RESUMO
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.
Assuntos
Canais de Cálcio/genética , Proteínas de Membrana/genética , Mutação , Rim Policístico Autossômico Dominante/genética , Animais , Anticorpos/farmacologia , Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Linhagem Celular , Membrana Celular/fisiologia , Feminino , Gadolínio/farmacologia , Humanos , Lantânio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/fisiologia , Placenta/fisiologia , Gravidez , Biossíntese de Proteínas , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Spodoptera , Canais de Cátion TRPP , Transfecção , Trofoblastos/fisiologiaRESUMO
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.
Assuntos
Vilosidades Coriônicas/fisiologia , Canais Iônicos/fisiologia , Lantânio/farmacologia , Metais Terras Raras/farmacologia , Placenta/fisiologia , Vilosidades Coriônicas/efeitos dos fármacos , Condutividade Elétrica , Eletrofisiologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Feminino , Humanos , Canais Iônicos/efeitos dos fármacos , Bicamadas Lipídicas , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , GravidezRESUMO
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.
Assuntos
Ativação do Canal Iônico/fisiologia , Animais , Echinococcus/química , Echinococcus/fisiologia , Cinética , Bicamadas Lipídicas/química , Canais de Potássio/análise , Canais de Potássio/fisiologiaRESUMO
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.
Assuntos
Canais de Cloreto/metabolismo , Trofoblastos/metabolismo , Vilosidades Coriônicas/metabolismo , Vilosidades Coriônicas/ultraestrutura , Humanos , Bicamadas Lipídicas , Técnicas de Patch-ClampRESUMO
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.
Assuntos
Echinococcus/metabolismo , Canais Iônicos/metabolismo , Bicamadas Lipídicas/metabolismo , Microssomos/metabolismo , Animais , Bário/farmacologia , Cálcio/metabolismo , Cátions , Fracionamento Celular , Condutividade Elétrica , Ativação do Canal Iônico , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/isolamento & purificação , Lítio/metabolismo , Potássio/metabolismo , Ovinos , Sódio/metabolismoRESUMO
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.
Assuntos
Echinococcus/fisiologia , Amilorida/farmacologia , Animais , Cloretos/farmacologia , Ácido Desoxicólico/farmacologia , Relação Dose-Resposta a Droga , Echinococcus/efeitos dos fármacos , Condutividade Elétrica , Eletrofisiologia , Potenciais da Membrana , Microeletrodos , Ouabaína/farmacologia , Potássio/farmacologia , Sódio/farmacologiaRESUMO
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.
Assuntos
Potenciais de Ação , Cloro/metabolismo , Pele/metabolismo , Animais , Epitélio/fisiologia , RanidaeRESUMO
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.