we find that low intracellular pH (pHi) facilitates the closure of the CLC-0's slow gate, thus generating current inhibition. The rate of low pHi-induced current inhibition increases with intracellular H+ concentration ([H+]i)-the time constants of current inhibition by low pHi = 4.5, 5.5 and 6 are roughly 0.1, 1 and 10 sec, respectively, at room temperature. In comparison, the time constant of the slow gate closure at pHi = 7.4 at room temperature is hundreds of seconds. The inhibition by low pHi is significantly less prominent in mutants favoring the slow-gate open state (such as C212S and Y512A), further supporting the fact that intracellular H+ enhances the slow-gate closure in CLC-0. A fast inhibition by low pHi causes an apparent inverted voltage-dependent activation in the wild-type CLC-0, a behavior similar to those in some channel mutants such as V490W in which only membrane hyperpolarization can open the channel. Interestingly, when V490W mutation is constructed in the background of C212S or Y512A mutation, the inverted voltage-dependent activation disappears. We propose that the slow kinetics of CLC-0's slow-gate closure may be due to low [H+]i rather than due to the proposed large conformational change of the channel protein. Our results also suggest that the inverted voltage-dependent opening observed in some mutant channels may result from fast closure of the slow gate by the mutations.

Cobalt ion interaction with TMEM16A calcium-activated chloride channel: Inhibition and potentiation.

TMEM16A, a Ca2+-sensitive Cl- channel, plays key roles in many physiological functions related to Cl- transport across lipid membranes. Activation of this channel is mediated via binding intracellular Ca2+ to the channel with a relatively high apparent affinity, roughly in the sub-µM to low µM concentration range. Recently available high-resolution structures of TMEM16 molecules reveal that the high-affinity Ca2+ activation sites are formed by several acidic amino acids, using their negatively charged sidechain carboxylates to coordinate the bound Ca2+. In this study, we examine the interaction of TMEM16A with a divalent cation, Co2+, which by itself cannot activate current in TMEM16A. This divalent cation, however, has two effects when applied intracellularly. It inhibits the Ca2+-induced TMEM16A current by competing with Ca2+ for the aforementioned high-affinity activation sites. In addition, Co2+ also potentiates the Ca2+-induced current with a low affinity. This potentiation effect requires high concentration (mM) of Co2+, similar to our previous findings that high concentrations (mM) of intracellular Ca2+ ([Ca2+]i) can induce more TMEM16A current after the Ca2+-activation sites are saturated by tens of µM [Ca2+]i. The degrees of potentiation by Co2+ and Ca2+ also roughly correlate with each other. Interestingly, mutating a pore residue of TMEM16A, Y589, alters the degree of potentiation in that the smaller the sidechain of the replaced residue, the larger the potentiation induced by divalent cations. We suggest that the Co2+ potentiation and the Ca2+ potentiation share a similar mechanism by increasing Cl- flux through the channel pore, perhaps due to an increase of positive pore potential after the binding of divalent cations to phospholipids in the pore. A smaller sidechain of a pore residue may allow the pore to accommodate more phospholipids, thus enhancing the current potentiation caused by high concentrations of divalent cations.

Defective Gating and Proteostasis of Human ClC-1 Chloride Channel: Molecular Pathophysiology of Myotonia Congenita.

The voltage-dependent ClC-1 chloride channel, whose open probability increases with membrane potential depolarization, belongs to the superfamily of CLC channels/transporters. ClC-1 is almost exclusively expressed in skeletal muscles and is essential for stabilizing the excitability of muscle membranes. Elucidation of the molecular structures of human ClC-1 and several CLC homologs provides important insight to the gating and ion permeation mechanisms of this chloride channel. Mutations in the human CLCN1 gene, which encodes the ClC-1 channel, are associated with a hereditary skeletal muscle disease, myotonia congenita. Most disease-causing CLCN1 mutations lead to loss-of-function phenotypes in the ClC-1 channel and thus increase membrane excitability in skeletal muscles, consequently manifesting as delayed relaxations following voluntary muscle contractions in myotonic subjects. The inheritance pattern of myotonia congenita can be autosomal dominant (Thomsen type) or recessive (Becker type). To date over 200 myotonia-associated ClC-1 mutations have been identified, which are scattered throughout the entire protein sequence. The dominant inheritance pattern of some myotonia mutations may be explained by a dominant-negative effect on ClC-1 channel gating. For many other myotonia mutations, however, no clear relationship can be established between the inheritance pattern and the location of the mutation in the ClC-1 protein. Emerging evidence indicates that the effects of some mutations may entail impaired ClC-1 protein homeostasis (proteostasis). Proteostasis of membrane proteins comprises of biogenesis at the endoplasmic reticulum (ER), trafficking to the surface membrane, and protein turn-over at the plasma membrane. Maintenance of proteostasis requires the coordination of a wide variety of different molecular chaperones and protein quality control factors. A number of regulatory molecules have recently been shown to contribute to post-translational modifications of ClC-1 and play critical roles in the ER quality control, membrane trafficking, and peripheral quality control of this chloride channel. Further illumination of the mechanisms of ClC-1 proteostasis network will enhance our understanding of the molecular pathophysiology of myotonia congenita, and may also bring to light novel therapeutic targets for skeletal muscle dysfunction caused by myotonia and other pathological conditions.

CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy.

Voltage-gated ClC-2 channels are essential for chloride homeostasis. Complete knockout of mouse ClC-2 leads to testicular degeneration and neuronal myelin vacuolation. Gain-of-function and loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the genetic diseases aldosteronism and leukodystrophy, respectively. The protein homeostasis (proteostasis) mechanism of ClC-2 is currently unclear. Here, we aimed to identify the molecular mechanism of endoplasmic reticulum-associated degradation of ClC-2, and to explore the pathophysiological significance of disease-associated anomalous ClC-2 proteostasis. In both heterologous expression system and native neuronal and testicular cells, ClC-2 is subject to significant regulation by cullin-RING E3 ligase-mediated polyubiquitination and proteasomal degradation. The cullin 4 (CUL4)-damage-specific DNA binding protein 1 (DDB1)-cereblon (CRBN) E3 ubiquitin ligase co-exists in the same complex with and promotes the degradation of ClC-2 channels. The CRBN-targeting immunomodulatory drug lenalidomide and the cullin E3 ligase inhibitor MLN4924 promotes and attenuates, respectively, proteasomal degradation of ClC-2. Analyses of disease-related ClC-2 mutants reveal that aldosteronism and leukodystrophy are associated with opposite alterations in ClC-2 proteostasis. Modifying CUL4 E3 ligase activity with lenalidomide and MLN4924 ameliorates disease-associated ClC-2 proteostasis abnormality. Our results highlight the significant role and therapeutic potential of CUL4 E3 ubiquitin ligase in regulating ClC-2 proteostasis.

Comparison of ion transport determinants between a TMEM16 chloride channel and phospholipid scramblase.

Two TMEM16 family members, TMEM16A and TMEM16F, have different ion transport properties. Upon activation by intracellular Ca2+, TMEM16A-a Ca2+-activated Cl- channel-is more selective for anions than cations, whereas TMEM16F-a phospholipid scramblase-appears to transport both cations and anions. Under saturating Ca2+ conditions, the current-voltage (I-V) relationships of these two proteins also differ; the I-V curve of TMEM16A is linear, while that of TMEM16F is outwardly rectifying. We previously found that mutating a positively charged lysine residue (K584) in the ion transport pathway to glutamine converted the linear I-V curve of TMEM16A to an outwardly rectifying curve. Interestingly, the corresponding residue in the outwardly rectifying TMEM16F is also a glutamine (Q559). Here, we examine the ion transport functions of TMEM16 molecules and compare the roles of K584 of TMEM16A and Q559 of TMEM16F in controlling the rectification of their respective I-V curves. We find that rectification of TMEM16A is regulated electrostatically by the side-chain charge on the residue at position 584, whereas the charge on residue 559 in TMEM16F has little effect. Unexpectedly, mutation of Q559 to aromatic amino acid residues significantly alters outward rectification in TMEM16F. These same mutants show reduced Ca2+-induced current rundown (or desensitization) compared with wild-type TMEM16F. A mutant that removes the rundown of TMEM16F could facilitate the study of ion transport mechanisms in this phospholipid scramblase in the same way that a CLC-0 mutant in which inactivation (or closure of the slow gate) is suppressed was used in our previous studies.