Mechanism of the defect in gap-junctional communication by expression of a connexin 26 mutant associated with dominant deafness.

Gap-junctional channels (connexin oligomers) are large-diameter aqueous pores formed by head-to-head association of two gap-junctional hemichannels, one from each of the adjacent cells. Profound hearing loss of genetic origin is common, and mutations of connexin 26 (Cx26) are the most frequent cause of this disorder. The Cx26 R75W mutant has been associated with disruption of cell-to-cell communication and profound hearing loss, but the mechanism of the gap-junctional defect is unknown. Here, we show that Cx26 R75W forms gap-junctional hemichannels that display altered voltage dependency and reduced permeability, and which cannot form functional gap-junctional channels between neighboring cells. The R75W phenotype is dominant at the gap-junction channel but not at the hemichannel level. Therefore, the absence of gap-junctional communication caused by R75W expression is due to defective gap-junction formation by functional hemichannels.

Do connexin 43 gap-junctional hemichannels activate and cause cell damage during ATP depletion of renal-tubule cells?

UNLABELLED: We review our evidence in favour of the hypothesis that gap-junctional hemichannels (GJH) are activated by depletion of adenosine triphosphate (ATP) in human renal proximal tubule cells in primary culture (hPT cells). Undocked GJH permit fluxes of ions and hydrophilic molecules up to 1 kDa, and thus their opening can cause alterations of cell composition conducive to cell damage. We show that hPT cells express connexin 43 (Cx43) (at the mRNA and protein levels) and that the protein is expressed on the plasma membrane. Moderate levels of pharmacological depletion of ATP increased plasma-membrane permeability, as shown by loading with the hydrophilic dye 5/6 carboxyfluorescein (CF, 376 Da) and other low-molecular weight dyes, but not with fluorescein-labelled dextran (>1500 Da). Roles for endocytosis and activation of purinergic-receptor channels were experimentally ruled out. Moderate ATP depletion also caused necrosis, assessed by cell permeabilization to propidium iodide. Prolonged exposure to gadolinium reduced both the dye loading and the necrosis induced by ATP depletion, i.e. it protected the cells. Cx43 overexpressed in insect cells, purified to homogeneity and reconstituted in proteoliposomes formed hemichannels that are activated by dephosphorylation of Ser368, a residue in a protein-kinase-C consensus phosphorylation sequence near the end of the C-terminal domain. CONCLUSIONS: (1) ATP depletion of hPT cells induces a Gd3+-sensitive permeability of the plasma membrane to hydrophilic dyes with a cut-off size consistent with Cx43 GJH. (2) ATP depletion also increases the percentage of necrotic cells, an effect also reduced by Gd3+. (3) The experiments with purified Cx43 reconstituted in liposomes suggest that dephosphorylation of Ser368 is sufficient to activate GJH, although other mechanisms may be involved in some cells.

Gap-junctional hemichannels are activated by ATP depletion in human renal proximal tubule cells.

We present evidence suggesting that gap-junctional hemichannels (GJH) may be involved in acute ischemic injury of human renal proximal tubule cells (hPT cells). Two GJH, from neighboring cells, join to form an intercellular gap junction channel (GJC). Undocked GJH are permeable to hydrophilic molecules up to 1 kDa, and their opening can significantly alter cell homeostasis. Both GJC and GJH formed by connexin 43 (Cx43) are activated by dephosphorylation. Hence, we tested whether GJH activation during ATP depletion contributes to cell damage in renal ischemia. We found that hPT cells in primary culture express Cx43 (RT-PCR and Western-blot analysis) at the plasma membrane region (immunofluorescence). Divalent-cation removal or pharmacological ATP depletion increased cell loading with the hydrophilic dye 5/6 carboxy-fluorescein (CF, 376 Da) but not with fluorescein-labeled dextran (>1500 Da). Endocytosis and activation of P2X channels were experimentally ruled out. Several GJC blockers inhibited the loading elicited by PKC inhibition. Double labeling (CF and propidium iodide) showed that both Ca(2+) removal and ATP depletion increase the percentage of necrotic cells. Gadolinium reduced both the loading and the degree of necrosis during divalent-cation removal or ATP depletion. In conclusion, GJH activation may play an important role in the damage of human renal proximal tubule cells during ATP depletion. These studies are the first to provide evidence supporting a role of GJH in causing injury in epithelial cells in general and in renal-tubule cells in particular.

Pregnancy outcomes and community health: the POUCH study of preterm delivery.

In light of the social/ethnic disparity in preterm delivery (PTD) rates, the Pregnancy Outcomes and Community Health (POUCH) Study takes a broad view of the determinants of PTD by attempting to link underlying biological and psychosocial factors. The relationships between placental pathology, maternal biomarkers, and antecedent psychosocial factors are evaluated in three hypothesised pathways of PTD - one characterised primarily by infection, one by maternal vascular disease, and one by premature elevations in corticotropin releasing hormone in the absence of histological evidence of placental pathology. Within each pathway, an emphasis is placed on understanding the roles of stress and of maternal serum alpha-fetoprotein, an early biomarker associated with PTD. The POUCH Study enrolls pregnant women from five Michigan communities. Information about these women and their environments is gathered through detailed interviews and collection of biological samples including hair, urine, saliva, blood, vaginal fluid, and vaginal smear at 15-26 weeks of gestation. We have chosen to focus on the second trimester--a time when pathological processes may have evolved to a detectable stage, but generally before the onset of biological changes that accompany labour. This focus is consistent with the long-range goal of early detection/intervention and prevention of PTD.

Cholera toxin-induced PGE(2) activity is reduced by chemical reaction with L-histidine.

Mediators of cholera toxin (CT)-induced fluid secretion include 3',5'-adenosine monophosphate (cAMP), prostaglandin E(2) (PGE(2)), and 5-hydroxytryptamine (5-HT). Administration of L-histidine significantly reduced the net secretory response of the small intestine of mice challenged with CT and reduced the capacity of PGE(2) to stimulate Na+ transport in Ussing chambers. We demonstrated that L-histidine chemically modified the structure of PGE(2) but had no direct effect on cAMP or 5-HT. L-Histidine and imidazole reacted with PGE(2) in vitro in cell-free mixtures incubated at 37 degrees C and pH 7.0 under an atmosphere of N(2) with the formation of PGE(2)-imidazole and PGE(2)-histidine covalent adducts. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) analysis of the purified adduct showed that imidazole catalyzed the dehydration of PGE(2). A Michael adduct then was formed between C11 of 11-deoxy-Delta(10) PGE(2) (PGA(2)) and the tau nitrogen in the imidazole ring of L-histidine. Importantly, the isolated PGE(2)-imidazole and PGE(2)-histidine adducts inhibited CT-induced fluid loss and cAMP accumulation in mouse intestinal loops. The protection provided by PGE(2)-imidazole, PGE(2)-histidine, and L-histidine against intestinal fluid loss could provide a basis for future therapy against cholera.

PKC-mediated stimulation of amphibian CFTR depends on a single phosphorylation consensus site. insertion of this site confers PKC sensitivity to human CFTR.

Mutations of the CFTR, a phosphorylation-regulated Cl(-) channel, cause cystic fibrosis. Activation of CFTR by PKA stimulation appears to be mediated by a complex interaction between several consensus phosphorylation sites in the regulatory domain (R domain). None of these sites has a critical role in this process. Here, we show that although endogenous phosphorylation by PKC is required for the effect of PKA on CFTR, stimulation of PKC by itself has only a minor effect on human CFTR. In contrast, CFTR from the amphibians Necturus maculosus and Xenopus laevis (XCFTR) can be activated to similar degrees by stimulation of either PKA or PKC. Furthermore, the activation of XCFTR by PKC is independent of the net charge of the R domain, and mutagenesis experiments indicate that a single site (Thr665) is required for the activation of XCFTR. Human CFTR lacks the PKC phosphorylation consensus site that includes Thr665, but insertion of an equivalent site results in a large activation upon PKC stimulation. These observations establish the presence of a novel mechanism of activation of CFTR by phosphorylation of the R domain, i.e., activation by PKC requires a single consensus phosphorylation site and is unrelated to the net charge of the R domain.

Cell-volume changes and ion conductances in amphibian gallbladder epithelium.

Cell-volume regulation after hyposmotic swelling is common in transporting epithelial cells. Acute regulatory volume decrease is mediated in several cell types by loss of K(+) and Cl(-) via channels either activated by the swelling phenomenon or active in the cell membrane prior to swelling. In the last two decades, it has become clear that many features of cell-volume regulation mediated by ion fluxes vary considerably among cell types. In some instances, the ion channel activation, although demonstrable, is insufficient to account, on the basis of ion fluxes, for the measured cell volume changes. Here we review the case of a salt-transporting epithelium in which hyposmotic cell swelling activates plasma membrane K(+) channels, but at the same time inhibits Cl(-) channels, thus preventing an acute volume-regulatory response. The sequence of events following cell swelling appears to be as follows: K(+) channels are activated by membrane stretch, the cell membrane hyperpolarizes (the voltage moves closer to the K(+) equilibrium potential) and this hyperpolarization reduces the Cl(-) conductance, likely by a reduction in open probability of voltage-sensitive Cl(-) channels. The significance of this phenomenon may be related to the preservation of intracellular Cl(-) rather than cell volume, or to a physiological 'need' to prevent shrinkage of cells stimulated by hormones or other mediators.

Inhibition of P-glycoprotein-mediated transport by a hydrophobic contaminant in commercial gluconate salts.

The substitution of gluconate for Cl- is commonly used to characterize Cl- transport or Cl--dependent transport mechanisms. We evaluated the effects of substituting gluconate for Cl- on the transport of the P-glycoprotein substrate rhodamine 123 (R123). The replacement of Ringer solution containing Cl- (Cl--Ringer) with gluconate-Ringer inhibited R123 efflux, whereas the replacement of Cl- by other anions (sulfate or cyclamate) had no effect. The inhibition of R123 efflux by gluconate-Ringer was absent after chloroform extraction of the sodium gluconate salt. The readdition of the sodium gluconate-chloroform extract to the extracted gluconate-Ringer or to cyclamate-Ringer inhibited R123 efflux, whereas its addition to Cl--Ringer had no effect. These observations indicate that the inhibition of P-glycoprotein-mediated R123 transport by gluconate is due to one or more chloroform-soluble contaminants and that the inhibition is absent in the presence of Cl-. The results are consistent with the fact that P-glycoprotein substrates are hydrophobic. Care should be taken when replacing ions to evaluate membrane transport mechanisms because highly pure commercial preparations may still contain potent contaminants that affect transport.

Stretch-activated single K+ channels account for whole-cell currents elicited by swelling.

Functionally significant stretch-activated ion channels have been clearly identified in excitable cells. Although single-channel studies suggest their expression in other cell types, their activity in the whole-cell configuration has not been shown. This discrepancy makes their physiological significance doubtful and suggests that their mechanical activation is artifactual. Possible roles for these molecules in nonexcitable cells are acute cell-volume regulation and, in epithelial cells, the complex adjustment of ion fluxes across individual cell membranes when the rate of transepithelial transport changes. We report the results of experiments on isolated epithelial cells expressing in the basolateral membrane stretch-activated K+ channels demonstrable by the cell-attached patch-clamp technique. In these cells, reversible whole-cell currents were elicited by both isosmotic and hyposmotic cell swelling. Cation selectivity and block by inorganic agents were the same for single-channel and whole-cell currents, indicating that the same entity underlies single-channel and whole-cell currents and that the single-channel events are not artifactual. In these cells, when the rate of apical-membrane NaCl entry increases, the cell Na+ content and volume also increase, stimulating the Na+,K+-ATPase at the basolateral membrane, i.e., both Na+ extrusion and K+ uptake increase. We speculate that, under these conditions, the parallel activation of basolateral K+ channels (by the swelling) elevates conductive K+ loss, tending to maintain the cell K+ content constant ("pump-leak parallelism"). This study describes a physiologically relevant stretch-activated channel, at both the single-channel and whole-cell levels, in a nonneural cell type.

Phosphorylation of P-glycoprotein by PKA and PKC modulates swelling-activated Cl- currents.

Several proteins belonging to the ATP-binding cassette superfamily can affect ion channel function. These include the cystic fibrosis transmembrane conductance regulator, the sulfonylurea receptor, and the multidrug resistance protein P-glycoprotein (MDR1). We measured whole cell swelling-activated Cl- currents (ICl,swell) in parental cells and cells expressing wild-type MDR1 or a phosphorylation-defective mutant (Ser-661, Ser-667, and Ser-671 replaced by Ala). Stimulation of protein kinase C (PKC) with a phorbol ester reduced the rate of increase in ICl,swell only in cells that express MDR1. PKC stimulation had no effect on steady-state ICl,swell. Stimulation of protein kinase A (PKA) with 8-bromoadenosine 3',5'-cyclic monophosphate reduced steady-state ICl, swell only in MDR1-expressing cells. PKA stimulation had no effect on the rate of ICl,swell activation. The effects of stimulation of PKA and PKC on ICl,swell were additive (i.e., decrease in the rate of activation and reduction in steady-state ICl,swell). The effects of PKA and PKC stimulation were absent in cells expressing the phosphorylation-defective mutant. In summary, it is likely that phosphorylation of MDR1 by PKA and by PKC alters swelling-activated Cl- channels by independent mechanisms and that Ser-661, Ser-667, and Ser-671 are involved in the responses of ICl,swell to stimulation of PKA and PKC. These results support the notion that MDR1 phosphorylation affects ICl,swell.

Isolated epithelial cells from amphibian urinary bladder express functional gap junctional hemichannels.

Exposure of the urinary bladder epithelium of Necturus maculosus (NUB) to protease and collagenase yields approximately 50% isolated polarized cells. These cells express a membrane current slowly activated by depolarization or by removal of external divalent cations. The biophysical and pharmacological properties of the current are largely consistent with those of gap junctional hemichannels. After removal of divalent cations, the cells can also be loaded with 5(6)-carboxyfluorescein, a hydrophilic fluorescent anionic dye, and exposure to dye reduces the current in a manner dependent on membrane voltage and side of application. In contrast, Necturus gallbladder (NGB) cells exhibit no membrane conductance attributable to gap junctional hemichannels, although previous studies reveal the persistence of gap junction plaques on the plasma membrane. We conclude that functional gap junctional hemichannels can be expressed on the surface of certain isolated epithelial cells and that this is not a necessary consequence of the isolation procedure. These structures may contribute to cell damage under pathological conditions involving cell detachment.

Swelling-activated chloride currents in a drug-sensitive cell line and a P glycoprotein-expressing derivative are underlied by channels with the same pharmacological properties.

It has been proposed that P glycoprotein (Pgp) expression is associated with swelling-activated Cl- currents in multidrug-resistant cells. The Pgp substrate vinblastine and the modulator verapamil produced a reversible concentration-dependent block of swelling-activated Cl- currents in both a drug-sensitive cell line (MCF-7) and a Pgp-expressing derivative (BC19/3). The similarity of the results obtained in both cell lines suggests that the mechanism of block is not related to Pgp expression and supports the hypothesis that Pgp expression is not necessary for the swelling activation of Cl- currents. In contrast to the results obtained with vinblastine, two other cytoskeleton-disrupting agents, colchicine and cytochalasin D, were not able to affect the swelling-activated Cl- currents in either cell line. The data provided no evidence for the involvement of the cytoskeleton in the swelling activation of Cl- channels in these cell lines. The Cl- channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid and 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, each produced a similar reversible concentration-dependent block in the swelling-activated currents in both the Pgp-expressing and nonexpressing cells. This strongly suggests that the Cl- channel(s) responsible for the swelling-dependent current in both cell lines are the same and, since MCF-7 cells do not express Pgp, that Pgp is not the channel responsible for the volume-activated Cl- currents in these cells.

P-glycoprotein is not a swelling-activated Cl- channel; possible role as a Cl- channel regulator.

1. The whole-cell configuration of the patch-clamp technique was used to determine if P-glycoprotein (Pgp) is a swelling-activated Cl- channel. 2. Hamster pgp1 cDNA was transfected into a mouse fibroblast cell line resulting in expression of functional Pgp in the plasma membrane. This cell line was obtained without exposure to chemotherapeutic agents. 3. Swelling-activated whole-cell Cl- current (ICl,swell) was elicited by lowering the bath osmolality. ICl,swell was characterized in detail in the pgp1-transfected mouse cell line and compared with that of its parental cell line. Expression of Pgp did not modify the magnitude or properties of ICl,swell, except that addition of the anti-Pgp antibody C219 to the pipette solution inhibited this current by 75% only in the Pgp-expressing cells. 4. ICl,swell in the mouse Pgp-expressing cell line was compared with that in a Pgp-expressing hamster fibroblast cell line. The characteristics of ICl,swell (voltage dependence, blocker sensitivity, anion selectivity sequence, requirement for hydrolysable ATP) in Pgp-expressing cells were different between the two cell lines. These results suggest that the channel(s) responsible for ICl,swell are different between the two cell lines. In addition, C219 inhibited ICl,swell in both Pgp-expressing cell lines, even though they seem to express different swelling-activated Cl- channels. 5. We conclude that firstly, Pgp is not a swelling-activated Cl- channel; secondly, it possibly functions as a Cl- channel regulator; and thirdly, ICl,swell is underlined by different Cl- channels in different cells.

Cell swelling activates the K+ conductance and inhibits the Cl- conductance of the basolateral membrane of cells from a leaky epithelium.

Necturus gallbladder epithelial cells bathed in 10 mM HCO3/1% CO2 display sizable basolateral membrane conductances for Cl- (GClb) and K+ (GKb). Lowering the osmolality of the apical bathing solution hyperpolarized both apical and basolateral membranes and increased the K+/Cl- selectivity of the basolateral membrane. Hyperosmotic solutions had the opposite effects. Intracellular free-calcium concentration ([Ca2+]i) increased transiently during hyposmotic swelling (peak at approximately 30 s, return to baseline within approximately 90 s), but chelation of cell Ca2+ did not prevent the membrane hyperpolarization elicited by the hyposmotic solution. Cable analysis experiments showed that the electrical resistance of the basolateral membrane decreased during hyposmotic swelling and increased during hyperosmotic shrinkage, whereas the apical membrane resistance was unchanged in hyposmotic solution and decreased in hyperosmotic solution. We assessed changes in cell volume in the epithelium by measuring changes in the intracellular concentration of an impermeant cation (tetramethylammonium), and in isolated polarized cells measuring changes in intracellular calcein fluorescence, and observed that these epithelial cells do not undergo measurable volume regulation over 10-12 min after osmotic swelling. Depolarization of the basolateral membrane voltage (Vcs) produced a significant increase in the change in Vcs elicited by lowering basolateral solution [Cl-], whereas hyperpolarization of Vcs had the opposite effect. These results suggest that: (a) Hyposmotic swelling increases GKb and decreases GClb. These two effects appear to be linked, i.e., the increase in GKb produces membrane hyperpolarization, which in turn reduces GClb. (b) Hyperosmotic shrinkage has the opposite effects on GKb and GClb. (c) Cell swelling causes a transient increase in [Ca2+]i, but this response may not be necessary for the increase in GKb during cell swelling.

Polarized expression of cAMP-activated chloride channels in isolated epithelial cells.

We have described a preparation of Necturus maculosus gallbladder (NGB) epithelium yielding isolated cells that retain structural and functional polarity ("figure-eight" cells). These cells have a normal membrane voltage and remain polarized for several hours after isolation. Apical and basolateral membrane domains are differentially labeled with hydrophobic fluorescent dyes; freeze-fracture electron microscopy reveals two distinct membrane domains separated by tight junctions; ZO-1, Na+/H+ exchanger (NHE3), and Na(+)-K(+)-ATPase are present in the junctional, apical, and basolateral region, respectively; and cell-attached patch-clamp experiments reveal different K+ currents in the two membrane domains [R. J. Torres, G. A. Altenberg, J. A. Copello, G. Zampighi, and L. Reuss, Am. J. Physiol. 270 (Cell Physiol. 39): C1864-C1874, 1996]. Here, we show that NGB epithelial cells express a protein cross-reactive with an antibody against human cystic fibrosis transmembrane conductance regulator (CFTR). In figure-eight cells, immunoreactivity was restricted to the apical membrane domain. Using intracellular microelectrodes and a novel method of regional superfusion, we found that control cells have high K+ conductances in both membranes and a small basolateral Cl- conductance, similar to findings in the epithelium. Activation of adenylate cyclase with forskolin elicited a large apical membrane Cl- conductance and membrane depolarization. Whole cell patch-clamp studies yielded a forskolin-activated linear Cl- current, with high Cl-/aspartate selectivity. In conclusion, 1) figure-eight cells maintain the conductive membrane properties present in the epithelium, including polarized expression of adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- channels, and 2) the cAMP-activated Cl- conductance is underlied by a CFTR homologue.