Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel.

Defects in polycystin-2, a ubiquitous transmembrane glycoprotein of unknown function, is a major cause of autosomal dominant polycystic kidney disease (ADPKD), whose manifestation entails the development of fluid-filled cysts in target organs. Here, we demonstrate that polycystin-2 is present in term human syncytiotrophoblast, where it behaves as a nonselective cation channel. Lipid bilayer reconstitution of polycystin-2-positive human syncytiotrophoblast apical membranes displayed a nonselective cation channel with multiple subconductance states, and a high perm-selectivity to Ca2+. This channel was inhibited by anti-polycystin-2 antibody, Ca2+, La3+, Gd3+, and the diuretic amiloride. Channel function by polycystin-2 was confirmed by patch-clamping experiments of polycystin-2 heterologously infected Sf9 insect cells. Further, purified insect cell-derived recombinant polycystin-2 and in vitro translated human polycystin-2 had similar ion channel activity. The polycystin-2 channel may be associated with fluid accumulation and/or ion transport regulation in target epithelia, including placenta. Dysregulation of this channel provides a mechanism for the onset and progression of ADPKD.

Single-channel characterization of a nonselective cation channel from human placental microvillus membranes. Large conductance, multiplicity of conductance states, and inhibition by lanthanides.

The rate-limiting step for the maternofetal exchange of low molecular-weight solutes in humans is constituted by transport across a single epithelial layer (syncytiotrophoblast) of the placenta. Other than the well-established presence of a large-conductance, multisubstate Cl- channel, the ionic channels occurring in this syncytial tissue are, for the most part, unknown. We have found that fusion of apical plasma membrane-enriched vesicle fractions with planar lipid bilayers leads, mainly (96% of 353 reconstitutions), to the reconstitution of nonselective cation channels. Here we describe the properties of this novel placental conductance at the single-channel level. The channel has a large (>200 pS) and variable conductance, is cation selective (P(Cl)/P(K) approximately or approximately equal 0.024), is reversibly inhibited (presumably blocked) by submillimolar La3+, has very unstable kinetics, and displays a large number (>10) of current sublevels with a "promiscuous" connectivity pattern. The occurrence of both "staircaselike" and "all-or-nothing" transitions between the minimum and maximum current levels was intriguing, particularly considering the large number of conductance levels spanned at a time during the concerted current steps. Single-channel data simulated according to a multistate linear reaction scheme, with rate constants that can vary spontaneously in time, reproduce many aspects of the recorded subconductance behavior. The channel's sensitivity to lanthanides is reminiscent of stretch-sensitive channels which, in turn, suggests a physiological role for this ion channel as a mechanotransducer during syncytiotrophoblast-volume regulation.

Electrodiffusional ATP movement through the cystic fibrosis transmembrane conductance regulator.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR), and of at least one other member of the ATP-binding cassette family of transport proteins, P-glycoprotein, is associated with the electrodiffusional movement of the nucleotide ATP. Evidence directly implicating CFTR expression with ATP channel activity, however, is still missing. Here it is reported that reconstitution into a lipid bilayer of highly purified CFTR of human epithelial origin enables the permeation of both Cl- and ATP. Similar to previously reported data for in vivo ATP current of CFTR-expressing cells, the reconstituted channels displayed competition between Cl- and ATP and had multiple conductance states in the presence of Cl- and ATP. Purified CFTR-mediated ATP currents were activated by protein kinase A and ATP (1 mM) from the "intracellular" side of the molecule and were inhibited by diphenylamine-2-carboxylate, glibenclamide, and anti-CFTR antibodies. The absence of CFTR-mediated electrodiffusional ATP movement may thus be a relevant component of the pleiotropic cystic fibrosis phenotype.

Interconverting gating modes of a nonselective cation channel from the tapeworm Echinococcus granulosus reconstituted on planar lipid bilayers.

A 107-pS (symmetrical 150 mM KCl), nonselective cation channel was reconstituted from a microsomal membrane fraction of the larval stage of the tapeworm Echinococcus granulosus. Most of the time, it displayed a high open probability (>>0.95) irrespective of either the applied voltage, Ca2+, Ba2+, or tetraethylammonium concentration. Nevertheless, in contrast with this "leaklike" behavior, less frequently this "all-the-time-open" channel reversibly entered two different kinetic modes. One of them was characterized by lower Po values and some voltage sensitivity (V(1/2) congruent with 129 mV, and an equilibrium constant for channel closing changing e-fold per 63-mV change) the kinetic analysis revealing that it resulted from the appearance of voltage-sensitivity in the mean closed times and a sixfold increase in the equilibrium constant for channel closing at 0 mV. The other mode was characterized by a very fast open-close activity leading to poorly resolved current levels and a Po around 0.6-0.7 which, occasionally and in a voltage-sensitive manner, entered a long-lived nonconducting state. However, the rare nature of these mode-shifting transitions precluded a more detailed analysis of their kinetics. The conductive properties of the channel were not affected by these switches. Model gating alone does not seem to ensure any physiological role of this channel and, instead, some other channel changes must occur if this phenomenon were to be of regulatory importance in vivo. Thus, mode-shifting might constitute an alternative target for channel activity modulation also in tapeworms.

Properties of two multisubstate Cl- channels from human syncytiotrophoblast reconstituted on planar lipid bilayers.

We describe the first successful reconstitution of placental ionic channels on planar lipid bilayers. An apical plasma membrane-enriched vesicle fraction from human syncytiotrophoblast at term was prepared by following isotonic agitation, differential centrifugation, and Mg2+-induced selective precipitation of nonapical membranes, and its purity was assessed by biochemical and morphological marker analysis. We have already reported that, unlike previous patch-clamp studies, nonselective cation channels were incorporated in most cases, a result consistent with the higher permeability for cations as compared with Cl- and with the low apical membrane potential difference at term revealed by fluorescent probe partition studies, and microelectrode techniques. In this paper, we report that Cl--selective channels were incorporated in 4% of successful reconstitutions (14 out of 353) and that their analysis revealed two types of activity. One of them was consistent with a voltage-dependent, 100-pS channel while the other was consistent with the lateral association of 47-pS conductive units, giving rise to multibarrelled, DIDS-sensitive channels of variable conductance (300 to 650 pS). The latter displayed a very complex behavior which included cooperative gating of conductive units, long-lived substates, voltage-dependent entry into an apparent inactivated state, and flickering activity. The role of the reported Cl- channels in transplacental ion transport and/or syncytium homeostasis remains to be determined.

Renal epithelial protein (Apx) is an actin cytoskeleton-regulated Na+ channel.

Apx, the amphibian protein associated with renal amiloride-sensitive Na+ channel activity and with properties consistent with the pore-forming 150-kDa subunit of an epithelial Na+ channel complex initially purified by Benos et al. (Benos, D. J., Saccomani, G., and Sariban-Sohraby, S.(1987) J. Biol. Chem. 262, 10613-10618), has previously failed to generate amiloride-sensitive Na+ currents (Staub, O., Verrey, F., Kleyman, T. R., Benos, D. J., Rossier, B. C., and Kraehenbuhl, J.-P.(1992) J. Cell Biol. 119, 1497-1506). Renal epithelial Na+ channel activity is tonically inhibited by endogenous actin filaments (Cantiello, H. F., Stow, J., Prat, A. G., and Ausiello, D. A.(1991) Am. J. Physiol. 261, C882-C888). Thus, Apx was expressed and its function examined in human melanoma cells with a defective actin-based cytoskeleton. Apx-transfection was associated with a 60-900% increase in amiloride-sensitive (Ki = 3 microM) Na+ currents. Single channel Na+ currents had a similar functional fingerprint to the vasopressin-sensitive, and actin-regulated epithelial Na+ channel of A6 cells, including a 6-7 pS single channel conductance and a perm-selectivity of Na+:K+ of 4:1. Na+ channel activity was either spontaneous, or induced by addition of actin or protein kinase A plus ATP to the bathing solution of excised inside-out patches. Therefore, Apx may be responsible for the ionic conductance involved in the vasopressin-activated Na+ reabsorption in the amphibian kidney.

Cellular ATP release by the cystic fibrosis transmembrane conductance regulator.

Recent studies from our laboratory indicate that members of the ATP-binding cassette (ABC) family of transporters, including P-glycoprotein and cystic fibrosis transmembrane conductance regulator (CFTR), are ATP-permeable channels. The physiological relevance of this novel transport mechanism is largely unknown. In the present study, intra- and extracellular ATP content, cellular ATP release, and O2 consumption before and after adenosine 3',5'-cyclic monophosphate (cAMP) stimulation were determined to assess the role of CFTR in the transport of ATP under physiological conditions. The functional expression of CFTR by the stable transfection of mouse mammary carcinoma cells, C1271, with human epithelial CFTR cDNA resulted in a stimulated metabolism, since both basal and cAMP-inducible O2 consumption were increased compared with mock-transfected cells. The stimulated (but not basal) O2 consumption was inhibited by diphenyl-2-carboxylic acid (DPC), a known inhibitor of CFTR. CFTR expression was also associated with the cAMP-activated and DPC-inhibitable release of intracellular ATP. The recovery of intracellular ATP from complete depletion after metabolic poisoning was also assessed under basal and cAMP-stimulated conditions. The various maneuvers indicate that CFTR may be an important contributor to the release of cellular ATP, which may help modify signal transduction pathways associated with secretory Cl- movement or other related processes. Changes in the CFTR-mediated delivery of nucleotides to the extracellular compartment may play an important role in the onset and reversal of the cystic fibrosis phenotype.

Echinococcus granulosus: partial characterization of the conductive properties of two cation channels from protoscoleces of the ovine strain, reconstituted on planar lipid bilayers.

Two cationic channels present in the microsomal fraction from Echinococcus granulosus protoscoleces of the sheep strain were studied in planar bilayer reconstitution experiments. A whole-worm homogenate was subjected to differential centrifugation and the postmitochondrial supernatant was laid on the top of a discontinuous sucrose gradient. The 15-30% (w/v) membrane fraction, enriched in NADPH-cytochrome c reductase, exhibited the highest fusion rate, two cationic channels being most frequently reconstituted. Both of them were highly cation-selective and had high conductances (244 and 107 pS in symmetrical 150 mM KCl). In most experiments, none of them displayed voltage dependence. The 244-pS channel was activated by Ca2+ and blocked by Ba2+, both in the micromolar range, thus partially resembling the Ca(2+)-activated K+ channel from more highly evolved animals. The 107-pS channel exhibited a Cs+ approximately equal to K+ > Na+ > Li+ > Ca2+ selectivity sequence (as measured by permeability ratios) and, most frequently, a high open probability (> 0.9) irrespective of the experimental conditions used, therefore sharing many properties of Schistosoma mansoni outer tegumental membrane cation channels.

The cystic fibrosis transmembrane conductance regulator is a dual ATP and chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to a superfamily of proteins implicated in the transport of ions, proteins, and hydrophobic substances. Recent studies have demonstrated that CFTR is a protein kinase A-sensitive anion channel regulated by ATP. In the present study, patch-clamp techniques were used to assess the role of CFTR in the transport of Cl- and ATP. The stable transfection of mouse mammary carcinoma cells, C127i, with the cDNA for human CFTR resulted in the appearance of a diphenylamine-2-carboxylate-inhibitable Cl- channel, which was activated by cAMP under whole-cell and cell-attached conditions and by protein kinase A plus ATP under excised, inside-out conditions. CFTR expression was also associated with the electrodiffusional movement of ATP as indicated by the cAMP activation of ATP currents measured under whole-cell conditions. In excised, inside-out patches, it was demonstrated that ATP currents were mediated by ATP-conductive channels, which were also activated by protein kinase A and blocked by the Cl- channel blocker diphenylamine-2-carboxylate under excised, inside-out conditions. Single-channel currents observed in the presence of asymmetrical Cl-/ATP concentrations indicated that the same conductive pathway was responsible for both ATP and Cl- movement. Thus, CFTR is a multifunctional protein with more than one anion transport capability and may modify signal transduction pathways for Cl- or other secretory processes by the selective delivery of nucleotides to the extracellular domain.

Echinococcus granulosus: characterization of the electrical potential of the syncytial tegument of protoscoleces incubated in vitro--effect of inhibitors.

Upon microelectrode impalements of the tegument of Echinococcus granulosus protoscoleces incubated in Ringer Krebs solution, electrical potential differences of -49 +/- 1 mV, using procedure I, and -53 +/- 1 mV, using procedure II, were recorded. The changes in the electrical potential difference as well as the structural alterations produced by sodium deoxicholate indicate that the observed potentials are established across the apical membrane of the syncytial tegument. The electrical potential difference is primarily dependent on the K+ gradient across the tegumental membrane: a 10-fold increase in the external K+ causes a 30-mV change in the electrical potential. External Na+ concentration changes also affected the electrical potential difference, but altering the external Cl- has no detectable effect. Amiloride, a very well-known blocker of Na+ epithelia channels, produced a reversible hyperpolarization that reached the maximum response at 10(-3) M. Ouabain, 10(-4) M, caused a depolarization in both fresh and Na-rich protoscoleces, although this depolarization was greater and showed a faster onset under the latter condition. It is concluded that the electrical potential difference of E. granulosus protoscolex is generated at the apical tegumental membrane and that it can be experimentally divided into two main components: One of them depends on ionic gradients and membrane permeabilities in accordance with the electrodiffusion predicted by the Goldman, Hodgkin, and Katz equation, while the other depends on the electrogenicity of an active Na+/K+ transport system.

External ATP and its analogs activate the cystic fibrosis transmembrane conductance regulator by a cyclic AMP-independent mechanism.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel activated by protein kinase A and regulated by ATP in a complex manner. We have applied patch-clamp techniques to C127i mouse mammary carcinoma cells transfected with human CFTR to assess the role of external ATP in the modulation of CFTR function. Extracellular ATP was sufficient to activate non-rectifying, Cl(-)-selective whole-cell currents in CFTR-transfected, but not mock-transfected cells. The ATP-mediated activation of CFTR was independent of protein kinase A since channel activation by ATP was preserved in cells that were (a) depleted of intracellular ATP, (b) incubated with the cAMP antagonist Rp-cAMPS, or (c) exposed to the protein kinase A inhibitor, 5-24 amide. In each of these conditions, 8-Br-cAMP was no longer capable of activating CFTR. The possibility that the extracellular ATP activation of Cl- currents in CFTR-expressing C127i cells was mediated by a P2-type purinergic receptor was supported by studies in which the effect of external ATP on the Cl- currents was mimicked by the ATP analogs, ATP gamma S and beta,gamma-methylene ATP, but not the uridine nucleotide, UTP. Single-channel analysis of ATP-activated Cl -currents under both cell-attached and excised, inside-out patch-clamp configurations indicated that this channel is only present in CFTR-transfected cells and indistinguishable from CFTR. External ATP also activated ATP currents in CFTR-transfected cells, a novel function of CFTR. These findings are consistent with the presence of a purinergic receptor signal transduction mechanism in C127i cells whose activation by external ATP is linked to the activation of CFTR in a cAMP-independent manner. The data provide additional support for the use of ATP and its analogs as alternative therapies in cystic fibrosis.

Membrane permeability of secondary hydatid cysts of Echinococcus granulosus. Determination of the water diffusional and osmotic permeability coefficients through a syncytial membrane.

Diffusional (Pw) and osmotic (Pf) water permeability coefficients were determined for the syncytial epithelium of larval Echinococcus granulosus. Pw was calculated from simultaneous influx measurements of tritiated water and n-[14C]butanol through the hydatid cyst wall. The total diffusional water permeability coefficient, P'w, was found to be 2.2 X 10(-4) cm s-1; which is similar to that previously reported by Rotunno et al. (1974, J. Parasitol. 60, 13-620). Nevertheless, when P'f is corrected for the unstirred water layer effects, a Pw value of 6.2 X 10(-4) cm s-1 is obtained. Thus, the unstirred water layer effects have a very important contribution to P'w. Total steady state osmotic permeability coefficient, P'f, was bound to be about 15 X 10(-4) cm s-1 and it is scarcely affected by those mechanisms that tend to distort the evaluation of Pf. The experimentally determined osmotic coefficient differs from the corrected Pf by only 6%. The Pf/Pw ratio was found to be 2.4. The present study clearly confirms that syncytial membranes can be highly permeable to water, in spite of the fact that they lack tight junctions. Thus, water permeability through epithelial syncytium must be exclusively controlled by the permeability of the apical and/or basocellular membranes.

[Localization of chlorine barrier by transepithelial transient electric potentials in rana skin]. / Localización de la barrera al cloruro mediante potenciales eléctricos transepiteliales transitorios en la piel de rana.

The localization of the outermost barrier to chloride influx in the abdominal skin of Leptodactylus ocellatus was investigated by a technique developed by Kidder et al. The method analyses the transient changes in transepithelial electrical potential differences produced when an impermeable anion (SO4(2) or gluconate) is rapidly replaced by Cl in the external bathing solution. The experimental results indicate that the Cl barrier is at the same level as the external Na barrier, that is, at the outward facing membrane of the cells of the stratum granulosum. Further experiments demonstrate that Br behaves like Cl, whereas I seems to behave as an impermeable anion, and that Na is needed for activating the anion permeation mechanism at the external barrier of the epithelium.

Localización de la barrera al cloruro mediante potenciales eléctricos transepiteliales transitorios en la piel de rana. / [Localization of chlorine barrier by transepithelial transient electric potentials in rana skin]

The localization of the outermost barrier to chloride influx in the abdominal skin of Leptodactylus ocellatus was investigated by a technique developed by Kidder et al. The method analyses the transient changes in transepithelial electrical potential differences produced when an impermeable anion (SO4(2) or gluconate) is rapidly replaced by Cl in the external bathing solution. The experimental results indicate that the Cl barrier is at the same level as the external Na barrier, that is, at the outward facing membrane of the cells of the stratum granulosum. Further experiments demonstrate that Br behaves like Cl, whereas I seems to behave as an impermeable anion, and that Na is needed for activating the anion permeation mechanism at the external barrier of the epithelium.