Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein. While 70% of CF chromosomes carry a deletion of the phenylalanine residue 508 (deltaF508) of CFTR, roughly 5% of all CF chromosomes carry a premature stop mutation. We reported that the aminoglycoside antibiotics G-418 and gentamicin can suppress two premature stop mutations [a stop codon in place of glycine residue 542 (G542X) and arginine residue 553 (R553X)] when expressed from a CFTR cDNA in HeLa cells. Suppression resulted in the synthesis of full-length CFTR protein and the appearance of a cAMP-activated anion conductance characteristic of CFTR function. However, it was unclear whether this approach could restore CFTR function in cells expressing mutant forms of CFTR from the nuclear genome. We now report that G-418 and gentamicin are also capable of restoring CFTR expression in a CF bronchial epithelial cell line carrying the CFTR W1282X premature stop mutation (a stop codon in place of tryptophan residue 1282). This conclusion is based on the reappearance of cAMP-activated chloride currents, the restoration of CFTR protein at the apical plasma membrane, and an increase in the abundance of CFTR mRNA levels from the W1282X allele.

Primary structure of an apical protein from Xenopus laevis that participates in amiloride-sensitive sodium channel activity.

High resistance epithelia express on their apical side an amiloride-sensitive sodium channel that controls sodium reabsorption. A cDNA was found to encode a 1,420-amino acid long polypeptide with no signal sequence, a putative transmembrane segment, and three predicted amphipathic alpha helices. A corresponding 5.2-kb mRNA was detected in Xenopus laevis kidney, intestine, and oocytes, with weak expression in stomach and eyes. An antibody directed against a fusion protein containing a COOH-terminus segment of the protein and an antiidiotypic antibody known to recognize the amiloride binding site of the epithelial sodium channel (Kleyman, T. R., J.-P. Kraehenbuhl, and S. A. Ernst. 1991. J. Biol. Chem. 266:3907-3915) immunoprecipitated a similar protein complex from [35S]methionine-labeled and from apically radioiodinated Xenopus laevis kidney-derived A6 cells. A single integral of 130-kD protein was recovered from samples reduced with DTT. The antibody also cross-reacted by ELISA with the putative amiloride-sensitive sodium channel isolated from A6 cells (Benos, D. J., G. Saccomani, and S. Sariban-Sohraby. 1987. J. Biol. Chem. 262:10613-10618). Although the protein is translated, cRNA injected into oocytes did not reconstitute amiloride-sensitive sodium transport, while antisense RNA or antisense oligodeoxynucleotides specific for two distinct sequences of the cloned cDNA inhibited amiloride-sensitive sodium current induced by injection of A6 cell mRNA. We propose that the cDNA encodes an apical plasma membrane protein that plays a role in the functional expression of the amiloride-sensitive epithelial sodium channel. It may represent a subunit of the Xenopus laevis sodium channel or a regulatory protein essential for sodium channel function.

Irreversible inhibition of sodium entry sites in frog skin by a photosensitive amiloride analog.

A photosensitive binding reaction is described in which an analog of amiloride is bound to sites that control sodium entry into frog skin. This reaction results in irreversible inhibition of net sodium transport.

Role of membrane curvature in mechanoelectrical transduction: ion carriers nonactin and valinomycin sense changes in integral bending energy.

We describe the phenomenon of mechanoelectrical transduction in macroscopic lipid bilayer membranes modified by two cation-selective ionophores, valinomycin and nonactin. We found that bulging these membranes, while maintaining the membrane tension constant, produced a marked supralinear increase in specific carrier-mediated conductance. Analyses of the mechanisms involved in mechanoelectrical transduction induced by the imposition of a hydrostatic pressure gradient or by an amphipathic compound chlorpromazine reveal similar changes in the charge carrier motility and carrier reaction rates at the interface(s). Furthermore, the relative change in membrane conductance was independent of membrane diameter, but was directly proportional to the square of membrane curvature, thus relating the observed phenomena to the bilayer bending energy. Extrapolated to biological membranes, these findings indicate that ion transport in cells can be influenced simply by changing shape of the membrane, without a change in membrane tension.

Acidification and sodium entry in frog skin epithelium.

Acidification of the external medium by isolated frog skin epithelium (Rana catesbeiana, Rana temporaria, and Caudiververa caudiververa) and its relationship to Na+ uptake was studied. Acidification was measured by the pH-stat technique under short-circuit or open-circuit conditions. The results of this study demonstrate that (a) acidification by these species of in vitro frog skins is not directly coupled to Na+ or anion transport; (b) acidification can be inhibited by the diuretic drug amiloride, but only at high external Na+ concentrations; (c) acidification rate in these species of frog skin is controlled in part by the metabolic production of CO2; and (d) the positive correlation between net Na+ absorption and net acidification observed in whole animal studies could not be replicated in the in vitro skin preparation, even when the frogs were first chronically stressed by salt depletion, a physiological state comparable to that used in the in vivo experiments.

Cation movements across mouse red blood cells.

This paper describes some features of the Na and K transport systems in red cells obtained from B10.A mice. When mouse erythrocytes were incubated in a plasma-like control medium, the scillaren-sensitive Na efflux was 3.6 +/- 0.4 mmol/l red blood cells per h while the scillaren-sensitive K influx was 3.1 +/- 0.3, values not significantly different from ech other. Scillaren had no significant effect on either Na influx or K efflux. There was a large (approx. 3 mmol/l red blood cells per h) scillaren-sensitive, Na-Na exchange diffusion component present under K-free conditions. When K was present in the incubation medium, this exchange system was suppressed.

A non-selective cation channel in the apical membrane of cultured A6 kidney cells.

The patch-voltage clamp technique was used to investigate the characteristics of a non-selective cation channel (NSCC) identified in the apical membrane of cultured A6 toad kidney cells. The NSCC was present in cell-attached and inside-out membrane patches. The characteristics of this NSCC are as follows: (a) linear current-voltage relationship with a channel conductance of 21 +/- 2 pS; (b) a low selectivity between Na+ and K+ (1.5:1); (c) a high selectivity of Na+ to Cl- (greater than 45:1); (d) this channel has a single open state and two closed states; (e) the open-time constant and the second closed-time constant of this channel are voltage dependent; and (f) this NSCC is insensitive to amiloride (10(-7) M). We conclude that the NSCC resembles previously described non-selective cation channels. The NSCC of the apical membrane of A6 cells may aid in the movement of Na+ and K+ in response to varying ionic concentrations across the apical membrane.

Amiloride fluxes across erythrocyte membranes.

Amiloride is known to inhibit both the influx of Na+ and the activation of mitogenesis in many cultured cell lines. This paper describes experiments in which the permeability coefficient of amiloride was determined from measurements of tracer fluxes across human erythrocytes and resealed ghosts. From an analysis of these fluxes, a permeability coefficient of 10(-7) cm/s for the uncharged form of amiloride was deduced. Based upon this measured permeability value, we present calculations of intracellular accumulation times of amiloride in cells of differing surface-to-volume ratio.

Membrane actions of male contraceptive gossypol tautomers.

The role of different gossypol tautomers in the interaction of this molecule with membranes has been investigated using the isolated hemiacetal moiety of gossypol and the pH dependency of the keto-enol tautomeric equilibrium. Our results indicate that: the actions of the hemiacetal tautomer cannot explain the effects of gossypol on mitochondrial oxidative phosphorylation, lipid membrane interfacial potentials, and proton conductance of lipid bilayers; the enolate forms of gossypol are the species that bind to the membrane interface and decrease the electrostatic interfacial potential; and the uncharged (keto and/or enol) species in equilibrium with the enolate forms of gossypol give the molecule the ability to carry protons across biological membranes.

Sodium flux ratio through the amiloride-sensitive entry pathway in frog skin.

The sodium flux ratio of the amiloride-sensitive Na+ channel in the apical membrane of in vitro Rana catesbeiana skin has been evaluated at different sodium concentrations and membrane potentials in sulfate Ringer solution. Amiloride-sensitive unidirectional influxes and effluxes were determined as the difference between bidirectional 22Na and 24Na fluxes simultaneously measured in the absence and presence of 10(-4) M amiloride in the external bathing solution. Amiloride-sensitive Na+ effluxes were induced by incorporation of cation-selective ionophores (amphotericin B or nystatin) into the normally Na+-impermeable basolateral membrane. Apical membrane potentials (Va) were measured with intracellular microelectrodes. We conclude that since the flux ratio exponent, n', is very close to 1, sodium movement through this channel can be explained by a free-diffusion model in which ions move independently. This result, however, does not necessarily preclude the possibility that this transport channel may contain one or more ion binding sites.

Regulation by Na+ and Ca2+ of renal epithelial Na+ channels reconstituted into planar lipid bilayers.

Purified bovine renal epithelial Na+ channels when reconstituted into planar lipid bilayers displayed a specific orientation when the membrane was clamped to -40 mV (cis-side) during incorporation. The trans-facing portion of the channel was extracellular (i.e., amiloride-sensitive), whereas the cis-facing side was intracellular (i.e., protein kinase A-sensitive). Single channels had a main state unitary conductance of 40 pS and displayed two subconductive states each of 12-13 pS, or one of 12-13 pS and the second of 24-26 pS. Elevation of the [Na+] gradient from the trans-side increased single-channel open probability (Po) only when the cis-side was bathed with a solution containing low [Na+] (< 30 mM) and 10-100 microM [Ca2+]. Under these conditions, Po saturated with increasing [Na+]trans. Buffering of the cis compartment [Ca2+] to nearly zero (< 1 nM) with 10 mM EGTA increased the initial level of channel activity (Po = 0.12 +/- 0.02 vs 0.02 +/- 0.01 in control), but markedly reduced the influence of both cis- and trans-[Na+] on Po. Elevating [Ca2+]cis at constant [Na+] resulted in inhibition of channel activity with an apparent [KiCa2+] of 10-100 microM. Protein kinase C-induced phosphorylation shifted the dependence of channel Po on [Ca2+]cis to 1-3 microM at stationary [Na+]. The direct modulation of single-channel Po by Na+ and Ca2+ demonstrates that the gating of amiloride-sensitive Na2+ channels is indeed dependent upon the specific ionic environment surrounding the channels.

On the mechanism of the amiloride-sodium entry site interaction in anuran skin epithelia.

The steady-state transport kinetics of the interaction between external sodium and the diuretic drug, amiloride, was studied in isolated anuran skin epithelia. We also investigated the effect of calcium on the amiloride-induced inhibition of short-circuit current (Isc) in these epithelial preparations. The major conclusions of this study are: (a) amiloride is a noncompetitive inhibitor of Na entry in bullfrog and grassfrog skin, but displays mixed inhibition in R. temporaria and the toad. A hypothesis which states that the interaction sites for amiloride and Na on the putative entry protein are spatially distinct in all of these species is proposed. (b) The stoichiometry of interaction between amiloride and the Na entry mechanism is not necessarily one-to-one. (c) The external Ca requirement for the inhibitory effect of amiloride is not absolute. Amiloride, at all concentrations, is equally effective in inhibiting Isc of bullfrog skin independently from the presence or absence of external Ca.

Cationic selectivity and competition at the sodium entry site in frog skin.

The cation selectivity of the Na entry mechanism located in the outer membrane of the bullfrog (Rana catesbeiana) skin epithelium was studied. This selectivity was determined by measuring the short-circuit current when all of the external sodium was replaced by another cation and, also, by noting the relative degree of inhibition that the alkali metal cations produced on Na influx. The ability of the Group Ia cations to permeate the apical membrane was determined from the tracer uptake experiments. The results demonstrate that (a) only Li and Na are actively transported through the epithelium; (b) the alkali cations K, Rb, and Cs do not enter the epithelium through the apical border and, therefore, Na and Li are the only alkali cations translocated through this membrane; (c) these impermeable cations are competitive inhibitors of Na entry; (d) the cations NH4 and Tl exhibit more complex behavior but, under well-defined conditions, also inhibit Na entry; and (e) the selectivity of the cation binding site is in the sequence Li congruent to Na > Tl > NH4 congruent to K > Rb > Cs, which corresponds to a high field strength site with tetrahedral symmetry.

Effect of amiloride and some of its analogues of cation transport in isolated frog skin and thin lipid membranes.

The inhibition of short-circuit current (Isc) in isolated frog skin and the induction of surface potentials in lipid bilayer membranes produced by the diuretic drug amiloride and a number of its chemical analogues was studied. The major conclusions of our study are: (a) The charged form of amiloride is the biologically active species. (b) Both the magnitude of Isc and the amiloride inhibitory effect are sensitive to the ionic milieu bathing the isolated skin, and these two features are modulated at separate and distinct regions on the transport site. (c) Amiloride is very specific in its inhibitory interaction with the Na+ transport site since slight structural modifications can result in significant changes in drug effectiveness. We found that substitutions at pyrazine ring position 5 greatly diminish drug activity, while changes at position 6 are less drastic. Alterations in the guanidinium moiety only diminish activity if the result is a change in the spatial orientation of the amino group carrying the positive charge. (d) Amiloride can bind to and alter the charge on membrane surfaces, but this action cannot explain its highly specific effects in biological systems.

Protein kinase regulation of a cloned epithelial Na+ channel.

We examined the regulation of a cloned epithelial Na+ channel (alpha beta gamma-rENaC) by protein kinase A (PKA) and protein kinase C (PKC). Experiments were performed in Xenopus oocytes and in planar lipid bilayers. At a holding potential of -100 mV, amiloride-sensitive current averaged -1,279 +/- 111 nA (n = 7) in alpha beta gamma-rENaC-expressing oocytes. Currents in water-injected oocytes were essentially unresponsive to 10 microM amiloride. A 1-h stimulation of PKC with 100 nM of PMA inhibited whole-cell currents in Xenopus oocytes to 17.1 +/- 1.8, and 22.1 +/- 2.6% of control (n = 7), at holding potentials of -100 and +40 mV, respectively. Direct injection of purified PKC resulted in similar inhibition to that observed with PMA. Additionally, the inactive phorbol ester, phorbol-12-myristate-13-acetate, 4-O-methyl, was without effect on alpha beta gamma-rENaC currents. Pretreatment with the microtubule inhibitor colchicine (100 microM) did not modify the inhibitory effect of PMA; however, pretreatment with 20 microM cytochalasin B decreased the inhibitory action of PMA to < 20% of that previously observed. In vitro-synthesized alpha beta gamma-rENaC formed an amiloride-sensitive Na(+)-selective channel when incorporated into planar lipid bilayers. Addition of PKC, diacyl-glycerol, and Mg-ATP to the side opposite that which amiloride blocked, decreased the channel's open probability (Po) from 0.44 +/- 0.06 to 0.13 +/- 0.03 (n = 9). To study the effects of PKA on alpha beta gamma-rENaC expressed in Xenopus oocytes, cAMP levels were elevated with 10 microM forskolin and 1 mM isobutyl-methyl-xanthine. This cAMP-elevating cocktail did not cause any stimulation of alpha beta gamma-rENaC currents in either the inward or outward directions. This lack of activation was also observed in oocytes preinhibited with PMA and in oocytes pretreated with cytochalasin B and PMA. Neither alpha-rENaC nor alpha beta gamma-rENaC incorporated into planar lipid bilayers could be activated with PKA and Mg-ATP added to either side of the membrane, as Po remained at 0.63 +/- 0.06 (n = 7) and 0.45 +/- 0.05 (n = 9), respectively. We conclude that: alpha beta gamma-rENaC is inhibited by PKC, and that alpha beta gamma-rENaC is not activated by PKA.

Differential gene expression profiling in human brain tumors.

Gene expression profiling of three human temporal lobe brain tissue samples (normal) and four primary glioblastoma multiforme (GBM) tumors using oligonucleotide microarrays was done. Moreover, confirmation of altered expression was performed by whole cell patch clamp, immunohistochemical staining, and RT-PCR. Our results identified several ion and solute transport-related genes, such as N-methyl-d-aspartate (NMDA) receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-2 receptors, GABA(A) receptor subunits alpha3, beta1, beta2, and beta3, the glutamate transporter, the glutamate/aspartate transporter II, the potassium channel K(V)2.1, hK(V)beta3, and the sodium/proton exchanger 1 (NHE-1), that are all downregulated in the tumors compared with the normal tissues. In contrast, aquaporin-1, possibly aquaporins-3 and -5, and GLUT-3 message appeared upregulated in the tumors. Our results also confirmed previous work showing that osteopontin, nicotinamide N-methyltransferase, murine double minute 2 (MDM2), and epithelin (granulin) are upregulated in GBMs. We also demonstrate for the first time that the cytokine and p53 binding protein, macrophage migration inhibitory factor (MIF), appears upregulated in GBMs. These results indicate that the modulation of ion and solute transport genes and heretofore unsuspected cytokines (i.e., MIF) may have profound implications for brain tumor cell biology and thus may identify potential useful therapeutic targets in GBMs.

Putative immunolocalization of the mechanoelectrical transduction channels in mammalian cochlear hair cells.

Hair cells bear an apical bundle of stereocilia arranged in serried rows. Deflection of the bundle controls the opening and closing of mechanoelectrical transduction channels, thereby altering the conductance across the apical plasma membrane. Two locations for these channels have been proposed in the bundle, either near the bases of the stereocilia or towards their tips. One hypothesis that is consistent with the latter possibility suggests that fine extracellular filaments, which run between the tips of the shorter stereocilia and the sides of the taller stereocilia behind, operate the channels. Determining the precise position of the channels is essential to test this hypothesis. We have therefore attempted to localize them immunocytochemically. Because hair-cell transduction is amiloride sensitive, the channels may have an amiloride-binding site associated with them. We have therefore used a polyclonal antibody raised against another amiloride-sensitive ion channel to hunt for them. This antibody recognizes a 62-64 kDa band in immunoblots of cochlear tissue, and produces discrete labelling in the hair bundle. This is most concentrated just below the tips of the shorter stereocilia, coinciding with a region of specialization in the closely apposed membranes of the short and tall stereocilia but not with either end of the tip link.

Current topic: transport mechanisms in preimplantation mammalian embryos.

The main theme of this brief review article is that the study of membrane transport from a developmental point of view can be equated with the study of regulation. Although the blastocyst develops primarily under the control of the genetic information it inherits, it may also need maternal signals at critical stages. The study of blastocyst physiology is thus relevant to understanding the events leading up to implantation and to future research on the mechanisms of exchange between the mother and embryo during the initial stages of placentation. This information may be of considerable importance to understanding some of the causes of embryonic loss, which is particularly high around the time of implantation, and to understand some of the basic physiology of the embryo to fill gaps in our knowledge of in vitro fertilization processes. We feel that the mammalian blastocyst provides a unique model for the study of the development of vectorial transport systems in epithelia and, in the future, of patterns of genes that regulate this development.

Macromolecular transport in rabbit blastocysts: evidence for a specific uteroglobin transport system.

Though uterine proteins are found within the blastocoel of the rabbit blastocyst, the mechanisms involved in protein entry into the blastocoel have not been studied. To investigate potential avenues of protein entry into the blastocoel, we have monitored uptake of a fluorescent, fluid-phase marker (lucifer yellow) into the rabbit blastocyst trophectodermal cell. In addition, we have measured the transtrophectodermal permeabilities of several uterine proteins (uteroglobin (UTG), rabbit serum albumin, rabbit IgG), and radiolabeled fluid-phase markers (sucrose, polyethylene glycol, dextran) in the 6- and 7-day post-coitus rabbit blastocyst. Extrablastocoelic lucifer yellow (LY) was rapidly endocytosed by the trophectodermal cell and was subsequently trapped in a perinuclear compartment for at least 30 min after removal of the extracellular dye. The slow rate of LY turnover within the endocytic compartments implies that the rate of fluid-phase transcytosis was negligible. Permeability coefficients of the fluid-phase markers and proteins, with the exception of UTG, decreased with increasing molecular weight of each compound tested. These data are consistent with the conclusion that these compounds traverse the trophectoderm via a 'leak' pathway, as opposed to a transcytotic pathway. In contrast, permeability of 125I-UTG when compared to the other compounds, was 10-fold greater than would be predicted based on its molecular weight. Furthermore, the flux of radioiodinated UTG displayed saturation kinetics with a Km of 19.5 micrograms/ml and a Vmax of 132 ng.cm-2.h-1. Sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated that the radioiodinated protein recovered from the blastocoel was of a lower molecular weight than native UTG and was not immunoreactive with goat anti-UTG antibody. Our data are consistent with the idea that UTG is transported across the trophectoderm by a receptor-mediated system, and UTG is modified intracellularly during transport to a protein of a lower molecular weight.

Transcellular and paracellular pathways in lingual epithelia and their influence in taste transduction.

The lingual epithelium is innervated by special sensory (taste) and general sensory (trigeminal) nerves that transmit information about chemical stimuli introduced into the mouth to the higher brain centers. Understanding the cellular mechanisms involved in eliciting responses from these nerves requires a detailed understanding of the contributions of both the paracellular and transcellular pathways. In this paper we focus on the contribution of these 2 pathways to the responses of salts containing sodium and various organic anions in the presence and absence of amiloride. Electrophysiological recordings from trigeminal nerves, chorda tympani nerves, and isolated lingual epithelia were combined with morphological studies investigating the location (and permeability) of tight junctions, the localization of amiloride-inhibitable channels, and Na-K-ATPase in taste and epithelial cells. Based on these measurements, we conclude that diffusion across tight junctions can modulate chorda tympani and trigeminal responses to sodium-containing salts and rationalize the enhancement of taste responses to saccharides by NaCl.