Biomedical subjects

H Oberleithner

Publications and source records attributed to H Oberleithner.

At least 55 records · Page 3Linked to original sources

PubMed1997

The atomic force microscope detects ATP-sensitive protein clusters in the plasma membrane of transformed MDCK cells.

Plasma membrane proteins are supposed to form clusters that allow 'functional cross-talk' between individual molecules within nanometre distance. However, such hypothetical protein clusters have not yet been shown directly in native plasma membranes. Therefore, we developed a technique to get access to the inner face of the plasma membrane of cultured transformed kidney (MDCK) cells. The authors applied atomic force microscopy (AFM) to visualize clusters of native proteins protruding from the cytoplasmic membrane surface. We used the K+ channel blocker iberiotoxin (IBTX), a positively charged toxin molecule, that binds with high affinity to plasma membrane potassium channels and to atomically flat mica. Thus, apical plasma membranes could be 'glued' with IBTX to the mica surface with the cytosolic side of the membrane accessible to the scanning AFM tip. The topography of these native inside-out membrane patches was imaged with AFM in electrolyte solution mimicking the cytosol. The plasma membrane could be clearly identified as a lipid bilayer with the characteristic height of 4.9 +/- 0.02 nm. Multiple proteins protruded from the lipid bilayer into the cytosolic space with molecule heights between 1 and 20 nm. Large protrusions were most likely protein clusters. Addition of the proteolytic enzyme pronase to the bath solution led to the disappearance of the proteins within minutes. The metabolic substrate ATP induced a shape-change of the protein clusters and smaller subunits became visible. ADP or the non-hydrolysable ATP analogue, ATP-gamma-S, could not exert similar effects. It is concluded that plasma membrane proteins (and/or membrane associated proteins) form 'functional clusters' in their native environment. The 'physiological' arrangement of the protein molecules within a cluster requires ATP.

Adenosine Triphosphate↗

PubMed1997

Using atomic force microscopy to investigate patch-clamped nuclear membrane.

Nuclear patch clamp is an emerging research field that aims to disclose the electrical phenomena underlying macromolecular transport across the nuclear envelope (NE), its properties as an ion barrier and its function as an intracellular calcium store. The authors combined the patch clamp technique with atomic force microscopy (AFM) to investigate the structure-function relationship of NE. In principle, patch clamp currents, recorded from the NE can indicate the activity of the nuclear pore complexes (NPCs) and/or of ion channels in the two biomembranes that compose the NE. However, the role of the NPCs is still nuclear because the observed NE current in patch clamp experiments is lower than expected from the known density of the NPCs. Therefore, AFM was applied to link patch clamp currents to structure. The membrane patch was excised from the nuclear envelope and, after electrical evaluation, transferred from the patch pipette to a substrate. We could identify the native nuclear membrane patches with AFM at a lateral and a vertical resolution of 3 nm and 0.1 nm, respectively. It was shown that complete NE together with NPCs can be excised from the nucleus after their functional identification in patch clamp experiments. However, we also show that membranes of the endoplasmic reticulum can contaminate the tip of the patch pipette during nuclear patch clamp experiments. This possibility must be considered carefully in nuclear patch clamp experiments.

Animals↗

PubMed1997

Rapid aldosterone-induced cell volume increase of endothelial cells measured by the atomic force microscope.

Atomic force microscopy (AFM) is a useful technique for imaging the surface of living cells in three dimensions. The authors applied AFM to obtain morphological information of individual cultured endothelial cells of bovine aorta under stationary and strain conditions and to simultaneously measure changes in cell volume in response to aldosterone. This mineralocorticoid hormone is known to have acute, non-genomic effects on intracellular pH, intracellular electrolytes and inositol-1,4,5-triphosphate production. In this study whether endothelial cells under tension change their volume in response to aldosterone was tested. Such changes were already shown in human leukocytes measured by Coulter counter. In contrast to leukocytes that are more or less spherical and live in suspension, endothelial cells exhibit a complex morphology and adhere to a substrate. Thus, measurements of discrete cell volume changes in endothelial cells under physiological condition is only feasible with more sophisticated techniques. By using AFM we could precisely measure the absolute cell volume of individual living endothelial cells. Before the addition of aldosterone the cell volume of mechanically stressed endothelial cells mimicking arterial blood pressure was 1827 +/- 172 fl. Cell volume was found to increase by 28% 5 min after hormone exposure. Twenty-five minutes later cell volume was back to normal despite the continuous presence of aldosterone in the medium. Amiloride, a blocker of the plasma membrane Na+/H+ exchanger prevented the initial aldosterone-induced volume increase. Taken together, AFM disclosed a transient swelling of endothelial cells induced by the activation of an aldosterone sensitive plasma membrane Na+/H+ exchanger.

Aldosterone↗

PubMed1997

Intracellular Ca2+ distribution in migrating transformed epithelial cells.

Migration of transformed Madin-Darby canine kidney (MDCK-F) cells depends on the polarized activity of a Ca2+-sensitive K+ channel. We tested whether a gradient of intracellular Ca2+-concentration ([Ca2+]i) underlies the horizontal polarization of K+ channel activity. [Ca2+]i was measured with the fluorescent dye fura-2/AM. Spatial analysis of [Ca2+]i indicated that a horizontal gradient exists, with [Ca2+]i being higher in the cell body than in the lamellipodium. Resting and maximal levels during oscillations of [Ca2+]i in the cell body were found to be 135 +/- 34 and 405 +/- 59 nml/l, respectively, whereas they were 79 +/- 18 and 307 +/- 102 nmol/l in the lamellipodium. This gradient can partially explain the preferential activation of K+ channels in the plasma membrane of the cell body. We applied a local superfusion technique during migration experiments and measurements of [Ca2+]i to test whether its maintenance is due to an uneven distribution of Ca2+ influx into migrating MDCK-F cells. Locally superfusing the cell body of migrating MDCK-F cells with La3+ alone or together with charybdotoxin, a specific blocker of Ca2+-sensitive K+ channels, slowed migration to 47 +/- 10% and 9 +/- 5% of control, respectively. Local blockade of Ca2+ influx into the cell body and the lamellipodium with la3+ was followed by a decrease of [Ca2+]i at both cell poles. This points to Ca2+ influx occurring over the entire cell surface. This conclusion was confirmed by locally superfusing Mn2+ over the cell body and the lamellipodium. Fura-2 fluorescence was quenched in both areas, the decrease of fluorescence being two to three times faster in the cell body than in the lamellipodium. However, this difference is insufficient to account for the observed gradient of [Ca2+]i. We hypothesize that the polarized distribution of intracellular Ca2+ stores contributes significantly to the generation of a gradient of [Ca2+]i.

Animals↗

PubMed1997

Imaging excised apical plasma membrane patches of MDCK cells in physiological conditions with atomic force microscopy.

We combined the patch-clamp technique with atomic force microscopy (AFM) to visualize plasma membrane proteins protruding from the extracellular surface of cultured kidney cells (MDCK cells). To achieve molecular resolution, patches were mechanically isolated from whole MDCK cells by applying the patch-clamp technique. The excised inside-out patches were transferred on freshly cleaved mica and imaged with the AFM in air and under physiological conditions (i. e. in fluid). Thus, the resolution could be increased considerably (lateral and vertical resolutions 5 and 0.1 nm, respectively) as compared to experiments on intact cells, where plasma membrane proteins were hardly detectable. The apical plasma membrane surface of the MDCK cells showed multiple protrusions which could be identified as membrane proteins through the use of pronase. These proteins had a density of about 90 per micron(2), with heights between 1 and 9 nm, and lateral dimensions of 20-60 nm. Their frequency distribution showed a peak value of 3 nm for the protein height. A simplified assumption - modelling plasma membrane proteins as spherical structures protruding from the lipid bilayer - allowed an estimation of the possible molecular weights of these proteins. They range from 50 kDa to 710 kDa with a peak value of 125 kDa. We conclude that AFM can be used to study the molecular structures of membranes which were isolated with the patch-clamp technique. Individual membrane proteins and protein clusters, and their arrangement and distribution in a native plasma membrane can be visualized under physiological conditions, which is a first step for their identification.

Animals↗

PubMed1997

Differential expression and activation of MAP kinases in dedifferentiated MDCK-focus cells.

Mitogen-activated protein kinases (MAPK) play a key role in the regulation of cellular processes such as cell growth, cell differentiation, and apoptosis. However, the specific function of single isoforms of the MAPK family in renal epithelial cell differentiation and/or proliferation has not been investigated so far. We now report stable reduction of extracellular signal-regulated kinase 1 (ERK1) protein expression and lack of serum-induced ERK1 activation in alkali-dedifferentiated Madin-Darby canine kidney-C7 focus (MDCK-C7F) cells compared with their parental epithelial MDCK-C7 cells. The changes in ERK1 protein expression and activation were accompanied by a small rise in c-jun NH2-terminal kinase 1 (JNK1) protein expression but slightly decreased basal and anisomycin-stimulated JNK1 activity. In contrast, ERK2 phosphorylation, as assessed by using an antibody which detects phosphorylated tyrosine 204 of both ERK1 and ERK2, as well as enzymatic ERK2 activity, was substantially increased in untreated and fetal calf serum-treated MDCK-C7F cells, although ERK2 protein expression remained unchanged. Differential expression and activation of ERK1, ERK2, and JNK1 were accompanied by an inhibition of serum-induced MDCK-C7F cell proliferation. Together, our results demonstrate an association between changes in the activation of certain MAPK and alkali-induced stable MDCK-C7 cell dedifferentiation. Moreover, these data provide evidence for distinct signaling functions of ERK1 and ERK2 in these cells.

Alkalies↗

PubMed1997

The mineralocorticoid aldosterone activates a proton conductance in cultured kidney cells.

The mineralocorticoid aldosterone is the most important hormone for the regulation of Na+ and K+ homeostasis in mammals and is thereby involved in the regulation of extracellular volume and blood pressure. Because aldosterone is a steroid hormone, the classical way of action involves transcription, translation, and protein synthesis. We previously reported a rapid, nongenomic, and Zn(2+)-sensitive action of aldosterone on Na+/H+ exchange in renal epithelial [Madin-Darby canine kidney (MDCK)] cells (M. Gekle, N. Golenhofen, H. Oberleithner, and S. Silbernagl. Proc. Natl. Acad. Sci. 93: 10500-10504, 1996). Here we show that, in the absence of Na+ (i.e., with inactive Na+/H+ exchange), aldosterone induces a membrane potential-dependent and Zn(2+)-sensitive cytoplasmic acidification in MDCK cells within 2-4 min. This aldosterone-induced activation of a proton conductance is insensitive to the inhibitor of the classical genomic pathway, spironolactone. Furthermore, the inhibitor of serine/threonine kinases and staurosporine, as well as the specific inhibitor of protein kinase C (PKC), calphostin C, prevented proton conductance activation. Activation of PKC by phorbol esters mimicked the effect of aldosterone. Furthermore, preincubation of the cells with pertussis toxin reduced the effect of aldosterone significantly. We propose a new nongenomic mechanism of action for aldosterone, independently of the intracellular type 1 mineralocorticoid receptor: G protein-dependent stimulation of PKC by aldosterone leads to the activation of a plasma membrane proton conductance that enhances the activity of Na+/H+ exchange. This rapid nongenomic effect could explain the observation that aldosterone may alter renal Na+ and K+ excretion within 5-10 min.

Aldosterone↗

PubMed1996

Rapid activation of Na+/H+ exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance.

There is increasing evidence for an additional acute, nongenomic action of the mineralocorticoid hormone aldosterone on renal epithelial cells, leading to a two-step model of mineralocorticoid action on electrolyte excretion. We investigated the acute effect of aldosterone on intracellular free Ca2+ and on intracellular pH in an aldosterone-sensitive Madin-Darby canine kidney cell clone. Within seconds of application of aldosterone, but not of the glucocorticoid hydrocortisone, there was a 3-fold sustained increase of intracellular Ca2+ at a half-maximal concentration of 10(-10) mol/liter. Omission of extracellular Ca2+ prevented this hormone response. In the presence of extracellular Ca2+ aldosterone led to intracellular alkalinization. The Na+/H+ exchange inhibitor ethyl-isopropanol-amiloride (EIPA) prevented the aldosterone-induced alkalinization but not the aldosterone-induced increase of intracellular Ca2+. Omission of extracellular Ca2+ also prevented aldosterone-induced alkalinization. Instead, aldosterone led to a Zn(2+)-dependent intracellular acidification in the presence of EIPA, indicative of an increase of plasma membrane proton conductance. Under control conditions, Zn2+ prevented the aldosterone-induced alkalinization completely. We conclude that aldosterone stimulated net-entry of Ca2+ from the extracellular compartment and a plasma membrane H+ conductance as prerequisites for the stimulation of plasma membrane Na+/H+ exchange which in turn modulates K+ channel acitivity. It is probable that the aldosterone-sensitive H+ conductance maintains Na+/H+ exchange activity by providing an acidic environment in the vicinity of the exchanger. Thus, genomic action of aldosterone determines cellular transport equipment, whereas the nongenomic action regulates transporter activity that requires responses within seconds or minutes, which explains the rapid effects on electrolyte excretion.

Aldosterone↗

PubMed1996

Imaging ROMK1 inwardly rectifying ATP-sensitive K+ channel protein using atomic force microscopy.

The inwardly rectifying K+ channel ROMK1 has been implicated as being significant in K+ secretion in the distal nephron. ROMK1 has been shown by immunocytochemistry to be expressed in relevant nephron segments. The development of the atomic force microscope has made possible the production of high resolution images of small particles, including a variety of biological macromolecules. Recently, a fusion protein of glutathione S-transferase (GST) and ROMK1 (ROMK1-GST) has been used to produce a polyclonal antibody for immunolocalization of ROMK1. We have used atomic force microscopy to examine ROMK1-GST and the native ROMK1 polypeptide cleaved from GST. Imaging was conducted with the proteins in physiological solutions attached to mica. ROMK1-GST appears in images as a particle composed of two units of similar size. Analyses of images indicate that the two units have volumes of approximately 118 nm3, which is close to the theoretical volume of a globular protein of approximately 65 kDa (the molecular mass of ROMK1-GST). Native GST exists as a dimer, and the images obtained here are consistent with the ROMK1-GST fusion protein's existence as a heterodimer. In experiments on ROMK1 in aqueous solution, single molecules appear to aggregate, but contact to the mica was maintained. Addition of ATP to the solution produced a change in height of the aggregates. This change (which was reversible) suggests that ATP induces a structural change in the ROMK1 protein. The data show that atomic force microscopy is a useful tool for examination of purified protein molecules under near-physiological conditions, and furthermore, that structural alterations in the proteins may be continuously investigated.

Adenosine Triphosphate↗

PubMed1996

Rapid activation of calcium-sensitive Na+/H+ exchange induced by 20-hydroxyecdysone in salivary gland cells of Drosophila melanogaster.

Ecdysteroids play an important role in the larval moulting process of insects. 20-Hydroxyecdysone (20E) causes the induction of specific 'puffs' in polytene chromosomes of Drosophila melanogaster salivary gland cells. Although it is known that inorganic ions control pretranscriptional processes in the cell nucleus, the intracellular mechanisms of gene activation are still unclear. Therefore, we examined the effects of 20E on plasma membrane ion transport of Drosophila melanogaster salivary gland cells. Isolated glands of the third larval stage were superfused with a solution mimicking the haemolymph. The relative K+ conductance of the cell membrane (tK+) was measured with microelectrodes by performing ion substitution experiments. Under control conditions tK+ averaged to 0.16 + 0.02 (n = 15). Addition of 5 x 10(-6) M 20E increased tK+ within 2 min by 19.1 +/- 4.2% (n = 15). This rapid response to 20E was elicited only in the presence of calcium. Moreover, starting from a steady-state intracellular pH of 7.20-7.60, 20E induced a rise in cytoplasmic pH by 0.27 +/- 0.06 (n = 6) within minutes. Amiloride (10(-3) M), a blocker of plasma membrane Na+/H+ exchange, prevented the 20E-induced intracellular alkalinization. We conclude that 20E activates a calcium-sensitive plasma membrane Na+/H+ exchange leading to a rise of plasma membrane K+ conductance and intracellular alkalinization both being prerequisites for steroid hormone induced gene activation.

Amiloride↗

PubMed1996

Characterization of hormone-stimulated Na+ transport in a high-resistance clone of the MDCK cell line.

The Madin-Darby canine kidney (MDCK) cell line forms an epithelial monolayer which expresses many of the morphological and functional properties of the renal collecting duct. The C7 subclone of the parent line forms an epithelium which expresses many of the characteristics of principal cells. The MDCK-C7 subclone forms a high-resistance epithelium that is capable of vectorial ion transport. We have found that this epithelium responds to aldosterone, antidiuretic hormone (ADH) and insulin like growth factor 1 (IGF1) with increases in amiloride-sensitive Na+ transport. The responses to aldosterone and ADH follow time-courses that are consistent with the action of these hormones in vivo. This is the first demonstration of IGF1-induced Na+ reabsorption in a mammalian model system. Interestingly, a maximal response to any one of these natriferic factors does not inhibit a subsequent response to another hormone. These studies indicate that the C7 subclone retains many of the natriferic responses of the native principal cells and is an ideal model for studying hormonal modulation of Na+ transport.

Aldosterone↗

PubMed1996

Aldosterone activates the nuclear pore transporter in cultured kidney cells imaged with atomic force microscopy.

Nuclear pore complexes (NPC), located in the nuclear envelope, functionally connect the cell nucleus with the cytoplasm and serve as a crucial pathway for macromolecule exchange. A Madin-Darby canine kidney (MDCK) clone that resembles principal cells of the collecting duct was shown recently to respond to sustained aldosterone exposure with a significant increase in the NPC number per nucleus. The present study elucidates the molecular nature of the NPC pathway and its regulation by aldosterone applying atomic force microscopy. We imaged individual NPC in situ and searched for a putative so-called transporter in the NPC centre. In aldosterone-depleted cells we found numerous macromolecules docked to individual NPC waiting for translocation into the nucleoplasm (standby mode=inactive pore). In contrast, in aldosterone-treated cells NPC were frequently found free of macromolecules, indicating that the translocation process kept pace with docking under hormone-stimulated conditions (transport mode=active pore). In the NPC centre we detected a ring-like structure with a central invagination. We assume that the ring is the putative transporter and that the invagination is the channel entrance used for translocation of macromolecules. Transporters were found in open and closed configurations. In conclusion, the results provide evidence for the existence of a nuclear transporter as part of the translocation machinery of an individual NPC. Aldosterone increases the activity of the nuclear transporter and thus facilitates steroid-mediated gene expression.

Aldosterone↗

PubMed1996

Atomic force microscopy visualizes ATP-dependent dissociation of multimeric TATA-binding protein before translocation into the cell nucleus.

The TATA-binding protein (TBP) is a universal transcription factor which plays an essential role in eukaryotic gene expression. As a karyophilic molecule, this cytosolic protein reaches its DNA-binding site through the transport channel of the nuclear pore complex. As occurs with other major cellular proteins, TBP forms multimers in solution, which is a limiting factor for nuclear translocation. While studying the nuclear translocation of TBP, we detected ATP-dependent multimerization of TBP with atomic force microscopy. In physiological solutions containing ATP, 14-molecule multimers dissociated into four-molecule multimers with a half-maximum dissociation constant of 10 microM. Electrophysiological experiments using isolated cell nuclei of cultured kidney cells revealed that TBP translocates into the cell nucleus only in the presence of ATP. When ATP was replaced with its slowly hydrolysing analogue, ATP[gamma-S] [i.e. adenosine 5'-o-(3-thiotriphosphate)], the aggregates remained intact and nuclear translocation was not possible. Taken together, our investigations suggest that TBP exhibits ATPase activity similar to that observed in relation to molecular chaperons. This activity secures physiological translocation of the transcription factor into the nucleus.

Adenosine Triphosphate↗

PubMed1996

Viewing the renal epithelium with the atomic force microscope.

The atomic force microscope (AFM) was invented in 1986. It created the possibility to study the surfaces of biological material at the molecular level. The AFM is not only a microscope with atomic resolution (at least when applied to suitable samples) but is also an instrument with a broad variety of other applications. We present five examples demonstating the application of the AFM technique in different ways, mainly in renal epithelium. First, it was used as a high-resolution near-field microscope which generated images of canine kidney chromosomes. Second, dynamic changes of the plasma membrane of canine kidney cells were visualized with a resolution at least 10-fold greater than that obtainable by light microscopy. Third, the AFM was used as a tool which interacted directly with the plasma membrane of canine kidney cells. Fourth, due to its excellent resolution in the third dimension (namely height), continuous measurements of changes in 'height' of multimers of cloned plasma membrane potassium channel proteins of rat kidney (ROMK1) in response to ATP were performed with a time resolution of about 100 ms in physiological buffer. Fifth, the AFM continuously followed dissociation processes of multimeric macromolecules (such as transcription factors) under physiological conditions and, on the basis of individual molecular volume measurements, allowed estimation of the number of monomers in a multimeric complex. Taken together, the AFM offers a wide spectrum of novel approaches for the experimental nephrologist.

Animals↗

PubMed1996

Plasticity of renal epithelial cells: the way a potassium channel supports migration.

In this article we review aspects of plasticity of renal epithelial cells. We focus on one particular feature, namely on cell migration. For normal renal and other epithelial cells migration is a motif of plasticity which can be activated after disrupture of epithelial integrity. In the case of transformed renal epithelial cells, however, migration is "pathophysiological" as it is no longer regulated as in normal cells. We studied migration in a permanently transformed Madin-Darby canine kidney cell line, called MDCK-F. Locomotion of these cells strictly depends-in addition to the cytoskeletal "migration machinery"-upon the oscillatory activity of a Ca(2+)-sensitive plasma membrane K+ channel. We propose that K+ channel activity is linked to migration via changes of cell volume. We deduced from patch-clamp experiments in combination with high resolution 3D-images obtained by atomic force microscopy that periods of high K+ channel activity are parallelled by cell shrinkage. By locally superfusing either cell body (rear part) or lamellipodium (front part of the cell) with specific K+ channel blockers we disclosed a polar distribution of K+ channel activity in MDCK-F cells. K+ channels are preferentially active at the rear part of MDCK-F cells. We discuss how localized K+ channel activity, in concert with other migration-relevant phenomena such as "tail contraction" or asymmetric cell-matrix interactions, may result in localized changes of cell volume supporting migration. Finally, we define cell polarization for a migrating epithelial cell. Whereas normal epithelial cells are "vertically" polarized, transformed cells are "horizontally" polarized, i.e., in the plane of movement. Such a distinct view could be helpful for better understanding the transition from a normal differentiated epithelial cell to a tumorigenic migrating cell.

Animals↗

PubMed1995

Phenotypically and karyotypically distinct Madin-Darby canine kidney cell clones respond differently to alkaline stress.

We isolated two cell clones from the wild-type Madin-Darby canine kidney cell line (MDCK) that resembles renal collecting duct epithelium. Morphology and karyotypes of the two cell clones were evaluated. The MDCK-C7 cell clone morphologically resembles principal cells (polygonal cell shape, flat), while the MDCK-C11 clone resembles intercalated cells (cuboidal cell shape, high). The diploid chromosome number of MDCK-C7 cells is 83.1 +/- 0.2 (n = 139); that for MDCK-C11 cells is 78.8 +/- 0.1 (n = 128). Culture of MDCK-C7 cells in alkaline medium (pH 7.7) induced irreversible phenotypical and genotypical alterations. Transformed MDCK-C7F cells are characterized by two abnormal (biarmed) chromosomes. In contrast, MDCK-C11 cells are not phenotypically altered by alkaline stress. In order to elucidate the role of intracellular pH (pHi) in the transformation process, we measured pHi under control conditions (pH 7.4), after 5 min exposure to alkaline stress ("acute experiment," pH 7.7) and after incubation of the cells in alkaline medium for two weeks ("chronic experiment," pH 7.7). Under control conditions, MDCK-C7 cells maintained pHi at 7.14 +/- 0.01 (n = 154) and MDCK-C11 cells at 7.01 +/- 0.01 (n = 147). Acute alkaline stress increased pHi of both cell types to similar steady-state values. Under chronic alkaline stress, MDCK-C7 cells were unable to maintain intracellular pH within normal limits exhibiting sustained alkalinization, whereas MDCK-C11 cells could successfully regulate pHi. We conclude that wild-type MDCK cells consist of two genetically distinct subpopulations with different morphology and function. Only the MDCK-C7 clone that resembles the principle cell type of renal collecting duct can be transformed by alkaline stress while the MDCK-C11 clone resists this treatment, due to efficient pHi control mechanisms.

Alkalies↗

PubMed1995

Patch clamp detection of transcription factor translocation along the nuclear pore complex channel.

Transcription factors (TFs) are cytoplasmic proteins that play an essential role in gene expression. These proteins form multimers and this phenomenon is thought to be one of the mechanisms that regulate transcription. TF molecules reach their DNA binding sites through the large central channel of the nuclear pore complex (NPC). However, the NPC channel is known to restrict the translocation of molecules > or = 20-70 kD. Therefore, during their translocation, TF molecules and/or their multimers may plug the NPC channel and thus, interrupt ion flow through the channel, with a concomitant reduction in the ion conductance of the channel (gamma). Here we show with patch clamp that gamma is reduced during translocation of three major TFs: c-Jun (40 kD), NF-kappa B (approximately equal to 50 kD), and SP1 (approximately equal to 100 kD). Within a minute, femtomolar concentrations of these proteins reduced gamma suggesting a purely mechanical interaction between single TF molecules and the inner wall of the NPC channel. NPCs remained plugged for 0.5-3 hr in the absence of ATP but when ATP was added, channel plugging was shortened to < 5 min. After unplugging, channel closures were rarely observed and the number of functional channels increased. The transcription factors also stabilized the NPCs as shown by the extended duration of the preparations which allowed recordings for up to 72 hr. These observations are the first direct demonstration of the important role of NPCs in mediating nuclear translocation of TFs and, therefore, in forming part of the mechanisms regulating gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1995

Patch clamp and atomic force microscopy demonstrate TATA-binding protein (TBP) interactions with the nuclear pore complex.

The universal TATA-binding protein, TBP, is an essential component of the multiprotein complex known as transcription factor IID (TFIID). This complex, which consists of TBP and TBP-associated factors (TAFs), is essential for RNA polymerase II-mediated transcription. The molecular size of human TBP (37.7 kD) is close to the passive diffusion limit along the transport channel of the nuclear pore complex (NPC). Therefore, the possibility exists that NPCs restrict TBP translocation to the nuclear interior. Here we show for the first time, with patch-clamp and atomic force microscopy (AFM), that NPCs regulate TBP movement into the nucleus and that TBP (10(-15)-10(-10)M) is capable of modifying NPC structure and function. The translocation of TBP was ATP-dependent and could be detected as a transient plugging of the NPC channels, with a concomitant transient reduction in single NPC channel conductance, gamma, to a negligible value. NPC unplugging was accompanied by permanent channel opening at concentrations greater than 250 pM. AFM images demonstrated that the TBP molecules attached to and accumulated on the NPC cytosolic side. NPC channel activity could be recorded for more than 48 hr. These observations suggest that three novel functions of TBP are: to stabilize NPC, to force the NPC channels into an open state, and to increase the number of functional channels. Since TBP is a major component of transcription, our observations are relevant to the understanding of the gene expression mechanisms underlying normal and pathological cell structure and function.

Animals↗