Distinct ClC-6 and ClC-7 Cl- sensitivities provide insight into ClC-7's role in lysosomal Cl- homeostasis.

ClC-6 and ClC-7 are closely related, intracellular Cl- /H+ antiporters belonging to the CLC family of channels and transporters. They localize to acidic late endosomes and lysosomes and probably function in ionic homeostasis of these contiguous compartments. ClC-7 transport function requires association with the accessory protein Ostm1, whereas ClC-6 transport does not. To elucidate their roles in endo-lysosomes, we measured Cl- - and pH-dependences of over-expressed wild-type ClC-6 and ClC-7, as well as disease-associated mutants, using high-resolution recording protocols. Lowering extracellular Cl- (corresponding to luminal Cl- in endo-lysosomes) reduced ClC-6 currents, whereas it increased transport activity of ClC-7/Ostm1. Low extracellular Cl- activated ClC-7/Ostm 1 under acidic extracellular conditions, as well as under conditions of low intracellular chloride. Activation is conserved in ClC-7Y713C , a variant displaying disrupted PI(3,5)P2 inhibition. Detailed biophysical analysis of disease-associated ClC-6 and ClC-7 gain-of-function (GoF) variants, ClC-6Y553C and ClC-7Y713C , and the ClC-7Y577C and ClC-6Y781C correlates, identified additional functional nuances distinguishing ClC-6 and ClC-7. ClC-7Y577C recapitulated GoF produced by ClC-6Y553C . ClC-6Y781C displayed transport activation qualitatively similar to ClC-7Y713C , although current density did not differ from that of wild-type ClC-6. Finally, rClC-7R760Q , homologous to hClC-7R762Q , an osteopetrosis variant with fast gating kinetics, appeared indifferent to extracellular Cl- , identifying altered Cl- sensitivity as a plausible mechanism underlying disease. Collectively, the present studies underscore the distinct roles of ClC-6 and ClC-7 within the context of their respective localization to late endosomes and lysosomes. In particular, we suggest the atypical inhibition of ClC-7 by luminal Cl- serves to limit excessive intraluminal Cl- accumulation. KEY POINTS: ClC-6 and ClC-7 are late endosomal and lysosomal 2 Cl- /1 H+ exchangers, respectively. When targeted to the plasma membrane, both activate slowly at positive voltages. ClC-6 activity is decreased in low extracellular (i.e. luminal) chloride, whereas ClC-7 is activated by low luminal chloride, even at acidic pH. The functional gain-of-function phenotypes of the ClC-6 and ClC-7 disease mutations ClC-6Y553C and ClC-7Y715C are maintained when introduced in their respective homologues, ClC-7Y577C and ClC-6Y781C , with all mutations retaining chloride dependence of the respective wild type (WT). An osteopetrosis mutation of ClC-7 displaying fast gating kinetics (R762Q) was less sensitive to extracellular chloride compared to WT. The opposing substrate dependences of ClC-6 and ClC-7 Cl- / H+ exchangers point to non-overlapping physiological functions, leading us to propose that inhibition of ClC-7 by luminal chloride and protons serves to prevent osmotic stress imposed by hyper-accumulation of chloride.

Altered voltage-dependence of slowly activating chloride-proton antiport by late endosomal ClC-6 explains distinct neurological disorders.

ClC-6 is an intracellularly localised member of the CLC family of chloride transport proteins. It presumably functions in the endolysosomal compartment as a chloride-proton antiporter, despite a paucity of biophysical studies in direct support. Observations of lysosomal storage disease, as well as neurodegenerative disorders, emerge with its disruption by knockout or mutation, respectively. An incomplete understanding of wild-type ClC-6 function obscures clear mechanistic insight into disease aetiology. Here, high-resolution recording protocols that incorporate extreme voltage pulses permit detailed biophysical measurement and analysis of transient capacitive, as well as ionic transport currents. This approach reveals that wild-type ClC-6 activation and transport require depolarisation to voltages beyond 140 mV. Mutant Y553C associated with early-onset neurodegeneration exerts gain-of-function by shifting the half-maximal voltage for activation to less depolarised voltages. Moreover, we show that the E267A proton glutamate mutant conserves transport currents, albeit reduced. Lastly, the positive shift in activation voltage shown by V580M, a mutant identified in a patient with late-onset lysosomal storage disease, can explain loss-of-function leading to disease. KEY POINTS: Ionic composition and pH within intracellular compartments, such as endolysosomes, rely on the activity of chloride/proton transporters including ClC-6. Distinct CLCN6 mutations were previously found in individuals with neurodegenerative disease, and also putatively associated with neuronal ceroid lipofuscinosis. Limited knowledge of wild-type ClC-6 transport function impedes understanding of mechanisms underlying these conditions. We resolved transient and transport currents that permit measurement of voltage- and pH-dependences, as well as kinetics, for wild-type and disease-associated mutant ClC-6s. These findings define wild-type ClC-6 function robustly, and reveal how alterations of the slow activation gating of the transporter cause different kinds of neurological diseases.

Iodide Binding in Sodium-Coupled Cotransporters.

Several apical iodide translocation pathways have been proposed for iodide efflux out of thyroid follicular cells, including a pathway mediated by the sodium-coupled monocarboxylate transporter 1 (SMCT1), which remains controversial. Herein, we evaluate structural and functional similarities between SMCT1 and the well-studied sodium-iodide symporter (NIS) that mediates the first step of iodide entry into the thyroid. Free-energy calculations using a force field with electronic polarizability verify the presence of a conserved iodide-binding pocket between the TM2, TM3, and TM7 segments in hNIS, where iodide is coordinated by Phe67, Gln72, Cys91, and Gln94. We demonstrate the mutation of residue Gly93 of hNIS to a larger amino acid expels the side chain of a critical tryptophan residue (Trp255) into the interior of the binding pocket, partially occluding the iodide binding site and reducing iodide affinity, which is consistent with previous reports associating mutation of this residue with iodide uptake deficiency and hypothyroidism. Furthermore, we find that the position of Trp255 in this hNIS mutant mirrors that of Trp253 in wild-type hSMCT1, where a threonine (Thr91) occupies the position homologous to that occupied by glycine in wild-type hNIS (Gly93). Correspondingly, mutation of Thr91 to glycine in hSMCT1 makes the pocket structure more like that of wild-type hNIS, increasing its iodide affinity. These results suggest that wild-type hSMCT1 in the inward-facing conformation may bind iodide only very weakly, which may have implications for its ability to transport iodide.

Cultured Pig Thyroid Follicular Cells: Electrical Evaluation of Epithelial Integrity.

Thyroid epithelial cells organize as enclosed follicles containing thyroid hormone precursor, iodinated thyroglobulin, with lumina bordered by the cellular apices. Transepithelial transport determines composition of compartmental milieu essential for both prohormone formation and its downstream conversion to thyroxine. Hence, not only do follicular lumina function as storage vessels but also as physiological reaction chambers into which reactive components, together with the proper salts and water, are secreted. Polarized, two-dimensional cultures of pig thyroid epithelia, prepared using established protocols, provide a convenient system for assessing transport processes subserving hormone production. This chapter details established methods for growing and evaluating integrity of primary pig thyroid cultures for downstream analysis of transport and other key physiological functions.

Novel role for pendrin in orchestrating bicarbonate secretion in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing airway serous cells.

In most HCO(3)(-)-secreting epithelial tissues, SLC26 Cl(-)/HCO(3)(-) transporters work in concert with the cystic fibrosis transmembrane conductance regulator (CFTR) to regulate the magnitude and composition of the secreted fluid, a process that is vital for normal tissue function. By contrast, CFTR is regarded as the only exit pathway for HCO(3)(-) in the airways. Here we show that Cl(-)/HCO(3)(-) anion exchange makes a major contribution to transcellular HCO(3)(-) transport in airway serous cells. Real-time measurement of intracellular pH from polarized cultures of human Calu-3 cells demonstrated cAMP/PKA-activated Cl(-)-dependent HCO(3)(-) transport across the luminal membrane via CFTR-dependent coupled Cl(-)/HCO(3)(-) anion exchange. The pharmacological and functional profile of the luminal anion exchanger was consistent with SLC26A4 (pendrin), which was shown to be expressed by quantitative RT-PCR, Western blot, and immunofluorescence. Pendrin-mediated anion exchange activity was confirmed by shRNA pendrin knockdown (KD), which markedly reduced cAMP-activated Cl(-)/HCO(3)(-) exchange. To establish the relative roles of CFTR and pendrin in net HCO(3)(-) secretion, transepithelial liquid secretion rate and liquid pH were measured in wild type, pendrin KD, and CFTR KD cells. cAMP/PKA increased the rate and pH of the secreted fluid. Inhibiting CFTR reduced the rate of liquid secretion but not the pH, whereas decreasing pendrin activity lowered pH with little effect on volume. These results establish that CFTR predominately controls the rate of liquid secretion, whereas pendrin regulates the composition of the secreted fluid and identifies a critical role for this anion exchanger in transcellular HCO(3)(-) secretion in airway serous cells.

Endogenous surface expression of ΔF508-CFTR mediates cAMP-stimulated Cl(-) current in CFTR(ΔF508/ΔF508) pig thyroid epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is both an anion channel and a regulator of other transport proteins. Mutations in the CFTR gene underlie the human disease, cystic fibrosis. The most common CFTR mutation, ΔF508, produces a misfolded protein which traffics improperly. The availability of transgenic CFTR(ΔF508/ΔF508) pigs allows measurement of the impact of ΔF508 in native tissue. Thyroid epithelia respond to cAMP-elevating agents by increasing anion transport, a process reliant on functional CFTR. To assess whether endogenous levels of ΔF508-CFTR mediate thyroid transport, primary thyroid epithelial cultures (pThECs) were grown from newborn CFTR(+/+) (wild-type) and CFTR(ΔF508/ΔF508) (ΔF) pig thyroids and the stimulated, secretory components of short-circuit current (I(sc)) compared. Surface biotinylation studies assessed the surface presentation of ΔF508-CFTR. Baseline I(sc) levels of both wild-type and ΔF pThECs consisted of an amiloride-sensitive component. In ΔF pThECs, this mirrored previous measurements in CFTR(-/-) (knockout) pThECs. Surprisingly, elevation of cAMP transiently increased I(sc) to peak levels ∼65% of those achieved by wild-type. In contrast, knockout pThECs were indifferent to cAMP activation. In ΔF pThECs, total ΔF508-CFTR expression was ∼9% that of wild-type, consistent with misfolding and enhanced degradation. Surface biotinylation studies indicated that ∼4% of the total ΔF508 resided at the surface and did not increase with cAMP elevation. The present findings show that low endogenous levels of pig ΔF508-CFTR can mediate substantial anion transport by thyroid epithelia. These data suggest that both wild-type and ΔF508-CFTR regulate additional thyroid transporters, and together co-ordinate the overall I(sc) response.

Thyroid iodide efflux: a team effort?

The thyroid hormones thyroxine (T(4)) and triiodothyronine (T(3)) play key roles in regulating development, growth and metabolism in pre- and postnatal life. Iodide (I(-)) is an essential component of the thyroid hormones and is accumulated avidly by the thyroid gland. The rarity of elemental iodine and I(-) in the environment challenges the thyroid to orchestrate a remarkable series of transport processes that ultimately ensure sufficient levels for hormone synthesis. In addition to actively extracting circulating I(-), thyroid follicular epithelial cells must also translocate I(-) into a central intrafollicular compartment, where thyroglobulin is iodinated to form the protein precursor to T(4) and T(3). In the last decade, several bodies of evidence render questionable the notion that I(-) exits thyrocytes solely via the Cl(-)/I(-) exchanger Pendrin (SLC26A4), therefore necessitating reconsideration of several other candidate I(-) conduits: the Cl(-)/H(+) antiporter, CLC-5, the cystic fibrosis transmembrane conductance regulator (CFTR) and the sodium monocarboxylic acid transporter (SMCT1).

Non-linear classification for on-the-fly fractional mass filtering and targeted precursor fragmentation in mass spectrometry experiments.

MOTIVATION: Mass spectrometry (MS) has become the method of choice for protein/peptide sequence and modification analysis. The technology employs a two-step approach: ionized peptide precursor masses are detected, selected for fragmentation, and the fragment mass spectra are collected for computational analysis. Current precursor selection schemes are based on data- or information-dependent acquisition (DDA/IDA), where fragmentation mass candidates are selected by intensity and are subsequently included in a dynamic exclusion list to avoid constant refragmentation of highly abundant species. DDA/IDA methods do not exploit valuable information that is contained in the fractional mass of high-accuracy precursor mass measurements delivered by current instrumentation. RESULTS: We extend previous contributions that suggest that fractional mass information allows targeted fragmentation of analytes of interest. We introduce a non-linear Random Forest classification and a discrete mapping approach, which can be trained to discriminate among arbitrary fractional mass patterns for an arbitrary number of classes of analytes. These methods can be used to increase fragmentation efficiency for specific subsets of analytes or to select suitable fragmentation technologies on-the-fly. We show that theoretical generalization error estimates transfer into practical application, and that their quality depends on the accuracy of prior distribution estimate of the analyte classes. The methods are applied to two real-world proteomics datasets. AVAILABILITY: All software used in this study is available from http://software.steenlab.org/fmf CONTACT: hanno.steen@childrens.harvard.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Altered ion transport by thyroid epithelia from CFTR(-/-) pigs suggests mechanisms for hypothyroidism in cystic fibrosis.

Subclinical hypothyroidism has been linked to cystic fibrosis, and the cystic fibrosis transmembrane conductance regulator (CFTR) shown to be expressed in the thyroid. The thyroid epithelium secretes Cl⁻ and absorbs Na(+) in response to cAMP. Chloride secretion may provide a counter-ion for the SLC26A4 (pendrin)-mediated I⁻ secretion which is required for the first step of thyroid hormonogenesis, thyroglobulin iodination. In contrast, few models exist to explain a role for Na(+) absorption. Whether CFTR mediates the secretory Cl⁻ current in thyroid epithelium has not been directly addressed. We used thyroids from a novel pig CFTR(-/-) model, generated primary pig thyroid epithelial cell cultures (pThECs), analysed these cultures for preservation of thyroid-specific transcripts and proteins, and monitored the following parameters: (1) the Cl⁻ secretory response to the cAMP agonist, isoprenaline; and (2) the amiloride-sensitive Na(+) current. Baseline short-circuit current (I(sc)) did not differ between CFTR(+/+) and CFTR(-/-) cultures. Serosal isoprenaline increased I(sc) in CFTR(+/+), but not CFTR(-/-), monolayers. Compared with CFTR(+/+) thyroid cultures, amiloride-sensitive Na(+) absorption measured in CFTR(-/-) pThECs represented a greater fraction of the resting I(sc). However, levels of transcripts encoding epithelial sodium channel (ENaC) subunits did not differ between CFTR(+/+) and CFTR(-/-) pThECs. Immunoblot analysis verified ENaC subunit protein expression, but quantification indicated no difference in expression levels. Our studies definitively demonstrate that CFTR mediates cAMP-stimulated Cl⁻ secretion in a well-differentiated thyroid culture model and that knockout of CFTR promotes increased Na(+) absorption by a mechanism other than increased ENaC expression. These findings suggest several models for the mechanism of cystic fibrosis-associated hypothyroidism.

Developmental delays consistent with cochlear hypothyroidism contribute to failure to develop hearing in mice lacking Slc26a4/pendrin expression.

Mutations of SLC26A4 cause an enlarged vestibular aqueduct, nonsyndromic deafness, and deafness as part of Pendred syndrome. SLC26A4 encodes pendrin, an anion exchanger located in the cochlea, thyroid, and kidney. The goal of the present study was to determine whether developmental delays, possibly mediated by systemic or local hypothyroidism, contribute to the failure to develop hearing in mice lacking Slc26a4 (Slc26a4(-/-)). We evaluated thyroid function by voltage and pH measurements, by array-assisted gene expression analysis, and by determination of plasma thyroxine levels. Cochlear development was evaluated for signs of hypothyroidism by microscopy, in situ hybridization, and quantitative RT-PCR. No differences in plasma thyroxine levels were found in Slc26a4(-/-) and sex-matched Slc26a4(+/-) littermates between postnatal day 5 (P5) and P90. In adult Slc26a4(-/-) mice, the transepithelial potential and the pH of thyroid follicles were reduced. No differences in the expression of genes that participate in thyroid hormone synthesis or ion transport were observed at P15, when plasma thyroxine levels peaked. Scala media of the cochlea was 10-fold enlarged, bulging into and thereby displacing fibrocytes, which express Dio2 to generate a cochlear thyroid hormone peak at P7. Cochlear development, including tunnel opening, arrival of efferent innervation at outer hair cells, endochondral and intramembraneous ossification, and developmental changes in the expression of Dio2, Dio3, and Tectb were delayed by 1-4 days. These data suggest that pendrin functions as a HCO3- transporter in the thyroid, that Slc26a4(-/-) mice are systemically euthyroid, and that delays in cochlear development, possibly due to local hypothyroidism, lead to the failure to develop hearing.

Isolation and Culture of Juvenile Pig Thyroid Follicular Epithelia.

Epithelial tissues are defined by their polarity and their ability to transport directionally. Thyroid is a tissue comprising functional epithelial units organized as enclosed follicles, with their luminal spaces defined by thyrocyte apices. Thus, the native arrangement of thyroid epithelia limits accessibility to the follicular space, presenting a challenge in studying transepithelial movements. This limitation can be overcome by studying thyrocytes grown as two-dimensional cultures. Herein we present methods for isolation of thyroid follicles from juvenile pigs and preparation of high-resistance, polarized cultures.

Apical iodide efflux in thyroid.

Thyroid follicular epithelial cells produce thyroxine (T4) and its physiologically active derivative, 3,3',5-triiodothyronine (T3), hormones that regulate critical developmental and metabolic functions. In order for the thyroid to form hormone precursor, iodide, the defining element in thyroid hormone, must cross both blood-facing and luminal sides of the follicular epithelium. The pathway for uptake from blood is well understood, but the mechanism(s) that enable iodide to cross the luminally facing apical membrane remain obscure. This chapter considers the physiological properties of several molecularly characterized anion transport proteins, all of which potentially contribute to the overall mechanism of apical iodide efflux.