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1.
Restricted ion flow at the nuclear envelope of cardiac myocytes.   总被引:2,自引:1,他引:1       下载免费PDF全文
Flow of small ions across the nuclear envelope (NE) is thought to occur without restriction through large diameter nuclear pore complexes (NPCs). However, investigations with electron and fluorescence microscopy, and with patch-clamp and microelectrode electrophysiology, suggest that in many animal and plant cell types small ions move through a barrier having the signature of large conductance nuclear ion channels (NICs). As nucleocytoplasmic transport and gene activity are regulated by cytoplasmic signals and as it has recently been shown by this investigator that cardiac NICs are sensitive to cAMP-dependent processes (1), it was considered relevant to further investigate the effects of various cytosolic signals on NIC activity. Ion species substitution demonstrated that K+ is the major species responsible for NIC currents. The Na-channel blocker tetrodotoxin (TTX, 100 microM) and the Ca-channel blocker diltiazem (100 microM) had no effect, indicating no relation of NICs to Na- or Ca-channels in transit to the cell surface membrane. Zn2+ (100 microM) blocked NIC activity, suggesting a dual role in nucleocytoplasmic transport and gene function. GTP did not produce measurable effect. However, its nonhydrolyzable analogue GTP-gamma-S (10 microM) suppressed NIC activity, suggesting a role for GTP hydrolysis in NIC function. Deoxynucleotides (dNTPs, 200 microM) produced a transient increase in NIC activity, pointing to a modulation of NIC function by nucleic acid substrates. These results indicate a role for NICs in mediating: (a) control of gene activity by transduction and other cytosolic signals, and (b) nuclear demands and response to such signals.  相似文献   

2.
3.
Nuclear ionic channels (NICs) represent ubiquitous structures of living cells, although little is known about their functional properties and encoding genes. To characterize NICs, liver nuclear membrane vesicles were reconstituted into either planar lipid bilayers or proteoliposomes. Reconstitution of nuclear envelope (NE) vesicles into planar lipid bilayer proceeded with low efficiency. NE vesicle reconstitution into proteoliposomes led to NIC observations by the patch-clamp technique. Large conductance, voltage-gated, K(+)-permeant and Cl(-)-permeant NICs were characterized. An 80-105-pS K(+)-permeant NIC with conducting sub-state was also recorded. Our data establish that NICs can be characterized upon reconstitution into giant proteoliposomes and retain biophysical properties consistent with those described for native NICs.  相似文献   

4.
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.The author thanks (in alphabetical order) the intellectual contributions of Drs. Christopher W. Akey, Gregory S. Beckler (Promega), Louis J. DeFelice, Colin Dingwall, Alexander Fabiato, Julio M. Fernández, Larry Gerace, John A. Hanover, Bertil Hille, Stuart L. Jacobson, W. Jonathan Lederer, Andrejs Liepins, Gilbert N. Ling, Michele Mazzanti, Ernst Niggli, Sanford M. Simon, Walter Stühmer, and W. Gil Wier. Special thanks are tendered to Drs. Dingwall, Gerace, Hanover and Liepins for their observations on nuclear electrophysiology within the context of cell/molecular biology. Thanks are also extended to Drs. Lederer and Wier for discussions on fluorescence microscopy of Ca2+ transients. Dr. Niggli provided the preprint of his paper, with P. Lipp, confirming previous observations that cardiomyocyte nuclei behave as a barrier to intracellular Ca2+ waves. Drs. DeFelice and Mazzanti provided a draft of their review on the biophysics of the nuclear envelope. This work is supported by the American Heart Association, Maryland Affiliate. Institutional support and facilities have come through Drs. C. William Balke, Michael R. Gold, W. Gil Wier and W. Jonathan Lederer, to whom the author is deeply grateful. This work is dedicated to my parents for introducing me to scientific curiosity and for their constant incentive and support. A special dedication to my father who recently passed away.  相似文献   

5.
The nuclear hourglass technique (NHT) was recently introduced as a novel technique that measures the electrical nuclear envelope (NE) conductance of isolated Xenopus laevis oocyte nuclei. The main conclusion drawn from NHT work so far is that nuclear pore complexes (NPCs) of oocytes are in an electrically open state under physiological conditions, with a mean conductance of 1.7 nS per NPC. Since nuclear patch-clamp data indicate that usually NPCs are electrically closed, our work has been challenged by the notion that NHT cannot assure a high resistance seal (``gigaseal') between glass wall and NE like that required for patch-clamp experiments. Thus, NHT could have dramatically underestimated NE electrical resistance. Here we demonstrate that NHT does not require a gigaseal for accurate NE conductance measurements. In addition, we present experimental conditions where mean single NPC electrical conductance is reduced 26-fold due to electrophoretic plugging by negatively charged nucleoplasmic macromolecules. In addition, data indicate that under physiological conditions (i.e., when macromolecules are offered in the cytosolic solution) the nuclear surface is heavily folded, underestimating ``true' NE surface by a factor of 2.6. When ``true' NE surface area is taken into consideration, modified values of mean single NPC conductances of 654 pS for electrically open conditions and 25 pS for electrically plugged conditions can be calculated. We conclude that the large overall NE conductance detected with the nuclear hourglass technique in intact Xenopus laevis oocyte nuclei can be explained by the sum of single NPC conductances in the pS range, as long as open probability is high. This confirms previous patch-clamp work concerning single NPC conductance, but disagrees with the view that mean open probability of NPC channels is usually low. Received: 27 March 2001/Revised: 3 July 2001  相似文献   

6.
In eukaryotic cells the nuclear envelope (NE) serves as a functional barrier between cytosol and nucleoplasm perforated by nuclear pore complexes (NPCs). Both active and passive transport of ions and macromolecules are thought to be mediated by the centrally located large NPC channel. However, 3-dimensional imaging of NPCs based on electron microscopy indicates the existence of additional small channels of unknown function located in the NPC periphery. By means of the recently developed nuclear hourglass technique that measures NE electrical conductance, we evaluated passive electrically driven transport through NPCs. In isolated Xenopus laevis oocyte nuclei, we varied ambient Ca2+ and ATP in the cytosolic solution and/or chelated Ca2+ in the perinuclear stores in order to assess the role of Ca2+ in regulating passive ion transport. We noticed that NE electrical conductance is large under conditions where macromolecule permeability is known to be low. In addition, atomic force microscopy applied to native NPCs detects multiple small pores in the NPC periphery consistent with channel openings. Peripheral pores were detectable only in the presence of ATP. We conclude that NPC transport of ions and macromolecules occurs through different routes. We present a model in which NE ion flux does not occur through the central NPC channel but rather through Ca2+- and ATP-activated peripheral channels of individual NPCs.  相似文献   

7.
8.
A J Matzke  T M Weiger  M A Matzke 《FEBS letters》1990,271(1-2):161-164
To determine whether the nuclear envelope of eukaryotic cells has the capability to regulate ion fluxes, we have used the patch-clamp technique to detect ion channels in this membrane system. Since possible sites for ion channels in the nuclear envelope include not only the nuclear pores, but also both the inner and outer nuclear membranes, we have patched giant liposomes composed of phosphatidylcholine and nuclear envelope fragments isolated from mature avian erythrocytes. A large, cation-selective channel with a maximum conductance of approximately 800 pS in symmetrical 100 mM KCl was detected. This channel is a possible candidate for a nuclear pore.  相似文献   

9.
Several types of ionic channels on the outer membrane of the nuclear envelope communicate with the nuclear cisternae. These are distinct from nucleocytoplasmic pathways, the nuclear pores that span the double membrane of the envelope and are the route for RNA and protein traffic in the nucleus. Recent data indicate that the nuclear pores may also function as ion channels. The most probable candidate for nucleocytoplasmic ion flux is a 300-400 pS pathway observed in many nuclear preparations. Morphological and functional studies of nuclear envelope suggest a tight relationship between the large conductance channel and the pore complex. However, there is no direct evidence for gating of the nuclear pore or its ability to open and close as a conventional channel. This study shows that in liver nuclei isolated from newborn mouse, there is a substantial correspondence between the number of pores and the number of channels recorded during patch-clamp. This is not the case for adult nuclei. Although pore density is comparable, some nuclear cytoskeletal components, such as actin and nonmuscle myosin, show a significant increase in the adult preparation. Previous studies demonstrate the presence of these two proteins in association with the pore complex. Here we show that by using actin filament disrupter, we were able to increase the number of active channels in adult isolated nuclei. We suggest that a functional interaction between actin filaments and the nuclear pore complex could regulate nucleocytoplasmic permeability.  相似文献   

10.
The role of the nuclear pore complex in adenovirus DNA entry.   总被引:20,自引:1,他引:19       下载免费PDF全文
Adenovirus targets its genome to the cell nucleus by a multistep process involving endocytosis, membrane penetration and cytoplasmic transport, and finally imports its DNA into the nucleus. Using an immunochemical and biochemical approach combined with inhibitors of nuclear import, we demonstrate that incoming viral DNA and DNA-associated protein VII enter the nucleus via nuclear pore complexes (NPCs). Depletion of calcium from nuclear envelope and endoplasmic reticulum cisternae by ionophores or thapsigargin blocked DNA and protein VII import into the nucleus, but had no effect on virus targeting to NPCs. Calcium-depleted cells were capable of disassembling incoming virus. In contrast, inhibitors of cytosolic O-linked glycoproteins of the NPC blocked virus attachment to the nuclear envelope, capsid disassembly and also nuclear import of protein VII. The data indicate that NPCs have multiple roles in adenovirus entry into cells: they contain a virus-binding and/or dissociation activity and provide a gateway for the incoming DNA genome into the nucleus.  相似文献   

11.
12.
Using a patch-clamp technique, we studied the biophysical properties of large-conductance channels in the nuclear envelope of rat cerebellar Purkinje neurons. Our experiments showed that channels with identical conductance, selectivity, and kinetics are expressed in the external and internal nuclear membranes of these cells. These channels connect the perinuclear space with the cyto-and nucleoplasm; they are not channels of the complex of the nuclear pores for passive diffusion of ions and small molecules, as was believed earlier [17]. We hypothesize that large-conductance cationic channels in the membranes of the nuclear envelope are identical to ion channels of the endoplasmic reticulum and are necessary for functioning of the intermembrane space of the envelope as a calcium store. Neirofiziologiya/Neurophysiology, Vol. 39, No. 2, pp. 113–118, March–April, 2007.  相似文献   

13.
Over 4 decades ago, microelectrode studies of in situ nuclei showed that, under certain conditions, the nuclear envelope (NE) behaves as a barrier opposing the nucleocytoplasmic flow of physiological ions. As the nuclear pore complexes (NPCs) of the NE are the only pathways for direct nucleocytoplasmic flow, those experiments implied that the NPCs are capable of restricting ion flow. These early studies validated electrophysiology as a useful approach to quantify some of the mechanisms by which NPCs mediate gene activity and expression. Since electron microscopy (EM) and other non-electrophysiological investigations, showed that the NPC lumen is a nanochannel, the opinion prevailed that the NPC could not oppose the flow of ions and, therefore, that electrophysiological observations resulted from technical artifacts. Consequently, the initial enthusiasm with nuclear electrophysiology faded out in less than a decade. In 1990, nuclear electrophysiology was revisited with patch-clamp, the most powerful electrophysiological technique to date. Patch-clamp has consistently demonstrated that the NE has intrinsic ion channel activity. Direct demonstrations of the NPC on-off ion channel gating behavior were published for artificial conditions in 1995 and for intact living nuclei in 2002. This on-off switching/gating behavior can be interpreted in terms of a metastable energy barrier. In the hope of advancing nuclear electrophysiology, and to complement the other papers contained in this special issue of the journal, here I review some of the main technical, experimental, and theoretical issues of the field, with special focus on NPCs.  相似文献   

14.
The membrane of the endoplasmic reticulum is, in fact, an extension of the nuclear envelope of eukaryotic cells; both these compartments can fulfill the function of intracellular calcium stores. Using a patch-clamp technique, we studied the biophysical properties of the channels expressed in the inner nuclear membrane of pyramidal neurons of the rat hippocampal CA1 area, in particular of large-conductance cationic channels and calcium channels of inositol trisphosphate receptors (the main channels in membranes of this type). As the results of the measurements showed, the activity of channels of both types demonstrates clearly pronounced voltage dependences. The probability of their open state (P o) depends on the voltage inside the nuclear envelope lumen. At positive potentials, the activity of these channels is significantly more intense than at negative potentials. Moreover, channels of both types are reversibly blocked at considerable negative potentials. We believe that this property of ion channels in the nuclear envelope is an important factor responsible for the control of calcium signals in the cell nucleus. We propose a hypothesis on the mechanism underlying termination of Ca2+ release from such intracellular stores, which is based on the specificity of the voltage dependence of ion channels of the above-mentioned types.  相似文献   

15.
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 unclear 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 3nm and 0.1nm, 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.  相似文献   

16.
The genetic apparatus of an eukaryotic cell is surrounded by two membranes of the nuclear envelope that forms a half-permeable barrier for the movement of molecules and ions. Using a patch-clamp technique in experiments on isolated nuclei of pyramidal neurons from the hippocampal CA1 area, we describe the biophysical properties of spontaneously active ion channels in the nuclear membranes of these cells. In the external nuclear membrane, we found anion channels with a unitary conductance of 156 pS and with very rapid kinetics of fluctuation, while in the inner membrane we recorded cationic channels with a unitary conductance of 248 pS and very slow kinetics. Channels of both types demonstrated clear voltage dependences. We hypothesize that the physiological importance of these channels is related to the function of the intermembrane space of the nuclear envelope of these cells forming a considerable calcium store. It seems possible that such channels in the nuclear membranes are necessary for the maintenance of the ion balance between the cytoplasm and perinuclear space and between the latter and karyoplasm, and also for neutralization of voltage shifts in the course of Ca2+ release. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 3–8, January–February, 2007.  相似文献   

17.
Nuclear pore complexes (NPCs) are channels within the nuclear envelope that mediate nucleocytoplasmic transport. NPCs form within the closed nuclear envelope during interphase or assemble concomitantly with nuclear envelope reformation in late stages of mitosis. Both interphase and mitotic NPC biogenesis require coordination of protein complex assembly and membrane deformation. During early stages of mitotic NPC assembly, a seed for new NPCs is established on chromatin, yet the factors connecting the NPC seed to the membrane of the forming nuclear envelope are unknown. Here, we report that the reticulon homology domain protein REEP4 not only localizes to high-curvature membrane of the cytoplasmic endoplasmic reticulum but is also recruited to the inner nuclear membrane by the NPC biogenesis factor ELYS. This ELYS-recruited pool of REEP4 promotes NPC assembly and appears to be particularly important for NPC formation during mitosis. These findings suggest a role for REEP4 in coordinating nuclear envelope reformation with mitotic NPC biogenesis.  相似文献   

18.
The outer nuclear membrane, endoplasmic reticulum, and mitochondrial membrane ion channels are poorly understood, although they are important in the control of compartmental calcium levels, cell division, and apoptosis. Few direct recordings of these ion channels have been made because of the difficulty of accessing these intracellular membranes. Using patch-clamp techniques on isolated nuclei, we measured distinct ion channel classes on the outer nuclear envelope of T-cell (human Jurkat) and BFL5 cell (murine promyelocyte) lines. We first imaged the nuclear envelopes of both Jurkat and FL5 cells with atomic force microscopy to determine the density of pore proteins. The nuclear pore complex was intact at roughly similar densities in both cell types. In patch-clamp recordings of Jurkat nuclear membranes, Cl channels (105 +/- 5 pS) predominated and inactivated with negative pipette potentials. Nucleotides transiently inhibited the anion channel. In contrast, FL5 nuclear channels were cation selective (52 +/- 2 pS), were inactivated with positive membrane potentials, and were insensitive to GTPgammaS applied to the bath. We hypothesize that T- and B-cell nuclear membrane channels are distinct, and that this is perhaps related to their unique roles in the immune system.  相似文献   

19.
Summary The observation that the nuclear envelope outer mem brane contains ion channels raises the question of whether these conductances communicate between the cytosol and the nuclear envelope cisternae or between the cytosol and the cytoplasm. Failure to detect large, nonselective holes using the patch-clamp technique has led to the speculation that ion channels and nuclear pores are in fact the same. In this paper we present evidence that the ionic channel, recorded in isolated liver nuclei with the patch-clamp configura tion of “nucleus-attached,” spans the double membrane of the envelope, providing a direct contact between nucleoplasm and cytoplasm.  相似文献   

20.
Nuclear‐pore complexes (NPCs) are large protein channels that span the nuclear envelope (NE), which is a double membrane that encloses the nuclear genome of eukaryotes. Each of the typically 2,000–4,000 pores in the NE of vertebrate cells is composed of multiple copies of 30 different proteins known as nucleoporins. The evolutionarily conserved NPC proteins have the well‐characterized function of mediating the transport of molecules between the nucleoplasm and the cytoplasm. Mutations in nucleoporins are often linked to specific developmental defects and disease, and the resulting phenotypes are usually interpreted as the consequences of perturbed nuclear transport activity. However, recent evidence suggests that NPCs have additional functions in chromatin organization and gene regulation, some of which might be independent of nuclear transport. Here, we review the transport‐dependent and transport‐independent roles of NPCs in the regulation of nuclear function and gene expression.  相似文献   

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