Calcium signalling and calcium transport in bone disease.

Calcium transport and calcium signalling mechanisms in bone cells have, in many cases, been discovered by study of diseases with disordered bone metabolism. Calcium matrix deposition is driven primarily by phosphate production, and disorders in bone deposition include abnormalities in membrane phosphate transport such as in chondrocalcinosis, and defects in phosphate-producing enzymes such as in hypophosphatasia. Matrix removal is driven by acidification, which dissolves the mineral. Disorders in calcium removal from bone matrix by osteoclasts cause osteopetrosis. On the other hand, although bone is central to management of extracellular calcium, bone is not a major calcium sensing organ, although calcium sensing proteins are expressed in both osteoblasts and osteoclasts. Intracellular calcium signals are involved in secondary control including cellular motility and survival, but the relationship of these findings to specific diseases is not clear. Intracellular calcium signals may regulate the balance of cell survival versus proliferation or anabolic functional response as part of signalling cascades that integrate the response to primary signals via cell stretch, estrogen, tyrosine kinase, and tumor necrosis factor receptors.

The Bax pore in liposomes, Biophysics.

The protein BAX of the Bcl-2-family is felt to be one of the two Bcl-2-family proteins that directly participate in the mitochondrial cytochrome c-translocating pore. We have studied the kinetics, stoichiometry and size of the pore formed by BAX in planar lipid bilayers and synthetic liposomes. Our data indicate that a cytochrome c-competent pore can be formed by in-membrane association of BAX monomers.

CLIC-1 functions as a chloride channel when expressed and purified from bacteria.

CLIC-1 is a member of a family of proteins related to the bovine intracellular chloride channel p64 which has been proposed to function as a chloride channel. We expressed CLIC-1 as a glutathione S-transferase fusion protein in bacteria. The fusion protein was purified by glutathione affinity, and CLIC-1 was released from its fusion partner by digestion with thrombin. After further purification, CLIC-1 was reconstituted into phospholipid vesicles by detergent dialysis. Chloride permeability of reconstituted vesicles was assessed using a valinomycin dependent chloride efflux assay, demonstrating increased vesicular chloride permeability with CLIC-1 compared with control. CLIC-1-dependent chloride permeability was inhibited by indanyloxyacetic acid-94 with an apparent IC(50) of 8.6 micrometer. The single channel properties of CLIC-1 were determined using the planar lipid bilayer technique. We found that CLIC-1 forms a voltage-dependent, Cl-selective channel with a rectifying current-voltage relationship and single channel conductances of 161 +/- 7.9 and 67.5 +/- 6.9 picosiemens in symmetric 300 and 150 mm KCl, respectively. The anion selectivity of this activity is Br approximately Cl > I. The open probability of CLIC-1 channels in planar bilayers was decreased by indanyloxyacetic acid-94 with an apparent IC(50) of 86 micrometer at 50 mV. These data convincingly demonstrate that CLIC-1 is capable of forming a novel, chloride-selective channel in the absence of other subunits or proteins.

Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c.

We review data supporting a model in which activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux. The BH3 domain of tBID is not required for targeting but remains on the mitochondrial surface where it is required to trigger BAK to release cytochrome c. tBID functions not as a pore-forming protein but as a membrane targeted and concentrated death ligand. tBID induces oligomerization of BAK, and both Bid and Bak knockout mice indicate the importance of this event in the release of cytochrome c. In parallel, the full pro-apoptotic member BAX, which is highly homologous to BAK, rapidly forms pores in liposomes that release intravesicular FITC-cytochrome c approximately 20A. A definable pore progressed from approximately 11A consisting of two BAX molecules to a approximately 22A pore comprised of four BAX molecules, which transported cytochrome c. Thus, an activation cascade of pro-apoptotic proteins from BID to BAK or BAX integrates the pathway from surface death receptors to the irreversible efflux of cytochrome c. Cell Death and Differentiation (2000) 7, 1166 - 1173

Parasitophorous vacuoles of Leishmania mexicana acquire macromolecules from the host cell cytosol via two independent routes.

The intracellular parasite Leishmania survives and proliferates in host macrophages. In this study we show that parasitophorous vacuoles of L. mexicana gain access to cytosolic material via two different routes. (1) Small anionic molecules such as Lucifer Yellow are rapidly transported into the vacuoles by an active transport mechanism that is sensitive to inhibitors of the host cell's organic anion transporter. (2) Larger molecules such as fluorescent dextrans introduced into the host cell cytosol are also delivered to parasitophorous vacuoles. This transport is slower and sensitive to modulators of autophagy. Infected macrophages were examined by two novel assays to visualize and quantify this process. Immunoelectron microscopy of cells loaded with digoxigenin-dextran revealed label in multivesicular endosomes, which appeared to fuse with parasitophorous vacuoles. The inner membranes of the multivesicular vesicles label strongly with antibodies against lysobisphosphatidic acid, suggesting that they represent a point of confluence between the endosomal and autophagosomal pathways. Although the rate of autophagous transfer was comparable in infected and uninfected cells, infected cells retained hydrolyzed cysteine proteinase substrate to a greater degree. These data suggest that L. mexicana-containing vacuoles have access to potential nutrients in the host cell cytosol via at least two independent mechanisms.

Ligand binding results in divalent cation displacement from the alpha 2 beta 1 integrin I domain: evidence from terbium luminescence spectroscopy.

The alpha 2 beta 1 integrin serves as a cell surface collagen or collagen/laminin receptor. Binding of the integrin to its ligands is largely mediated by the alpha 2 subunit I domain and requires the presence of divalent cations. Terbium ion (Tb3+), a fluorescent trivalent cation that often binds divalent cation-binding sites on proteins, supported binding of the I domain to collagen with half-maximal binding occurring at 5.2 +/- 1.7 microM Tb3+. By fluorescence resonance energy transfer spectroscopy, Tb3+ showed specific and saturable binding to the recombinant I domain with a Kd of 27 +/- 4 microM. Although both Mg2+ and Mn2+ were capable of quenching Tb3+ fluorescence, Mn2+ was much more effective than Mg2+. The alpha 2 beta 1 integrin also binds the pro-alpha 1(I) collagen carboxyl-terminal propeptide in a Mg2+-dependent manner via the I domain. Recombinant propeptide was used to examine the effect of ligand on the Tb3+ binding properties of the alpha 2 integrin I domain. As propeptide bound to the I domain, Tb3+ fluorescence progressively diminished suggesting that as ligand binds to the I domain, either Tb3+ is displaced or its fluorescence is quenched. Consistent with the former possibility, little dissociation of collagen-bound I domain occurred upon the addition of EDTA and subsequent incubation. These data support a model in which (1) the divalent cation is required for initial ligand-binding activity of the I domain and (2) ligand binding results in subsequent metal ion displacement to generate a metal-free I domain-ligand complex.

Functional expression of p64, an intracellular chloride channel protein.

p64 is a protein identified as a chloride channel by biochemical purification from kidney microsomes. We expressed p64 in HeLa cells using a recombinant vaccinia virus/T7 RNA polymerase driven system. Total cell membranes were prepared from infected/transfected cells and fused to a planar lipid bilayer. A novel chloride channel activity was found in cells expressing p64 and not in control cells. The p64-associated activity shows strong anion over cation selectivity. Single channels show prominent outward rectification with single channel conductance at positive potentials of 42 pS. The chloride channel activity is activated by treatment of the membranes with alkaline phosphatase and inhibited by DNDS and by TS-TM calix(4)arene. Whole membrane anion permeability was determined by a chloride efflux assay, revealing that membranes from cells expressing p64 showed a small but highly significant increase in chloride permeability, consistent with expression of a novel chloride channel activity.

Cytokine activation leads to acidification and increases maturation of Mycobacterium avium-containing phagosomes in murine macrophages.

Mycobacterium avium (MAC) organisms multiply in phagosomes that have restricted fusigenicity with lysosomes, do not acidify due to a paucity of vacuolar proton-ATPases, yet remain accessible to recycling endosomes. During the course of mycobacterial infections, IFN-gamma-mediated activation of host and bystander macrophages is a key mechanism in the regulation of bacterial growth. Here we demonstrate that in keeping with earlier studies, cytokine activation of host macrophages leads to a decrease in MAC viability, demonstrable by bacterial esterase staining with fluorescein diacetate as well as colony-forming unit counts from infected cells. Analysis of the pH of MAC phagosomes demonstrated that the vacuoles in activated macrophages equilibrate to pH 5.2, in contrast to pH 6.3 in resting phagocytes. Biochemical analysis of MAC phagosomes from both resting and activated macrophages confirmed that the lower intraphagosomal pH correlated with an increased accumulation of proton-ATPases. Furthermore, the lower pH is reflected in the transition of MAC phagosomes to a point no longer accessible to transferrin, a marker of the recycling endosomal system. These alterations parallel the coalescence of bacterial vacuoles from individual bacilli in single vacuoles to communal vacuoles with multiple bacilli. These data demonstrate that bacteriostatic and bactericidal activities of activated macrophages are concomitant with alterations in the physiology of the mycobacterial phagosome.

Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2.

The BCL-2 family of proteins is composed of both pro- and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-XL monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion efflux. Release is maximum at approximately pH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings revealed a minimal channel conductance for BAX of 22 pS that evolved to channel currents with at least three subconductance levels. The final, apparently stable BAX channel had a conductance of 0.731 nS at pH 4. 0 that changed to 0.329 nS when shifted to pH 7.0 but remained mildly Cl- selective and predominantly open. When BAX-incorporated lipid vesicles were fused to planar lipid bilayers at pH 7.0, a Cl--selective (PK/PCl = 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K+-selective (PK/PCl = 3.9) channels with a most prominent initial conductance of 80 pS that increased to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also revealed mild K+ selectivity (PK/PCl = 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites.

Characterization of the osteoclast ruffled border chloride channel and its role in bone resorption.

Bone resorption by osteoclasts requires massive transcellular acid transport, which is accomplished by the parallel action of a V-type proton pump and a chloride channel in the osteoclast ruffled border. We have studied the molecular basis for the appearance of acid transport as avian bone marrow mononuclear cells acquire a bone resorptive phenotype in vitro. We demonstrate a critical role for regulated expression of a ruffled border chloride channel as the cells become competent to resorb bone. Molecular characterization of the chloride channel shows that it is related to the renal microsomal chloride channel, p64. In planar bilayers, the ruffled border channel is a stilbene sulfonate-inhibitable, outwardly rectifying chloride channel. A mechanism by which outward rectification of the single channel chloride current could allow efficient regulation of acidification by the channel is discussed.

A novel pathway for Ca++ entry into Plasmodium falciparum-infected blood cells.

Growth of the human malaria parasite, Plasmodium falciparum, within the red blood cell (RBC) requires external Ca++ and is associated with a markedly elevated intracellular Ca++ concentration, [Ca++]i. We used 45Ca++ flux studies and patch clamp recordings to examine the mechanisms responsible for this increased [Ca++]i. The 45Ca++ flux studies indicated that net Ca++ entry into parasitized RBCs (PRBCs) is 18 times faster than into unparasitized ATPase that keeps the [Ca++]i of unparasitized RBCs exceedingly low. Acceleration of the preexisting Ca++ entry, ATPase that keeps the [Ca++] of unparasitized RBCs exceedingly low. Acceleration of the preexisting Ca++ entry, mediated by a divalent cation carrier, also cannot explain Ca++ accumulation in PRBCs: there are fundamental differences in substrate preference and in the effects of external Ca++ on 45Ca++ efflux between unparasitized RBCs and PRBCs. Patch clamp of intact PRBC surface membranes revealed rare unitary channel openings not observed on unparasitized RBCs. With 80 mM of CaCl2 in the patch pipette, this channel carried inward current, suggesting Ca++ entry at a rate comparable with the observed 45Ca++ flux. These data indicate that the malaria parasite induces a novel pathway in the host RBC membrane for Ca++ entry and suggest that this pathway is a Ca++-permeable channel.

Avian cathepsin B cDNA: sequence and demonstration that mRNAs of two sizes are produced in cell types producing large quantities of the enzyme.

Overlapping cDNA fragments encoding avian cathepsin B were cloned from an osteoclast cDNA library and sequenced. The primary structure of the prepro enzyme deduced from this sequence has 340 amino acids. The mature portion of the enzyme is 80% identical with murine cathepsin B; regions found in other papain superfamily enzymes are conserved. In osteoclasts and cultured macrophages, which produce large quantities of cathepsin B, mRNAs of 1.8 and 2.4 kb are produced in approximately equal quantities, while cells producing smaller quantities of the enzyme produce predominantly the 2.4 kb form. This variation in mRNAs suggests transcriptional differences related to production of large quantities of the enzyme.

Isolation and reconstitution of a vacuolar-type proton pump of osteoclast membranes.

A vacuolar-type proton-translocating ATPase was extracted from ruffled membranes of chicken osteoclasts with 1% polyoxyethylene 9-lauryl ether (C12E9) and was purified 13-fold by glycerol gradient centrifugation. The isolated pump appears by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a subunit composition similar to that of the clathrin-coated vesicle proton pump, in that subunits of apparent molecular masses of 116, 71, 57, 40, 39, 33, and 17 kDa are present in the osteoclast pump preparation. In addition, the 116-, 71-, 57-, and 40-kDa components were shown to cross-react with specific antisera generated against the homologous subunits of the clathrin-coated vesicle proton pump. The isolated osteoclast H(+)-ATPase was reconstituted into liposomes prepared from purified lipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol) by a cholate-dilution, freeze-thaw method. Proton transport catalyzed by the reconstituted pump was inhibited by bafilomycin A1 (10 nM) and N-ethylmaleimide (1 mM) but was insensitive to vanadate. We propose that osteoclast-mediated bone resorption is effected by a vacuolar-type proton pump with functional and structural similarities to that isolated from clathrin-coated vesicles.

Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase.

The success of Mycobacterium species as pathogens depends on their ability to maintain an infection inside the phagocytic vacuole of the macrophage. Although the bacteria are reported to modulate maturation of their intracellular vacuoles, the nature of such modifications is unknown. In this study, vacuoles formed around Mycobacterium avium failed to acidify below pH 6.3 to 6.5. Immunoelectron microscopy of infected macrophages and immunoblotting of isolated phagosomes showed that Mycobacterium vacuoles acquire the lysosomal membrane protein LAMP-1, but not the vesicular proton-adenosine triphosphatase (ATPase) responsible for phagosomal acidification. This suggests either a selective inhibition of fusion with proton-ATPase-containing vesicles or a rapid removal of the complex from Mycobacterium phagosomes.

Osteoclastic acid transport: mechanism and implications for physiological and pharmacological regulation.

In order to solubilize bone mineral and degrade the organic matrix of bone osteoclasts must secrete 1-2 protons for every Ca2+ liberated. This transport is a major metabolic activity of osteoclasts requiring an electrogenic H(+)-ATPase, a conductive chloride channel, a chloride-bicarbonate exchanger, carbonic anhydrase, and functional/morphological polarization of the cell. The osteoclast H(+)-ATPase is electrically coupled to a chloride channel in the ruffled membrane as are similar transport activities found in acidic intracellular vesicles, but the vanadate sensitivity of the osteoclast proton pump is intermediated between that of the E- and v-type proton pumps. The carbonic anhydrase and chloride-bicarbonate exchange provide an interface with pH regulation and integrate bone resorption into systemic acid-base balance. With the molecular mediators of bone resorption being known we may consider the control of bone resorption with an eye to mechanism and specificity that has not previously been possible. The effects of systemic acidosis to increase bone resorption and the effects of carbonic anhydrase deficiency are consistent with our mechanism of osteoclast ion transport.

Recent advances toward understanding osteoclast physiology.

Osteoclasts develop from precursor cells of the monocyte series. However, specialized differentiation for efficient bone degradation separates the osteoclast from the macrophage. The physical reasons for these differences are emerging from the study of osteoclastic physiology and biochemistry. Key osteoclast specializations are multinucleation, formation of a tightly sealed extracellular compartment on bone, and high-capacity secretion of HCl and acid proteases into this extracellular site. Multinucleation increases efficiency of extracellular attachment processes. The attachment process is mediated by cell membrane integrins, and is sensitive to changes in intracellular or extracellular calcium. Acid production exploits carbonic acid as the source of acid and conjugate base equivalents, reflected in abundant osteoclastic carbonic anhydrase type II expression. Secretion of acid involves extremely high expression of vacuolar-type H(+)-ATPase and a chloride channel in the cell's specialized acid secreting organelle, the ruffled membrane, which is polarized to the osteoclast's bone attachment. Acid secretion is balanced by chloride-bicarbonate exchange in the cell's nonbone attached membranes; this functionally resembles the band 3 chloride-bicarbonate exchanger of the red cell carbon dioxide transport system. Bone collagen is degraded by acid proteases secreted into the acid degradation site via the mannose-6-phosphate receptor system, which is targeted to lysosomes in other cells. Functional deficits, as in osteopetrosis, may affect any of the elements involved in osteoclast differentiation. Furthermore, new antiosteoclastic therapeutic agents may inhibit osteoclast biochemistry intentionally, such as for the control of hypercalcemia of malignancy.