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  • Gene activity  (3)
  • Nuclear envelope  (2)
  • 1
    Electronic Resource
    Electronic Resource
    Open states of nuclear envelope ion channels in cardiac myocytes (1994)
    Springer
    The journal of membrane biology 138 (1994), S. 77-89 
    Details
    ISSN: 1432-1424
    Keywords: Cardiac ; Nucleus ; Nuclear envelope ; Nuclear pore complex ; Nuclear ion channels ; Channel conductance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Prevalent nucleocytoplasmic transport theory views flow of monoatomic ions as completely unrestricted, resulting from the presence of large diameter pore complexes (NPCs) that perforate, but hold together, the two separate membranes of the nuclear envelope (NE). However, three lines of investigations indicate that, at least in some cell types, monoatomic ion flow is restricted. (i) Patch clamp reveals quantized, ion channel-like activity in several NE preparations; activity thought to result from nuclear ion channels (NICs) connected to NPCs. (ii) Ratiometric fluorescence microscopy demonstrates that ions, as well as small molecules relevant to signal transduction, do distribute as if there is a NE barrier. (iii) Electron microscopy shows that NPCs contain material that behaves like a plug. NICs' large conductance (up to 1,000 pS) makes them a major determinant of nuclear ion concentrations which, in turn, influence nuclear processes. Therefore, NICs are an important modulating force of gene and transcriptional activities—two major determinants of gene expression. As nuclear processes may take from seconds (e.g., signaling) to minutes (e.g., transcription), the time the channels dwell in the ion-conducting open state is relevant to understanding NICs' role in nuclear function. Consequently, dwell-times and lifetimes of open NIC states were studied in 61 patch-clamped adult mouse cardiac myocyte nuclei. Upon voltage stimulation, NICs opened to main states of large conductance (281 ± 198 pS, range = 120–490 pS, n = 55) and wide-range mean dwell-times (∼100 msec, 1–10 sec, and min). Closed states (0 pS) also had widely distributed mean dwell-times (∼100 msec, 1–10 sec, and min). Putative open substates (37 ±11 pS, range = 25–50, pS, n = 6) of high bursting frequency (〈1 msec) were observed without intervening main states (≈5% of patches). Fast (∼0.1 msec) and slow (∼10 msec) statetransitions were also detected. These observations suggest a role for NICs in mediating cytoplasmic signal control of cardiomyocyte gene expression.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00211071
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  • 2
    Electronic Resource
    Electronic Resource
    Nuclear electrophysiology (1994)
    Springer
    The journal of membrane biology 138 (1994), S. 105-112 
    Details
    ISSN: 1432-1424
    Keywords: Nucleus ; Nuclear membrane ; Nuclear envelope ; Nucleocytoplasmic transport ; Nuclear pore complex ; Ions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Conclusions Patch-clamp, fluorescence microscopy and high-resolution EM have yielded new data which question current concepts of ion transport across the nuclear envelope. The current challenge is to prove that NICs play an important role in nuclear function either through their identity with NPCs or parts thereof. Electrophysiological designs must incorporate cell biology approaches as done for putative protein-conducting channels of the ER (Simon & Blobel, 1991, 1992). Preliminary studies (J.O. Bustamante et al., in preparation), illustrated in Fig. 1, confirm that, as is the case of NPCs, NICs cannot function in an extracellular environment deprived of cytosolic factors. Our current efforts aim at clarifying if the lysate factors required for macromolecular transport through NPCs (e.g., Adam et al., 199la,b) are those required for NIC open-shut gating. Monoclonal antibodies to identified NPC proteins should be helpful in furthering the identification of NICs with NPCs. Our observation of blockade of NIC activity with wheat germ agglutinin, discussed above, supports the idea that NPCs are the structural foundation for NICs. Should NICs be identified with NPCs or otherwise proven essential to nucleocytoplasmic transport, NIC response to cytoplasmic signals would suggest that they are relevant to mediating gene control by transduction and other cytosolic signals (Karin, 1991; Davis, 1992). NIC influence on intranuclear free ion concentrations is potentially important to controlling gene activation, repression, as well as the efficiency and fidelity of gene expression (e.g., Kroeger, 1963; Lezzi & Gilbert, 1970; Leake et al., 1972; Morgan & Curran, 1986; Li & Rokita, 1991; Lippard, 1993). As electrophysiological and cell/molecular biology approaches merge, the prospects improve for the field of nuclear electrophysiology.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00232638
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  • 3
    Electronic Resource
    Electronic Resource
    Patch clamp detection of transcription factor translocation along the nuclear pore complex channel (1995)
    Springer
    The journal of membrane biology 146 (1995), S. 253-261 
    Details
    ISSN: 1432-1424
    Keywords: Nuclear pore complex ; Nuclear ion channels ; Gene activity ; Control of gene expression ; Transcription factors ; Oncogenes ; Proto-oncogenes ; AP-1 ; c-Jun ; NF-κB ; SP1 ; Patch clamp ; Cardiac myocytes ; Cell nucleus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract 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 ⩾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 (γ). Here we show with patch clamp that γ is reduced during translocation of three major TFs: c-Jun (40 kD), NF-κB (≈50 kD), and SP1 (≈100 kD). Within a minute, femtomolar concentrations of these proteins reduced γ 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. The studies also demonstrate the potential of the patch clamp technique in quantifying TF translocation to the nucleus, mRNA export, and other processes governing gene expression.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233945
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  • 4
    Electronic Resource
    Electronic Resource
    The ion channel behavior of the nuclear pore complex (1995)
    Springer
    The journal of membrane biology 146 (1995), S. 239-251 
    Details
    ISSN: 1432-1424
    Keywords: Nuclear pore complex ; Nuclear ion channels ; Gene activity ; Control of gene expression ; Patch clamp ; Cardiac myocytes ; Cell nucleus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Macromolecule-conducting pores have been recently recognized as a distinct class of ion channels. The poor role of macromolecules as electrical charge carriers can be used to detect their movement along electrolyte-filled pores. Because of their negligible contribution to electrical ion currents, translocating macromolecules reduce the net conductivity of the medium inside the pore, thus decreasing the measured pore ion conductance. In the extreme case, a large translocating macromolecule can interrupt ion flow along the pore lumen, reflected as a negligible pore conductance. Therefore, ion conductance serves as a measurement of macromolecular transport, with lesser values indicating greater macromolecular translocation (in size and/or number). Such is the principle of operation of the widely used Coulter counter, an instrument for counting and sizing particles. It has long been known that macromolecules translocate across the central channel of nuclear pore complexes (NPCs). Recently, large conductance ion channel activity (100–1000 pS) was recorded from the nuclear envelope (NE) of various preparations and it was suggested that NPCs may be the source of this activity. Despite its significance to understanding the regulation of transcription, replication, mRNA export, and thus gene expression of normal and pathological states, no report has appeared demonstrating that this channel activity corresponds to ion flow along the central channel of the NPC. Here we present such a demonstration in adult mouse cardiac myocyte nuclei. In agreement with concepts introduced for macromolecule-conducting channels, our patch clamp experiments showed that ion conductance is reduced, and thus that ion flow is restricted during translocation of macromolecules containing nuclear targeting signals. Ion flow was blocked by mAb414, a monoclonal antibody raised against a major NPC glycoprotein and known to localize on the NPC channel where it blocks macromolecular transport. These results also establish patch clamp as a useful technique for the measurement of macromolecular translocation along the large central channel of the NPC and provide a basis for the design of future investigations of nuclear signaling for control of gene activity, mRNA export for gene expression, as well as other processes subservient to NPC-mediated nucleocytoplasmic exchange.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233944
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  • 5
    Electronic Resource
    Electronic Resource
    Patch clamp and atomic force microscopy demonstrate TATA-binding protein (TBP) interactions with the nuclear pore complex (1995)
    Springer
    The journal of membrane biology 146 (1995), S. 263-272 
    Details
    ISSN: 1432-1424
    Keywords: Nuclear pore complex ; Nuclear ion channels ; Gene activity ; Control of gene expression ; TATA-binding protein ; TBP ; Patch clamp ; Atomic force microscopy ; Cell nucleus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract 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, γ, 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233946
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