Chloride/proton antiporters ClC3 and ClC5 support bone formation in mice.

Acid transport is required for bone synthesis by osteoblasts. The osteoblast basolateral surface extrudes acid by Na+/H+ exchange, but apical proton uptake is undefined. We found high expression of the Cl-/H+ exchanger ClC3 at the bone apical surface. In mammals ClC3 functions in intracellular vesicular chloride transport, but when we found Cl- dependency of H+ transport in osteoblast membranes, we queried whether ClC3 Cl-/H+ exchange functions in bone formation. We used ClC3 knockout animals, and closely-related ClC5 knockout animals: In vitro studies suggested that both ClC3 and ClC5 might support bone formation. Genotypes were confirmed by total exon sequences. Expression of ClC3, and to a lesser extent of ClC5, at osteoblast apical membranes was demonstrated by fluorescent antibody labeling and electron microscopy with nanometer gold labeling. Animals with ClC3 or ClC5 knockouts were viable. In ClC3 or ClC5 knockouts, bone formation decreased ~40 % by calcein and xylenol orange labeling in vivo. In very sensitive micro-computed tomography, ClC5 knockout reduced bone relative to wild type, consistent with effects of ClC3 knockout, but varied with specific histological parameters. Regrettably, ClC5-ClC3 double knockouts are not viable, suggesting that ClC3 or ClC5 activity are essential to life. We conclude that ClC3 has a direct role in bone formation with overlapping but probably slightly smaller effects of ClC5. The mechanism in mineral formation might include ClC H+ uptake, in contrast to ClC3 and ClC5 function in cell vesicles or other organs.

Epithelial-like transport of mineral distinguishes bone formation from other connective tissues.

We review unique properties of bone formation including current understanding of mechanisms of bone mineral transport. We focus on formation only; mechanism of bone degradation is a separate topic not considered. Bone matrix is compared to other connective tissues composed mainly of the same proteins, but without the specialized mechanism for continuous transport and deposition of mineral. Indeed other connective tissues add mechanisms to prevent mineral formation. We start with the epithelial-like surfaces that mediate transport of phosphate to be incorporated into hydroxyapatite in bone, or in its ancestral tissue, the tooth. These include several phosphate producing or phosphate transport-related proteins with special expression in large quantities in bone, particularly in the bone-surface osteoblasts. In all connective tissues including bone, the proteins that constitute the protein matrix are mainly type I collagen and γ-carboxylate-containing small proteins in similar molar quantities to collagen. Specialized proteins that regulate connective tissue structure and formation are surprisingly similar in mineralized and non-mineralized tissues. While serum calcium and phosphate are adequate to precipitate mineral, specialized mechanisms normally prevent mineral formation except in bone, where continuous transport and deposition of mineral occurs.

Current challenges and future directions for engineering extracellular vesicles for heart, lung, blood and sleep diseases.

Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue-specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large-scale manufacturing of therapeutic-grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases. To address these challenges, a strategic workshop with multidisciplinary experts in EV biology and U.S. Food and Drug Administration (USFDA) officials was convened by the National Heart, Lung and Blood Institute. The presentations and discussions were focused on summarizing the current state of science and technology for engineering therapeutic EVs for HLBS diseases, identifying critical knowledge gaps and regulatory challenges and suggesting potential solutions to promulgate translation of therapeutic EVs to the clinic. Benchmarks to meet the critical quality attributes set by the USFDA for other cell-based therapeutics were discussed. Development of novel strategies and approaches for scaling-up EV production and the quality control/quality analysis (QC/QA) of EV-based therapeutics were recognized as the necessary milestones for future investigations.

Phagosomal chloride dynamics in the alveolar macrophage.

Acidification in intracellular organelles is tightly linked to the influx of Cl- counteracting proton translocation by the electrogenic V-ATPase. We quantified the dynamics of Cl- transfer accompanying cargo incorporation into single phagosomes in alveolar macrophages (AMs). Phagosomal Cl- concentration and acidification magnitude were followed in real time with maximal acidification achieved at levels of approximately 200 mM. Live cell confocal microscopy verified that phagosomal Cl- influx utilized predominantly the Cl- channel CFTR. Relative levels of elemental chlorine (Cl) in hard X-ray fluorescence microprobe (XFM) analysis within single phagosomes validated the increase in Cl- content. XFM revealed the complex interplay between elemental K content inside the phagosome and changes in Cl- during phagosomal particle uptake. Cl- -dependent changes in phagosomal membrane potential were obtained using second harmonic generation (SHG) microscopy. These studies provide a mechanistic insight for screening studies in drug development targeting pulmonary inflammatory disease.

Real time imaging of single extracellular vesicle pH regulation in a microfluidic cross-flow filtration platform.

Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo. Their complex cargo requires pH stability in EVs while traversing diverse body fluids. We used a filtration-based platform to capture and stabilize EVs based on their size and studied their pH regulation at the single EV level. Dead-end filtration facilitated EV capture in the pores of an ultrathin (100 nm thick) and nanoporous silicon nitride (NPN) membrane within a custom microfluidic device. Immobilized EVs were rapidly exposed to test solution changes driven across the backside of the membrane using tangential flow without exposing the EVs to fluid shear forces. The epithelial sodium-hydrogen exchanger, NHE1, is a ubiquitous plasma membrane protein tasked with the maintenance of cytoplasmic pH at neutrality. We show that NHE1 identified on the membrane of EVs is functional in the maintenance of pH neutrality within single vesicles. This is the first mechanistic description of EV function on the single vesicle level.

Growth and mineralization of osteoblasts from mesenchymal stem cells on microporous membranes: Epithelial-like growth with transmembrane resistance and pH gradient.

Osteoblasts in vivo form an epithelial-like layer with tight junctions between cells. Bone formation involves mineral transport into the matrix and acid transport to balance pH levels. To study the importance of the pH gradient in vitro, we used Transwell inserts composed of polyethylene terephthalate (PET) membranes with 0.4 µm pores at a density of (2 ± 0.4) x 106 pores per cm2. Mesenchymal stem cells (MSCs) prepared from murine bone marrow were used to investigate alternative conditions whereby osteoblast differentiation would better emulate in vivo bone development. MSCs were characterized by flow cytometry with more than 90% CD44 and 75% Sca-1 labeling. Mineralization was validated with paracellular alkaline phosphatase activity, collagen birefringence, and mineral deposition confirming MSCs identity. We demonstrate that MSCs cultured and differentiated on PET inserts form an epithelial-like layer while mineralizing. Measurement of the transepithelial resistance was ∼1400 Ω•cm2 at three weeks of differentiation. The pH value of the media above and under the cells were measured while cells were in proliferation and differentiation. In mineralizing cells, a difference of 0.145 pH unit was observed between the medium above and under the cells indicating a transepithelial gradient. A significant difference in pH units was observed between the medium above and below the cells in proliferation compared to differentiation. Data on pH below membranes were confirmed by pH-dependent SNARF1 fluorescence. Control cells in proliferative medium did not form an epithelial-like layer, displayed low transepithelial resistance, and there was no significant pH gradient. By transmission electron microscopy, membrane attached osteoblasts in vitro had abundant mitochondria consistent with active transport that occurs in vivo by surface osteoblasts. In keeping with osteoblastic differentiation, scanning electron microscopy identified deposition of extracellular collagen surrounded by hydroxyapatite. This in vitro model is a major advancement in modeling bone in vivo for understanding of osteoblast bone matrix production.

Kinetic Separation of Oxidative and Non-oxidative Metabolism in Single Phagosomes from Alveolar Macrophages: Impact on Bacterial Killing.

The relative contribution of the two phagosomal catabolic processes, oxidative and metabolic, was assessed in the killing of Pseudomonas aeruginosa in phagosomes of alveolar macrophages (AMs) from wild-type (p47-phox +/+ ) or NOX-defective (p47-phox -/- ) mice. Free radical release and degradative acidification within AM phagosomes is sequential and separable. The initial NOX activity, identifiable as a transient alkalinization, leads to fast bacterial wall permeabilization by ROS. This is followed by V-ATPase-induced acidification and enzymatic bacterial degradation contributed through phagosomal-lysosomal fusion. The alkalinization/acidification ratio was variable among phagosomes within single cells of a given genotype and not as a function of macrophage M1 or M2 classification, possibly owing to uneven distribution of phagosomal transporter proteins. Irregular, excessive NOX activity prevents phago-lysosomal fusion, and the lack of V-ATPase-induced acidification leads to bacterial stasis in the phagosome. Thus, efficient phagosomal bacterial killing is a result of tightly balanced activity between two processes.

Phylogeny and chemistry of biological mineral transport.

Three physiologically mineralizing tissues - teeth, cartilage and bone - have critical common elements and important evolutionary relationships. Phylogenetically the most ancient densely mineralized tissue is teeth. In jawless fishes without skeletons, tooth formation included epithelial transport of phosphates, a process echoed later in bone physiology. Cartilage and mineralized cartilage are skeletal elements separate from bone, but with metabolic features common to bone. Cartilage mineralization is coordinated with high expression of tissue nonspecific alkaline phosphatase and PHOSPHO1 to harvest available phosphate esters and support mineralization of collagen secreted locally. Mineralization in true bone results from stochastic nucleation of hydroxyapatite crystals within the cross-linked collagen fibrils. Mineral accumulation in dense collagen is, at least in major part, mediated by amorphous aggregates - often called Posner clusters - of calcium and phosphate that are small enough to diffuse into collagen fibrils. Mineral accumulation in membrane vesicles is widely suggested, but does not correlate with a definitive stage of mineralization. Conversely mineral deposition at non-physiologic sites where calcium and phosphate are adequate has been shown to be regulated in large part by pyrophosphate. All of these elements are present in vertebrate bone metabolism. A key biological element of bone formation is an epithelial-like cellular organization which allows control of phosphate, calcium and pH during mineralization.

Cellular and extracellular matrix of bone, with principles of synthesis and dependency of mineral deposition on cell membrane transport.

Bone differs from other connective tissues; it is isolated by a layer of osteoblasts that are connected by tight and gap junctions. This allows bone to create dense lamellar type I collagen, control pH, mineral deposition, and regulate water content forming a compact and strong structure. New woven bone formed after degradation of mineralized cartilage is rapidly degraded and resynthesized to impart structural order for local bone strength. Ossification is regulated by thickness of bone units and by patterning via bone morphogenetic receptors including activin, other bone morphogenetic protein receptors, transforming growth factor-ß receptors, all part of a receptor superfamily. This superfamily interacts with receptors for additional signals in bone differentiation. Important features of the osteoblast environment were established using recent tools including osteoblast differentiation in vitro. Osteoblasts deposit matrix protein, over 90% type I collagen, in lamellae with orientation alternating parallel or orthogonal to the main stress axis of the bone. Into this organic matrix, mineral is deposited as hydroxyapatite. Mineral matrix matures from amorphous to crystalline hydroxyapatite. This process includes at least two-phase changes of the calcium-phosphate mineral as well as intermediates involving tropocollagen fibrils to form the bone composite. Beginning with initiation of mineral deposition, there is uncertainty regarding cardinal processes, but the driving force is not merely exceeding the calcium-phosphate solubility product. It occurs behind a epithelial-like layer of osteoblasts, which generate phosphate and remove protons liberated during calcium-phosphate salt deposition. The forming bone matrix is discontinuous from the general extracellular fluid. Required adjustment of ionic concentrations and water removal from bone matrix are important details remaining to be addressed.

Support of bone mineral deposition by regulation of pH.

Osteoblasts secrete collagen and isolate bone matrix from extracellular space. In the matrix, alkaline phosphatase generates phosphate that combines with calcium to form mineral, liberating 8 H+ per 10 Ca+2 deposited. However, pH-dependent hydroxyapatite deposition on bone collagen had not been shown. We studied the dependency of hydroxyapatite deposition on type I collagen on pH and phosphate by surface plasmon resonance in 0-5 mM phosphate at pH 6.8-7.4. Mineral deposition saturated at <1 mM Ca2+ but was sensitive to phosphate. Mineral deposition was reversible, consistent with amorphous precipitation; stable deposition requiring EDTA removal appeared with time. At pH 6.8, little hydroxyapatite deposited on collagen; mineral accumulation increased 10-fold at pH 7.4. Previously, we showed high expression Na+/H+ exchanger (NHE) and ClC transporters in osteoblasts. We hypothesized that, in combination, these move protons across osteoblasts to the general extracellular space. We made osteoblast membrane vesicles by nitrogen cavitation and used acridine orange quenching to characterize proton transport. We found H+ transport dependent on gradients of chloride or sodium, consistent with apical osteoblast ClC family Cl-,H+ antiporters and basolateral osteoblast NHE family Na+/H+ exchangers. Little, if any, active H+ transport, supported by ATP, occurred. Major transporters include cariporide-sensitive NHE1 in basolateral membranes and ClC3 and ClC5 in apical osteoblast membranes. The mineralization inhibitor levamisole reduced bone formation and expression of alkaline phosphatase, NHE1, and ClC5. We conclude that mineral deposition in bone collagen is pH-dependent, in keeping with H+ removal by Cl-,H+ antiporters and Na+/H+-exchangers. Periodic orientation hydroxyapatite is organized on type I collagen-coiled coils.

Osteoblast Differentiation and Bone Matrix Formation In Vivo and In Vitro.

We review the characteristics of osteoblast differentiation and bone matrix synthesis. Bone in air breathing vertebrates is a specialized tissue that developmentally replaces simpler solid tissues, usually cartilage. Bone is a living organ bounded by a layer of osteoblasts that, because of transport and compartmentalization requirements, produce bone matrix exclusively as an organized tight epithelium. With matrix growth, osteoblasts are reorganized and incorporated into the matrix as living cells, osteocytes, which communicate with each other and surface epithelium by cell processes within canaliculi in the matrix. The osteoblasts secrete the organic matrix, which are dense collagen layers that alternate parallel and orthogonal to the axis of stress loading. Into this matrix is deposited extremely dense hydroxyapatite-based mineral driven by both active and passive transport and pH control. As the matrix matures, hydroxyapatite microcrystals are organized into a sophisticated composite in the collagen layer by nucleation in the protein lattice. Recent studies on differentiating osteoblast precursors revealed a sophisticated proton export network driving mineralization, a gene expression program organized with the compartmentalization of the osteoblast epithelium that produces the mature bone matrix composite, despite varying serum calcium and phosphate. Key issues not well defined include how new osteoblasts are incorporated in the epithelial layer, replacing those incorporated in the accumulating matrix. Development of bone in vitro is the subject of numerous projects using various matrices and mesenchymal stem cell-derived preparations in bioreactors. These preparations reflect the structure of bone to variable extents, and include cells at many different stages of differentiation. Major challenges are production of bone matrix approaching the in vivo density and support for trabecular bone formation. In vitro differentiation is limited by the organization and density of osteoblasts and by endogenous and exogenous inhibitors.

Modulating Innate and Adaptive Immunity by (R)-Roscovitine: Potential Therapeutic Opportunity in Cystic Fibrosis.

(R)-Roscovitine, a pharmacological inhibitor of kinases, is currently in phase II clinical trial as a drug candidate for the treatment of cancers, Cushing's disease and rheumatoid arthritis. We here review the data that support the investigation of (R)-roscovitine as a potential therapeutic agent for the treatment of cystic fibrosis (CF). (R)-Roscovitine displays four independent properties that may favorably combine against CF: (1) it partially protects F508del-CFTR from proteolytic degradation and favors its trafficking to the plasma membrane; (2) by increasing membrane targeting of the TRPC6 ion channel, it rescues acidification in phagolysosomes of CF alveolar macrophages (which show abnormally high pH) and consequently restores their bactericidal activity; (3) its effects on neutrophils (induction of apoptosis), eosinophils (inhibition of degranulation/induction of apoptosis) and lymphocytes (modification of the Th17/Treg balance in favor of the differentiation of anti-inflammatory lymphocytes and reduced production of various interleukins, notably IL-17A) contribute to the resolution of inflammation and restoration of innate immunity, and (4) roscovitine displays analgesic properties in animal pain models. The fact that (R)-roscovitine has undergone extensive preclinical safety/pharmacology studies, and phase I and II clinical trials in cancer patients, encourages its repurposing as a CF drug candidate.

Chloride-hydrogen antiporters ClC-3 and ClC-5 drive osteoblast mineralization and regulate fine-structure bone patterning in vitro.

Osteoblasts form an epithelium-like layer with tight junctions separating bone matrix from extracellular fluid. During mineral deposition, calcium and phosphate precipitation in hydroxyapatite liberates 0.8 mole of H(+) per mole Ca(+2). Thus, acid export is needed for mineral formation. We examined ion transport supporting osteoblast vectorial mineral deposition. Previously we established that Na/H exchangers 1 and 6 are highly expressed at secretory osteoblast basolateral surfaces and neutralize massive acid loads. The Na/H exchanger regulatory factor-1 (NHERF1), a pdz-organizing protein, occurs at mineralizing osteoblast basolateral surfaces. We hypothesized that high-capacity proton transport from matrix into osteoblast cytosol must exist to support acid transcytosis for mineral deposition. Gene screening in mineralizing osteoblasts showed dramatic expression of chloride-proton antiporters ClC-3 and ClC-5. Antibody localization showed that ClC-3 and ClC-5 occur at the apical secretory surface facing the bone matrix and in membranes of buried osteocytes. Surprisingly, the Clcn3(-/-) mouse has only mildly disordered mineralization. However, Clcn3(-/-) osteoblasts have large compensatory increases in ClC-5 expression. Clcn3(-/-) osteoblasts mineralize in vitro in a striking and novel trabecular pattern; wild-type osteoblasts form bone nodules. In mesenchymal stem cells from Clcn3(-/-) mice, lentiviral ClC-5 shRNA created Clcn3(-/-), ClC-5 knockdown cells, validated by western blot and PCR. Osteoblasts from these cells produced no mineral under conditions where wild-type or Clcn3(-/-) cells mineralize well. We conclude that regulated acid export, mediated by chloride-proton exchange, is essential to drive normal bone mineralization, and that CLC transporters also regulate fine patterning of bone.

TRPC6 channel translocation into phagosomal membrane augments phagosomal function.

Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H(+) into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired.

Claudin-2-dependent paracellular channels are dynamically gated.

Intercellular tight junctions form selectively permeable barriers that seal the paracellular space. Trans-tight junction flux has been measured across large epithelial surfaces, but conductance across individual channels has never been measured. We report a novel trans-tight junction patch clamp technique that detects flux across individual claudin-2 channels within the tight junction of cultured canine renal tubule or human intestinal epithelial monolayers. In both cells, claudin-2 channels display conductances of ~90 pS. The channels are gated, strictly dependent on claudin-2 expression, and display size- and charge-selectivity typical of claudin-2. Kinetic analyses indicate one open and two distinct closed states. Conductance is symmetrical and reversible, characteristic of a passive, paracellular process, and blocked by reduced temperature or site-directed mutagenesis and chemical derivatization of the claudin-2 pore. We conclude that claudin-2 forms gated paracellular channels and speculate that modulation of tight junction channel gating kinetics may be an unappreciated mechanism of barrier regulation.

HEK-293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying regulation of Cl- transport.

The Human Embryonic Kidney 293 cell line (HEK-293) readily lends itself to genetic manipulation and is a common tool for biologists to overexpress proteins of interest and study their function and molecular regulation. Although these cells have some limitations, such as an inability to form resistive monolayers necessary for studying transepithelial ion transport, they are nevertheless valuable in studying individual epithelial ion transporters. We report the use of HEK-293 cells to study the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. While HEK-293 cells endogenously express mRNA for the Cl(-) channels, ClC-2 and TMEM16A, they neither express CFTR mRNA nor protein. Therefore, we stably transfected HEK-293 cells with EGFP-CFTR (HEK-CFTR) and demonstrated CFTR function by measuring forskolin-stimulated iodide efflux. This efflux was inhibited by CFTRinh172, and the protein kinase A inhibitor H89, but not by Ca(2+) chelation. In contrast to intestinal epithelia, forskolin stimulation does not increase surface CFTR expression and does not require intact microtubules in HEK-CFTR. To investigate the role of an endogenous GαS-coupled receptor, we examined the bile acid receptor, TGR5. Although HEK-CFTR cells express TGR5, the potent TGR5 agonist lithocholic acid (LCA; 5-500 µmol/L) did not activate CFTR. Furthermore, forskolin, but not LCA, increased [cAMP]i in HEK-CFTR suggesting that endogenous TGR5 may not be functionally linked to GαS. However, LCA did increase [Ca(2+)]i and interestingly, abolished forskolin-stimulated iodide efflux. Thus, we propose that the stable HEK-CFTR cell line is a useful model to study the multiple signaling pathways that regulate CFTR.

Quantifying insulin sensitivity and entero-insular responsiveness to hyper- and hypoglycemia in ferrets.

Ferrets are an important emerging model of cystic fibrosis related diabetes. However, there is little documented experience in the use of advanced techniques to quantify aspects of diabetes pathophysiology in the ferret. Glycemic clamps are the gold standard technique to assess both insulin sensitivity and insulin secretion in humans and animal models of diabetes. We therefore sought to develop techniques for glycemic clamps in ferrets. To assess insulin sensitivity, we performed euglycemic hyperinsulinemic clamps in 5-6 week old ferrets in the anesthetized and conscious states. To assess insulin secretion, we performed hyperglycemic clamps in conscious ferrets. To evaluate responsiveness of ferret islet and entero-insular hormones to low glucose, a portion of the hyperglycemic clamps were followed by a hypoglycemic clamp. The euglycemic hyperinsulinemic clamps demonstrated insulin responsiveness in ferrets similar to that previously observed in humans and rats. The anesthetic isoflurane induced marked insulin resistance, whereas lipid emulsion induced mild insulin resistance. In conscious ferrets, glucose appearance was largely suppressed at 4 mU/kg/min insulin infusion, whereas glucose disposal was progressively increased at 4 and 20 mU/kg/min insulin. Hyperglycemic clamp induced first phase insulin secretion. Hypoglycemia induced a rapid diminishment of insulin, as well as a rise in glucagon and pancreatic polypeptide levels. The incretins GLP-1 and GIP were affected minimally by hyperglycemic and hypoglycemic clamp. These techniques will prove useful in better defining the pathophysiology in ferrets with cystic fibrosis related diabetes.

CLC-3 chloride channels moderate long-term potentiation at Schaffer collateral-CA1 synapses.

The chloride channel CLC-3 is expressed in the brain on synaptic vesicles and postsynaptic membranes. Although CLC-3 is broadly expressed throughout the brain, the CLC-3 knockout mouse shows complete, selective postnatal neurodegeneration of the hippocampus, suggesting a crucial role for the channel in maintaining normal brain function. CLC-3 channels are functionally linked to NMDA receptors in the hippocampus; NMDA receptor-dependent Ca(2+) entry, activation of Ca(2+)/calmodulin kinase II and subsequent gating of CLC-3 link the channels via a Ca(2+)-mediated feedback loop. We demonstrate that loss of CLC-3 at mature synapses increases long-term potentiation from 135 ± 4% in the wild-type slice preparation to 154 ± 7% above baseline (P < 0.001) in the knockout; therefore, the contribution of CLC-3 is to reduce synaptic potentiation by ∼40%. Using a decoy peptide representing the Ca(2+)/calmodulin kinase II phosphorylation site on CLC-3, we show that phosphorylation of CLC-3 is required for its regulatory function in long-term potentiation. CLC-3 is also expressed on synaptic vesicles; however, our data suggest functionally separable pre- and postsynaptic roles. Thus, CLC-3 confers Cl(-) sensitivity to excitatory synapses, controls the magnitude of long-term potentiation and may provide a protective limit on Ca(2+) influx.

Presynaptic CLC-3 determines quantal size of inhibitory transmission in the hippocampus.

The absence of the chloride channel CLC-3 in Clcn3(-/-) mice results in hippocampal degeneration with a distinct temporal-spatial sequence that resembles neuronal loss in temporal lobe epilepsy. We examined how the loss of CLC-3 might affect GABAergic synaptic transmission in the hippocampus. An electrophysiological study of synaptic function in hippocampal slices taken from Clcn3(-/-) mice before the onset of neurodegeneration revealed a substantial decrease in the amplitude and frequency of miniature inhibitory postsynaptic currents compared with those in wild-type slices. We found that CLC-3 colocalized with the vesicular GABA transporter VGAT in the CA1 region of the hippocampus. Acidification of inhibitory synaptic vesicles induced by Cl(-) showed a marked dependence on CLC-3 expression. The decrease in inhibitory transmission in Clcn3(-/-) mice suggests that the neurotransmitter loading of synaptic vesicles was reduced, which we attribute to defective vesicular acidification. Our observations extend the role of Cl(-) in inhibitory transmission from that of a postsynaptic permeant species to a presynaptic regulatory element.

Bepridil and amiodarone simultaneously target the Alzheimer's disease beta- and gamma-secretase via distinct mechanisms.

The two proteases beta-secretase and gamma-secretase generate the amyloid beta peptide and are drug targets for Alzheimer's disease. Here we tested the possibility of targeting the cellular environment of beta-secretase cleavage instead of the beta-secretase enzyme itself. beta-Secretase has an acidic pH optimum and cleaves the amyloid precursor protein in the acidic endosomes. We identified two drugs, bepridil and amiodarone, that are weak bases and are in clinical use as calcium antagonists. Independently of their calcium-blocking activity, both compounds mildly raised the membrane-proximal, endosomal pH and inhibited beta-secretase cleavage at therapeutically achievable concentrations in cultured cells, in primary neurons, and in vivo in guinea pigs. This shows that an alkalinization of the cellular environment could be a novel therapeutic strategy to inhibit beta-secretase. Surprisingly, bepridil and amiodarone also modulated gamma-secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target beta- and gamma-secretase through distinct molecular mechanisms. In addition to Alzheimer's disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins.