Competing Lipid-Protein and Protein-Protein Interactions Determine Clustering and Gating Patterns in the Potassium Channel from Streptomyces lividans (KcsA).

There is increasing evidence to support the notion that membrane proteins, instead of being isolated components floating in a fluid lipid environment, can be assembled into supramolecular complexes that take part in a variety of cooperative cellular functions. The interplay between lipid-protein and protein-protein interactions is expected to be a determinant factor in the assembly and dynamics of such membrane complexes. Here we report on a role of anionic phospholipids in determining the extent of clustering of KcsA, a model potassium channel. Assembly/disassembly of channel clusters occurs, at least partly, as a consequence of competing lipid-protein and protein-protein interactions at nonannular lipid binding sites on the channel surface and brings about profound changes in the gating properties of the channel. Our results suggest that these latter effects of anionic lipids are mediated via the Trp(67)-Glu(71)-Asp(80) inactivation triad within the channel structure and its bearing on the selectivity filter.

Na⁺, K⁺-ATPase and Ca²âº-ATPase activities in basal and microvillous syncytiotrophoblast membranes from preeclamptic human term placenta.

OBJECTIVE: The aim of this study is to evaluate the effect of preeclampsia on the level of lipid peroxidation, activity and expression of both plasma membrane Ca(2+)- and Na(+), K(+)-ATPases in syncytiotrophoblast. METHODS: The level of lipid peroxidation was estimated by measuring TBARS. ATPase activities were quantified by a colorimetric method measuring the amount of inorganic phosphate during the assay. Expression of Ca(2+)- and Na(+), K(+)-ATPases in syncytiotrophoblast plasma membranes and term placenta tissue sections was investigated using Western blot and immunohistochemistry, respectively. RESULTS: Our results show a higher level of lipid peroxidation of syncytiotrophoblast plasma membranes from preeclamptic, as compared to uncomplicated pregnant women. Preeclampsia also significantly reduced the activity of Ca(2+)- and Na(+), K(+)-ATPases; however, expression of both ATPases was unaffected. CONCLUSION: Our findings suggest that the reduction of Ca(2+)- and Na(+), K(+)-ATPase activities during preeclampsia could be at least partially due to an increased level of lipid peroxidation of the syncytiotrophoblast plasma membranes.

Differential expression of potassium channels in placentas from normal and pathological pregnancies: targeting of the K(ir) 2.1 channel to lipid rafts.

Potassium channels play important physiological roles in human syncytiotrophoblasts (hSTBs) from placenta, an epithelium responsible for maternal-fetal exchange. Basal and apical plasma membranes differ in their lipid and protein composition, and the latter contains cholesterol-enriched microdomains. In placental tissue, the specific localization of potassium channels is unknown. Previously, we described two isolated subdomains from the apical membrane (MVM and LMVM) and their respective microdomains (lipid rafts). Here, we report on the distribution of K(ir)2.1, K(v)2.1, TASK-1, and TREK-1 in hSTB membranes and the lipid rafts that segregate them. Immunoblotting experiments showed that these channels are present mainly in the apical membrane from healthy hSTBs. Apical expression versus basal membrane was 84 and 16% for K(ir)2.1 and K(v)2.1, 60 and 30% for TREK-1, and 74 and 26% for TASK-1. Interestingly, K(v)2.1 showed differences between apical membrane subdomains: 26 ± 8% was located in the LMVM and 59 ± 9% in MVM. In pathological placentas, the expression distribution changed in the basal membrane: preeclampsia shifted to 50% and intrauterine growth restriction to 42% for TASK-1 and both pathologies increased to 25% for K(ir)2.1 and K(v)2.1, K(ir)2.1 appeared to be associated with rafts that were sensitive to cholesterol depletion in healthy, but not in pathological, placentas. K(v)2.1 and TREK-1 emerged in the nonraft fractions. The precise membrane localization of ion channels in hSTB membranes is necessary to understand the physiological events.

Expression and distribution of nucleoside transporter proteins in the human syncytiotrophoblast.

The plasma membrane distribution and related biological activity of nucleoside transporter proteins (NTs) were investigated in human syncytiotrophoblast from term placenta using a variety of approaches, including nucleoside uptake measurements into vesicles from selected plasma membrane domains, NT immunohistochemistry, and subcellular localization (basal, heavy, and light apical membranes as well as raft-enriched membranes from the apical domain). In contrast with other epithelia, in this epithelium, we have identified the high-affinity pyrimidine-preferring human concentrative nucleoside transporter (hCNT) 1 as the only hCNT-type protein expressed at both the basal and apical membranes. hCNT1 localization in lipid rafts is also dependent on its subcellular localization in the apical plasma membrane, suggesting a complex cellular and regional expression. Overall, this result favors the view that the placenta is a pyrimidine-preferring nucleoside sink from both maternal and fetal sides, and hCNT1 plays a major role in promoting pyrimidine salvage and placental growth. This finding may be of pharmacological relevance, because hCNT1 is known to interact with anticancer nucleoside-derived drugs and other molecules, such as nicotine and caffeine, for which a great variety of harmful effects on placental and fetal development, including intrauterine growth retardation, have been reported.

Lipid rafts and cytoskeletal proteins in placental microvilli membranes from preeclamptic and IUGR pregnancies.

Intrauterine growth restriction (IUGR) and preeclampsia (PE) are leading causes of perinatal and maternal morbidity and mortality. Previously we reported the expression of lipid rafts in classical microvillous membrane (MVM) and light microvillous membrane (LMVM), two subdomains in apical membrane from the human placental syncytiotrophoblast (hSTB), which constitute the epithelium responsible for maternal-fetal transport. Here the aim was to study the raft and cytoskeletal proteins from PE and IUGR. Microdomains from MVM and LMVM were tested with raft markers (placental alkaline phosphatase, lipid ganglioside, and annexin 2) and a nonraft marker (hTf-R). No changes were detected with those markers in whole purified apical membranes in normal, PE, and IUGR pregnancies; however, their patterns of distribution in lipid rafts were different in PE and IUGR. Cholesterol depletion modified their segregation, confirming their presence in lipid rafts, although unlike normal placenta, in these pathologies there is only one type of microdomain. Additionally, the cytoskeleton proteins actin, ezrin, and cytokeratin-7 showed clear differences between normal and pathological membranes. Cytokeratin-7 expression decreased to 50% in PE, and the distribution between LMVM and MVM (~43 and 57%, respectively) changed in both PE and IUGR, in contrast with the asymmetrical enrichment obtained in normal LMVM (~62%). In conclusion, lipid rafts from IUGR and PE have different features compared to rafts from normal placentae, and this is associated with alterations in the expression and distribution of cytoskeletal proteins.

Electrophysiological characterization of potassium conductive pathways in Trypanosoma cruzi.

Potassium channels (K(+) channels) are members of one of the largest and most diverse families of membrane proteins, widely described from bacteria to humans. Their functions include voltage-membrane potential maintenance, pH and cell volume regulation, excitability, organogenesis and cell death. K(+) channels are involved in sensing and responding to environmental changes such as acidification, O(2) pressure, osmolarity, and ionic concentration. Trypanosoma cruzi is a parasitic protozoan, causative agent of Chagas disease (American trypanosomiasis) an endemic pathology in Latin America, where up 200,000 new cases are reported annually. In protozoan parasites, the presence of K(+) channels has been suggested, but functional direct evidence supporting this hypothesis is limited, mainly due to the difficulty of employing conventional electrophysiological methods to intact parasites. In T. cruzi, K(+) conductive pathways are thought to contribute in the regulatory volume decrease observed under hypoosmotic stress, the steady state pH and the compensatory response to extracellular acidification and the maintenance of plasma membrane potential. In this work we describe the isolation of plasma membrane enriched fractions from T. cruzi epimastigotes, their reconstitution into giant liposomes and the first functional characterization by patch-clamp of K(+) conductive pathways in protozoan parasites.

Endothelial epithelial sodium channel inhibition activates endothelial nitric oxide synthase via phosphoinositide 3-kinase/Akt in small-diameter mesenteric arteries.

Recent studies have shown that the epithelial sodium channel (ENaC) is expressed in vascular tissue. However, the role that ENaC may play in the responses to vasoconstrictors and NO production has yet to be addressed. In this study, the contractile responses of perfused pressurized small-diameter rat mesenteric arteries to phenylephrine and serotonin were reduced by ENaC blockade with amiloride (75.1+/-3.2% and 16.9+/-2.3% of control values, respectively; P<0.01) that was dose dependent (EC(50)=88.9+/-1.6 nmol/L). Incubation with benzamil, another ENaC blocker, had similar effects. alpha, beta, and gamma ENaC were identified in small-diameter rat mesenteric arteries using RT-PCR and Western blot with specific antibodies. In situ hybridization and immunohistochemistry localized ENaC expression to the tunica media and endothelium of small-diameter rat mesenteric arteries. Patch-clamp experiments demonstrated that primary cultures of mesenteric artery endothelial cells expressed amiloride-sensitive sodium currents. Mechanical ablation of the endothelium or inhibition of eNOS with N(omega)-nitro-L-arginine inhibited the reduction in contractility caused by ENaC blockers. ENaC inhibitors increased eNOS phosphorylation (Ser 1177) and Akt phosphorylation (Ser 473). The presence of the phosphoinositide 3-kinase inhibitor LY294002 blunted Akt phosphorylation and eNOS phosphorylation and the decrease in the response to phenylephrine caused by blockers of ENaC, indicating that the phosphoinositide 3-kinase/Akt pathway was activated after ENaC inhibition. Finally, we observed that the effects of blockers of ENaC were flow dependent and that the vasodilatory response to shear stress was enhanced by ENaC blockade. Our results identify a previously unappreciated role for ENaC as a negative modulator of eNOS and NO production in resistance arteries.

Distinct lipid rafts in subdomains from human placental apical syncytiotrophoblast membranes.

We report on the characteristics of raft domains in the apical membrane from human placental syncytiotrophoblast (hSTB), an epithelium responsible for maternal-fetal exchange. Previously, we described two isolated fractions of the hSTB apical membrane: a classical microvillous membrane (MVM) and a light microvillous membrane (LMVM). Detergent-resistant microdomains (DRMs) from MVM and LMVM were prepared with Triton X-100 followed by flotation in a sucrose gradient and tested by Western and dot blot with raft markers (placental alkaline phosphatase, lipid ganglioside, annexin 2) and transferrin receptor as a nonraft marker. DRMs from both fractions showed a consistent peak for these markers, except that the DRMs from MVM had no annexin 2 mark. Cholesterol depletion modified the segregation in both groups of DRMs. Our results show two distinguishable lipid raft subsets from MVM and LMVM. Additionally, we found significant differences between MVM and LMVM in cholesterol content and in expression of cytoskeletal proteins. MVM is enriched in ezrin and beta-actin; in contrast, cholesterol and cytokeratin-7 are more abundant in LMVM. These differences may explain the distinct properties of the lipid raft subtypes.

Equidad en educación superior: experiencia de un programa especial de ingreso a carreras de la salud / Equity in higher education: experience from special program for admission to health careers

Propósito: Describir los resultados de los primeros 5 años de un programa de ingreso diferenciado de estudiantes destacados de nivel socioeconómico bajo a carreras de la salud de la Facultad de Medicina de la Universidad de Chile. Material y Método: Se incorporaron anualmente 10 a 20 alumnos de tercero y cuarto Medio de escasos recursos por sus antecedentes académicos y psicosociales. Durante uno o dos años recibieron refuerzo en ramos científicos y habilidades para la vida, tutoría y orientación vocacional. Rindieron también la PAA/PSU. De los preseleccionados, se eligieron hasta 5 por año para ingresar a la Facultad de acuerdo a sus antecedentes académicos, vocacionales y condiciones psicosociales de adaptación. Cursaron las carreras con beca completa y otros apoyos económicos. A aquellos que no ingresaron se les facilitaron otras instancias de educación superior. Resultados: A la fecha, han participado 57 estudiantes, 43,9% mujeres y 56,1% hombres. Su promedio de notas de enseñanza media fue x igual 6,1. 43,5% no tenía expectativas previas de ingresar a la universidad. Durante el apoyo inicial 41,1% evidenció déficit de hábitos y técnicas de estudio, 67,3% desorientación vocacional, 33,3% psicopatología y 38,6% conductas de riesgo. 15,8% desertó y 17,5% fue excluido. La deserción se debió principalmente a problemas vocacionales y stress, y la exclusión a mal rendimiento y psicopatología. 47,4% de los que finalizaron la etapa previa fue seleccionado para cursar carreras en la Facultad. Sus puntajes ponderados estuvieron bajo los mínimos para el ingreso regular. Su desempeño posterior fue en general satisfactorio. Conclusiones: El programa generó oportunidades de acceso a la universidad fuera de las expectativas de este grupo de jóvenes. Su realidad psicosocial dificultó su desempeño. El apoyo en esta área es muy importante en este tipo de programas...

Purpose: To describe the results from the first 5 years of a differentiated admission program designed for outstanding students of low socioeconomic income admitted to health careers of the Faculty of Medicine, University of Chile. Materials and Methods: Annually, 10 to 20 low-income- students from 3rd and 4th years of secondary school entered the program in virtue of their academic and psychosocial background. For one or two years, they received extra support in scientific subjects and abilities for life, tutoring and vocational guidance. They also took the PAA/PUU (general post-secondary school test). Of the students who succeeded the pre-selection, up to 5 per year were chosen for admission to University according to their academic and vocational background and their psychosocial adaptation conditions. They studied their careers under full bursary coverage and other financial supports. Those who failed admission were helped to access other instances of higher education. Results: To date, 57 students have participated in the program. Of these, 43,9% are females and 56,1% are men. Their average secondary school grade was x = 6,1. In total, 43,5% had no prior expectations on being admitted to university. During the initial support delivery, 41,1% evidenced a lack of study habits and techniques, 33,3%showed a psychopathological condition and 38,6% evidenced risk behaviors. In 15,8% of the cases students dropped out and in 17,5% there was student exclusion. Drop out was due mainly to vocational problems and stress, and exclusion was due to low performance and psychopathological conditions. Of the students who completed the previous stage, 47,4% was selected to study careers at the Faculty. Their test scores were below the minimum for regular admission. In general, their subsequent performance was satisfactory. Conclusions: The program generated university access opportunities that...

Apical Maxi-chloride channel from human placenta: 12 years after the first electrophysiological recordings.

The Maxi-chloride channel was the first ion channel described by electrophysiological methods in placenta. Because it is difficult to access a complex epithelium such as the placenta for electrophysiological procedures, the studies of ion channels from placental membranes have been performed only very recently. It was only in 1993 that a direct demonstration of a high-conductance chloride channel in apical membranes of intact trophoblastic epithelium was mentioned, and two years later, the description of this channel was reported from purified placental apical membranes reconstituted into artificial lipid membranes suitable for patch-clamp recordings. This brief review comments on the work done with regard to the electrophysiological characterization and regulation of the large-conductance or "Maxi" chloride channel and its contribution to the development of a cellular model for syncytiotrophoblast ion transport.

Clustering and coupled gating modulate the activity in KcsA, a potassium channel model.

Different patterns of channel activity have been detected by patch clamping excised membrane patches from reconstituted giant liposomes containing purified KcsA, a potassium channel from prokaryotes. The more frequent pattern has a characteristic low channel opening probability and exhibits many other features reported for KcsA reconstituted into planar lipid bilayers, including a moderate voltage dependence, blockade by Na(+), and a strict dependence on acidic pH for channel opening. The predominant gating event in this low channel opening probability pattern corresponds to the positive coupling of two KcsA channels. However, other activity patterns have been detected as well, which are characterized by a high channel opening probability (HOP patterns), positive coupling of mostly five concerted channels, and profound changes in other KcsA features, including a different voltage dependence, channel opening at neutral pH, and lack of Na(+) blockade. The above functional diversity occurs correlatively to the heterogeneous supramolecular assembly of KcsA into clusters. Clustering of KcsA depends on protein concentration and occurs both in detergent solution and more markedly in reconstituted membranes, including giant liposomes, where some of the clusters are large enough (up to micrometer size) to be observed by confocal microscopy. As in the allosteric conformational spread responses observed in receptor clustering (Bray, D. and Duke, T. (2004) Annu. Rev. Biophys. Biomol. Struct. 33, 53-73) our tenet is that physical clustering of KcsA channels is behind the observed multiple coupled gating and diverse functional responses.

Puroindoline-a and alpha1-purothionin form ion channels in giant liposomes but exert different toxic actions on murine cells.

Puroindoline-a (PIN-a) and alpha1-purothionin (alpha1-PTH), isolated from wheat endosperm of Triticum aestivum sp., have been suggested to play a role in plant defence mechanisms against phytopathogenic organisms. We investigated their ability to form pores when incorporated into giant liposomes using the patch-clamp technique. PIN-a formed cationic channels (approximately 15 pS) with the following selectivity K(+) > Na(+) >> Cl(-). Also, alpha1-PTH formed channels of approximately 46 pS and 125 pS at +100 mV, the selectivity of which was Ca(2+) > Na(+) approximately K(+) >> Cl(-) and Cl(-) >> Na(+), respectively. In isolated mouse neuromuscular preparations, alpha1-PTH induced muscle membrane depolarization, leading to blockade of synaptic transmission and directly elicited muscle twitches. Also, alpha1-PTH caused swelling of differentiated neuroblastoma NG108-15 cells, membrane bleb formation, and disorganization of F-actin. In contrast, similar concentrations of PIN-a had no detectable effects. The cytotoxic actions of alpha1-PTH on mammalian cells may be explained by its ability to induce cationic-selective channels.

Apical Maxi-chloride channel from human placenta: 12 years after the first electrophysiological recordings

The Maxi-chloride channel was the first ion channel described by electrophysiological methods in placenta. Because it is difficult to access a complex epithelium such as the placenta for electrophysiological procedures, the studies of ion channels from placental membranes have been performed only very recently. It was only in 1993 that a direct demonstration of a high-conductance chloride channel in apical membranes of intact trophoblastic epithelium was mentioned, and two years later, the description of this channel was reported from purified placental apical membranes reconstituted into artificial lipid membranes suitable for patch-clamp recordings. This brief review comments on the work done with regard to the electrophysiological characterization and regulation of the large-conductance or "Maxi" chloride channel and its contribution to the development of a cellular model for syncytiotrophoblast ion transport.

Proteomic analysis of apical microvillous membranes of syncytiotrophoblast cells reveals a high degree of similarity with lipid rafts.

Brush borders (microvilli) are cell membrane specialized structures that function mainly as high-throughput absortive/secretory areas. It has been well-established that brush borders are particularly rich in membrane lipids characteristic to lipid rafts. Here, we report 57 proteins identified from microvillous membranes (MVM) isolated from human syncytiotrophoblast cells using an experimental method that avoids the use of nonionic detergents. About 60% of the proteins reported here have been described previously as lipid-raft specific. Well-known lipid raft-markers such as Annexin A2 and alkaline phosphatase were identified. Cytoskeleton structural constituents and proteins related with the control and modulation of the cytoskeletal architecture as well as the regulation of the interaction of cytoskeletal constituents with the cell membrane and particularly with lipid raft domains were found (Ezrin, IQGAP1 and 2, EBP50). Other proteins identified include signal transduction molecules, such as Ras-related protein Rab-1B and Rab-7, and ADP-ribosylation factor 1. Several proteins harbor putative post-translational modifications that favor its localization in the lipid-raft environment, such as GPI (alkaline phosphatase and 5'-nucleotidase) and myristoylation (BASP1 and MARCKS). On the whole, this extensive description demonstrates from the protein composition point of view that brush border membranes are indeed highly enriched in lipid raft microdomains.

Annexin 6 modulates the maxi-chloride channel of the apical membrane of syncytiotrophoblast isolated from human placenta.

The syncytiotrophoblast separates the maternal and fetal blood and constitutes the primary barrier for maternal-fetal transport. The Maxi-chloride channel from the apical membrane of the syncytiotrophoblast plays a role in the chloride conductance. Annexins can play an important role in the regulation of membrane events. In this study we evaluate the role of annexin 6 in the Maxichloride channel properties. The results showed that annexin 6 is bound in the apical placenta membranes in a calcium-dependent phospholipid-binding manner but also in a calcium-independent fashion. The neutralization of annexin 6 decreased the total current by 39 +/- 1.9% in the range of +/-80 mV, and the currents decrease with the time. The single-channel slope conductance was decreased from 253 +/- 7.4 pS (control) to 105 +/- 13 pS, and the amplitude decreased by 50%. The open probability was also affected when higher voltage steps were used, changes in either the positive or negative direction induced the channel to close, and the open probability (P(o)) did not decrease. In channels with neutralized annexin 6, it was maintained at 1 at +/-40 mV and at +/-80 mV. These results suggest that endogenous annexin 6 could regulate the Maxi-chloride channel. The results obtained with normal placentae, in which annexin 6 was neutralized, are similar to those described for the Maxi-chloride channel isolated from pre-eclamptic placenta. Together these data suggest that annexin 6 could play an important role in ion transport of the placenta.

Neuronal and muscular alterations caused by two wheat endosperm proteins, puroindoline-a and alpha1-purothionin, are due to ion pore formation.

Using the patch-clamp technique it was found that the toxicity of the two wheat endosperm proteins puroindoline-a and alpha1-purothionin probably results from the dissipation of ion concentration gradients essential for the maintenance of cellular homeostasis.