Search

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 Ca 2+ 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.

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.

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.

Insulin secretion from pancreatic beta cells is dependent on maturation and acidification of the secretory granule, processes necessary for prohormone convertase cleavage of proinsulin. Previous studies in isolated beta cells revealed that acidification may be dependent on the granule membrane chloride channel ClC-3, in a step permissive for a regulated secretory response. In this study, immuno-EM of beta cells revealed colocalization of ClC-3 and insulin on secretory granules. Clcn3(-/-) mice as well as isolated islets demonstrate impaired insulin secretion; Clcn3(-/-) beta cells are defective in regulated insulin exocytosis and granular acidification. Increased amounts of proinsulin were found in the majority of secretory granules in the Clcn3(-/-) mice, while in Clcn3(+/+) cells, proinsulin was confined to the immature secretory granules. These results demonstrate that in pancreatic beta cells, chloride channels, specifically ClC-3, are localized on insulin granules and play a role in insulin processing as well as insulin secretion through regulation of granular acidification.

Ins(3,4,5,6)P(4) inhibits plasma membrane Cl(-) flux in secretory epithelia [1]. However, in most other mammalian cells, receptor-dependent elevation of Ins(3,4,5,6)P(4) levels is an "orphan" response that lacks biological significance [2]. We set out to identify Cl(-) channel(s) and/or transporter(s) that are regulated by Ins(3,4,5,6)P4 in vivo. Several candidates [3-5] were excluded through biophysical criteria, electrophysiological analysis, and confocal immunofluorescence microscopy. Then, we heterologously expressed ClC-3 in the plasma membrane of HEK293-tsA201 cells; whole-cell patch-clamp analysis showed Ins(3,4,5,6)P4 to inhibit Cl(-) conductance through ClC-3. Next, we heterologously expressed ClC-3 in the early endosomal compartment of BHK cells; by fluorescence ratio imaging of endocytosed FITC-transferrin, we recorded intra-endosomal pH, an in situ biosensor for Cl(-) flux across endosomal membranes [6]. A cell-permeant, bioactivatable Ins(3,4,5,6)P4 analog elevated endosomal pH from 6.1 to 6.6, reflecting inhibition of ClC-3. Finally, Ins(3,4,5,6)P(4) inhibited endogenous ClC-3 conductance in postsynaptic membranes of neonatal hippocampal neurones. Among other ClC-3 functions that could be regulated by Ins(3,4,5,6)P4 are tumor cell migration [7], apoptosis [8], and inflammatory responses [9]. Ins(3,4,5,6)P4 is a ubiquitous cellular signal with diverse biological actions.

It is well established that ligand-gated chloride flux across the plasma membrane modulates neuronal excitability. We find that a voltage-dependent Cl(-) conductance increases neuronal excitability in immature rodents as well, enhancing the time course of NMDA receptor-mediated miniature excitatory postsynaptic potentials (mEPSPs). This Cl(-) conductance is activated by CaMKII, is electrophysiologically identical to the CaMKII-activated CLC-3 conductance in nonneuronal cells, and is absent in clc-3(-/-) mice. Systematically decreasing [Cl(-)](i) to mimic postnatal [Cl(-)](i) regulation progressively decreases the amplitude and decay time constant of spontaneous mEPSPs. This Cl(-)-dependent change in synaptic strength is absent in clc-3(-/-) mice. Using surface biotinylation, immunohistochemistry, electron microscopy, and coimmunoprecipitation studies, we find that CLC-3 channels are localized on the plasma membrane, at postsynaptic sites, and in association with NMDA receptors. This is the first demonstration that a voltage-dependent chloride conductance modulates neuronal excitability. By increasing postsynaptic potentials in a Cl(-) dependent fashion, CLC-3 channels regulate neuronal excitability postsynaptically in immature neurons.

CLC-3, a member of the CLC family of chloride channels, mediates function in many cell types in the body. The multifunctional calcium-calmodulin-dependent protein kinase II (CaMKII) has been shown to activate recombinant CLC-3 stably expressed in tsA cells, a human embryonic kidney cell line derivative, and natively expressed channel protein in a human colonic tumour cell line T84. We examined the CaMKII-dependent regulation of CLC-3 in a smooth muscle cell model as well as in the human colonic tumour cell line, HT29, using whole-cell voltage clamp. In CLC-3-expressing cells, we observed the activation of a Cl(-) conductance following intracellular introduction of the isolated autonomous CaMKII into the voltage-clamped cell via the patch pipette. The CaMKII-dependent Cl(-) conductance was not observed following exposure of the cells to 1 microm autocamtide inhibitory peptide (AIP), a selective inhibitor of CaMKII. Arterial smooth muscle cells express a robust CaMKII-activated Cl(-) conductance; however, CLC-3(-/-) cells did not. The N-terminus of CLC-3, which contains a CaMKII consensus sequence, was phosphorylated by CaMKII in vitro, and mutation of the serine at position 109 (S109A) abolished the CaMKII-dependent Cl(-) conductance, indicating that this residue is important in the gating of CLC-3 at the plasma membrane.

The multifunctional calcium/calmodulin-dependent protein kinase II, CaMKII, has been shown to regulate chloride movement and cellular function in both excitable and non-excitable cells. We show that the plasma membrane expression of a member of the ClC family of Cl(-) channels, human CLC-3 (hCLC-3), a 90-kDa protein, is regulated by CaMKII. We cloned the full-length hCLC-3 gene from the human colonic tumor cell line T84, previously shown to express a CaMKII-activated Cl(-) conductance (I(Cl,CaMKII)), and transfected this gene into the mammalian epithelial cell line tsA, which lacks endogenous expression of I(Cl,CaMKII). Biotinylation experiments demonstrated plasma membrane expression of hCLC-3 in the stably transfected cells. In whole cell patch clamp experiments, autonomously active CaMKII was introduced into tsA cells stably transfected with hCLC-3 via the patch pipette. Cells transfected with the hCLC-3 gene showed a 22-fold increase in current density over cells expressing the vector alone. Kinase-dependent current expression was abolished in the presence of the autocamtide-2-related inhibitory peptide, a specific inhibitor of CaMKII. A mutation of glycine 280 to glutamic acid in the conserved motif in the putative pore region of the channel changed anion selectivity from I(-) > Cl(-) to Cl(-) > I(-). These results indicate that hCLC-3 encodes a Cl(-) channel that is regulated by CaMKII-dependent phosphorylation.

1. We have studied the regulation of whole-cell chloride current in T84 colonic epithelial cells by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4). New information was obtained using (a) microcystin and okadaic acid to inhibit serine/threonine protein phosphatases, and (b) a novel functional tetrakisphosphate analogue, 1, 2-bisdeoxy-1,2-bisfluoro-Ins(3,4,5,6)P4 (i.e. F2-Ins(3,4,5,6)P4). 2. Calmodulin-dependent protein kinase II (CaMKII) increased chloride current 20-fold. This current (ICl,CaMK) continued for 7 +/- 1.2 min before its deactivation, or running down, by approximately 60 %. This run-down was prevented by okadaic acid, whereupon ICl,CaMK remained near its maximum value for >= 14.3 +/- 0.6 min. 3. F2-Ins(3, 4,5,6)P4 inhibited ICl,CaMK (IC50 = 100 microM) stereo-specifically, since its enantiomer, F2-Ins(1,4,5,6)P4 had no effect at >= 500 microM. Dose-response data (Hill coefficient = 1.3) showed that F2-Ins(3,4,5,6)P4 imitated only the non-co-operative phase of inhibition by Ins(3,4,5,6)P4, and not the co-operative phase. 4. Ins(3,4,5,6)P4 was prevented from blocking ICl,CaMK by okadaic acid (IC50 = 1.5 nM) and microcystin (IC50 = 0.15 nM); these data lead to the novel conclusion that, in situ, protein phosphatase activity is essential for Ins(3,4,5,6)P4 to function. The IC50 values indicate that more than one species of phosphatase was required. One of these may be PP1, since F2-Ins(3,4,5,6)P4-dependent current blocking was inhibited by okadaic acid and microcystin with IC50 values of 70 nM and 0.15 nM, respectively.

Increases in astroglial Cl- conductance accompany changes in cell morphology and disassembly of cytoskeletal actin, but Cl- channels underlying these conductance increases have not been described. We characterize an outwardly rectifying Cl- channel in rodent neocortical cultured astrocytes and describe how cell shape and cytoskeletal actin modulate channel gating. In inside-out patch-clamp recordings from cultured astrocytes, outwardly rectifying Cl- channels either were spontaneously active or inducible in quiescent patches by depolarizing voltage steps. Average single-channel conductance was 36 pS between -60 and -80 mV and was 75 pS between 60 and 80 mV in symmetrical (150 mM NaCl) solutions. The permeability ratio (PNa/PCl) was 0.14 at lower ionic strength but increased at higher salt concentrations. Both ATP and 4, 4-diisothiocyanostilbene-2,2'-disulfonic acid produced a flicker block, whereas Zn2+ produced complete inhibition of channel activity. The frequency of observing both spontaneous and inducible Cl- channel activity was markedly higher in stellate than in flat, polygonally shaped astrocytes. In addition, cytoskeletal actin modulated channel open-state probability (PO) and conductance at negative membrane potentials, controlling the degree of outward rectification. Direct application of phalloidin, which stabilizes actin, preserved low PO and promoted lower conductance levels at negative potentials. Lower PO also was induced by direct application of polymerized actin. The actions of phalloidin and actin were reversed by coapplication of gelsolin and cytochalasin D, respectively. These results provide the first report of an outwardly rectifying Cl- channel in neocortical astrocytes and demonstrate how changes in cell shape and cytoskeletal actin may control Cl- conductance in these cells.

The mechanism by which inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5, 6)P4) regulates chloride (Cl-) secretion was evaluated in the colonic epithelial cell line T84 using whole cell voltage clamp techniques. Our studies focused on the calcium-dependent chloride conductance (gClCa) that was activated either by mobilizing intracellular calcium (Cai) stores with thapsigargin or by introduction of the autonomous, autophosphorylated calmodulin-dependent protein kinase II (CaMKII) into the cell via the patch pipette. Basal concentrations of Ins(3,4,5,6)P4 (1 microM) present in the pipette solution had no significant effect on Cl- current; however, as the concentration of the polyphosphate was increased there was a corresponding reduction in anion current, with near complete inhibition at 8-10 microM Ins(3,4,5,6)P4. Corresponding levels are found in cells after sustained receptor-dependent activation of phospholipase C. The Ins(3,4,5, 6)P4-induced inhibition of gClCa was isomer specific; neither Ins(1, 3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, nor Ins(1,3,4,5,6)P5 induced current inhibition at concentrations of up to 100 microM. Annexin IV also plays an inhibitory role in modulating gClCa in T84 cells. When 2 microM annexin IV was present in the pipette solution, a concentration that by itself has no effect on gClCa, the potency of Ins(3,4,5,6)P4 was approximately doubled. The combination of Ins(3,4,5,6)P4 and annexin IV did not alter the in vitro activity of CaMKII. These data demonstrate that Ins(3,4,5,6)P4 is an additional cellular signal that participates in the control of salt and fluid secretion, pH balance, osmoregulation, and other physiological activities that depend upon gClCa activation. Ins(3,4,5,6)P4 metabolism and action should also be taken into account when designing treatment strategies for cystic fibrosis.

Ca(2+)-activated Cl- current (ICl,Ca) in colonic T84 cells is inhibited by the specific peptide inhibitor of Ca2+/calmodulin-dependent kinase II (CaM KII). Annexin IV, a Ca(2+)-dependent phospholipid binding protein also inhibits Ca(2+)-dependent anion current activation (Kaetzel, M.A., Chan, H.-C., Dubinsky, W.P., Dedman, J.R., and Nelson, D.J. (1994) J. Biol. Chem. 269, 5297-5302). Intracellular injection of antibodies against annexin IV enhances current activation; this activation is inhibited by the peptide inhibitor of CaM KII. Intracellular application of autonomously active CaM KII in the presence of ATP induced a Cl- current similar to that activated by the Ca2+ ionophore A23187. Current activation by the exogenous kinase was completely inhibited in the presence of purified annexin IV. In vitro, annexin IV does not inhibit CaM KII activity nor does it act as a substrate for CaM KII. Thus, it appears that annexin IV inhibits phosphorylation-dependent anion conductance activation by preventing CaM KII-ion channel interaction rather than by direct interaction with the enzyme itself. These findings suggest a novel mechanism by which Ca(2+)-dependent membrane binding proteins, cytoplasmic kinases, and ion channels interact to regulate membrane conductance. The characterization of unique channel regulatory pathways in Cl- transporting epithelia may identify potential avenues of alternate therapy to compensate for the loss of functional Cl- channels in the disease of cystic fibrosis.

The cellular function of annexin IV was evaluated by correlating tissue expression, cellular localization, and whole-cell electrophysiology. Immunolocalization and biochemical data demonstrate that annexin IV is concentrated along the apical membranes of many epithelia. Introduction of purified exogenous annexin IV into colonic T84 cells through a patch pipette specifically prevented Ca(2+)-dependent Cl- current activation. Affinity-purified antibody against annexin IV applied in the same manner enhanced the activation. Reduction of the endogenous level of annexin IV with a derivatized oligodeoxynucleotide antisense to annexin IV mRNA lowered the threshold for the Ca(2+)-induced current response, mimicking the enhancement of current activation exerted by anti-annexin IV antibody. The inhibitory effect of annexin IV on Ca(2+)-dependent Cl- conductance represents a novel mechanism by which Ca(2+)-binding proteins modulate membrane channel activity.

We describe the activation of a K+ current and inhibition of a Cl- current by a cyanoguanidine activator of ATP-sensitive K+ channels (KATP) in the smooth muscle cell line A10. The efficacy of U83757, an analogue of pinacidil, as an activator of KATP was confirmed in single channel experiments on isolated ventricular myocytes. The effects of U83757 were examined in the clonal smooth muscle cell line A10 using voltage-sensitive dyes and digital fluorescent imaging techniques. Exposure of A10 cells to U83757 (10 nM to 1 microM) produced a rapid membrane hyperpolarization as monitored by the membrane potential-sensitive dye bis-oxonol ([diBAC4(3)], 5 microM). The U83757-induced hyperpolarization was antagonized by glyburide and tetrapropylammonium (TPrA) but not by tetraethlyl-ammonium (TEA) or charybdotoxin (ChTX). The molecular basis of the observed hyperpolarization was studied in whole-cell, voltage-clamp experiments. Exposure of voltage-clamped cells to U83757 (300 nM to 300 microM) produced a hyperpolarizing shift in the zero current potential; however, the hyperpolarizing shift in reversal potential was associated with either an increase or decrease in membrane conductance. In solutions where EK = -82 mV and ECl = 0 mV, the reversal potential of the U83757-sensitive current was approximately -70 mV in those experiments where an increase in membrane conductance was observed. In experiments in which a decrease in conductance was observed, the reversal potential of the U83757-sensitive current was approximately 0 mV, suggesting that U83757 might be acting as a Cl- channel blocker as well as a K+ channel opener. In experiments in which Cl- current activation was specifically brought about by cellular swelling and performed in solutions where Cl- was the major permeant ion, U83757 (300 nM to 300 microM) produced a dose-dependent current inhibition. Taken together these results (i) demonstrate the presence of a K(+)-selective current which is sensitive to KATP channel openers in A10 cells and (ii) indicate that the hyperpolarizing effects of K+ channel openers in vascular smooth muscle may be due to both the inhibition of Cl- currents as well as the activation of a K(+)-selective current.