Electrophoretic Plugging of Nuclear Pores by Using the Nuclear Hourglass Technique

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     

Nuclear electrophysiology

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.     

Restricted ion flow at the nuclear envelope of cardiac myocytes.

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.     

The two halves of CFTR form a dual-pore ion channel

The cystic fibrosis transmembrane conductance regulator (CFTR) exhibits two conductance states, 9 picosiemens (pS) and 3 pS. To investigate the origin of these two distinct conductance states, we measured the single-channel activity of three truncated forms of CFTR. These include: TNR, which contains the first transmembrane domain, the first nucleotide binding domain, and the R domain; RT2N2, which contains the R domain, the second transmembrane domain, and the second nucleotide-binding domain; and T2N2, which contains only the second transmembrane domain and the second nucleotide-binding domain. The results show that TNR exhibits only the large conductance of 9.2 pS, whereas RT2N2 and T2N2 exhibit only the small conductance (3.8-4.0 pS). Co-expression of TNR with T2N2 resulted in a mixed pattern of two conductance states, which is similar to that observed in wild-type CFTR. In further studies, a "dual-R mutant," R334W and R347P in the transmembrane segment 6 of the first half of CFTR, severely impaired the large conductance channel without affecting the small conductance channel. The ion selectivity and gating behavior of the two conductance channels are different regardless of whether they are measured in wild-type CFTR or in truncated CFTRs. The ion selectivity of the large conductance channel is Br(-) > Cl(-) > I(-), whereas the ion selectivity of the small conductance channel is Br(-) = Cl(-) = I(-). The open probability (P(o)) of the large conductance is about 4-fold higher than that of the small conductance. Transition from closed to open states of the small conductance is not dependent upon the open or closed states of the large conductance. The independent behaviors of the two conductances in CFTR strongly suggest that CFTR may have two distinct pores. Thus, like ClC0, CFTR is likely to be a double-barreled ion channel, with the first half of CFTR forming the large conductance and the second half forming the small conductance.     

Reconstitution of the influenza virus M2 ion channel in lipid bilayers

M₂, an integral membrane protein of influenza A virus, was purified from either influenza A virus-infected CV-1 cells or from Spodoptera frugiperda (Sf9) cells infected with a recombinant-M₂ baculovirus. The purified protein, when incorporated into phospholipid bilayer membranes, produced ion-permeable channels with the following characteristics: (1) The channels appeared in bursts during which unit conductances of diverse magnitudes (25–500 pS) were observed. (2) The most probable open state was usually the lowest unit conductance (25–90 pS). (3) The channels were selective for cations; t _Na = 0.75 when 150 mm NaCl bathed both sides of the membrane. (4) Amantadine reduced the probability of opening of the high conductance state and also the conductance of the most probable state. (5) Reducing pH increased the mean current through the open channel as well as the conductance of the most probable state. (6) The sequence of selectivity for group IA monovalent cations was Rb > K > Cs ～ Na > Li. The pH activation, amantadine block and ion selectivity of the M₂ protein ion channel in bilayers are consistent with those observed on expression of the M₂ protein in oocytes of Xenopus laevis as well as for those predicted for the proposed role of an ion channel in the uncoating process of influenza virus. The finding that the M₂ protein has intrinsic ion channel activity supports the hypothesis that it has ion channel activity in the influenza virus particle.     

Early embryonic requirement for nucleoporin Nup35/NPP-19 in nuclear assembly

Nuclear pore complexes (NPCs) are gateways for transport between the nucleus and cytoplasm of eukaryotic cells and play crucial roles in regulation of gene expression. NPCs are composed of multiple copies of ∼ 30 different nucleoporins (nups) that display both ubiquitous and cell type specific functions during development. Vertebrate Nup35 (also known as Nup53) was previously described to interact with Nup93, Nup155 and Nup205 and to be required for nuclear envelope (NE) assembly in vitro. Here, we report the first in vivo characterization of a Nup35 mutation, npp-19(tm2886), and its temperature-dependent effects on Caenorhabditis elegans embryogenesis. At restrictive temperature, npp-19(tm2886) embryos exhibit chromosome missegregation, nuclear morphology defects and die around mid-gastrulation. Depletion of Nup35/NPP-19 inhibits NE localization of Nup155/NPP-8, NPC assembly and nuclear lamina formation. Consequently, nuclear envelope function, including nucleo-cytoplasmic transport, is impaired. In contrast, recruitment of Nup107/NPP-5, LEM-2 and nuclear membranes to the chromatin surface is Nup35/NPP-19-independent, suggesting an uncoupling of nuclear membrane targeting and NPC assembly in the absence of Nup35/NPP-19. We propose that Nup35/NPP-19 has an evolutionary conserved role in NE formation and function, and that this role is particularly critical during the rapid cell divisions of early embryogenesis.     

Patch-clamp study of liver nuclear ionic channels reconstituted into giant proteoliposomes

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.     

Evidence for Ca2+- and ATP-sensitive peripheral channels in nuclear pore complexes.

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.     

A carboxy-terminal fragment of colicin Ia forms ion channels

A carboxy-terminal, 18 kD fragment of colicin Ia, a bacterial toxin, forms ion channels in artificial phospholipid bilayers. This fragment, which comprises a quarter of the intact 70 kD molecule, is resistant to extensive protease digestion and probably constitutes a structural domain of the protein. The ion channels formed by the 18 kD fragment are functionally heterogeneous, having conductances that range from 15 to 30 pS at positive voltages and from 70 to 250 pS at negative voltages, and open lifetimes that range from at least 25 msec to 5 sec. In contrast, ion channels formed by whole colicin Ia open only at negative voltages, at which their conductances range from 6 to 30 pS, and their open lifetimes range from 1 sec to 3 min. Additionally, the open state of the 18 kD fragment channel is characterized by noisy fluctuations in current, while the open state of the whole molecule ion channel is often marked by numerous, stable subconductance states. Since the properties of the fragment channel differ substantially from those of the whole molecule channel, we suggest that portions of the molecule outside of the 18 kD fragment are involved in forming the whole molecule ion channel.     

Single acetylcholine-activated channel currents in developing muscle cells

The properties of single acetylcholine-activated ion channels in developing rat myoblasts and myotubes in tissue culture have been investigated using the gigaohm seal patch clamp technique. Two classes of ACh-activated channels were identified. The major class of channels (accounting for >95% of all channel openings) has a conductance of 35 pS and a mean open time of 15 msec (at room temperature and ?80 mV). The minor class of channels has a larger conductance (55 pS) and a briefer mean open time (2–3 msec). Functional ACh-activated channels are present in undifferentiated mononucleated myoblasts 1–2 days in culture, although the channel density on such cells is low. Over the next week in culture, as the myoblasts fuse to form multinucleate myotubes, there is a marked increase in channel density and an increase in the proportion of large conductance channels. No significant change, however, occurs in channel conductance or open time (within a given class of channels) during this period. At high concentrations of ACh, channels desensitize and channel openings occur in groups, similar to what has been previously described in adult muscle. The rate of channel opening within a group of openings increases with increasing agonist concentration while mean open time is independent of agonist concentration, as expected from simple models of drug action. During a group of openings, the channel is open for half the time (i.e., channel opening rate is equal to channel closing rate) at a concentration of approximately 6 μm ACh.     

Saccharomyces cerevisiae Ndc1p Is a Shared Component of Nuclear Pore Complexes and Spindle Pole Bodies

We report a novel connection between nuclear pore complexes (NPCs) and spindle pole bodies (SPBs) revealed by our studies of the Saccharomyces cerevisiae NDC1 gene. Although both NPCs and SPBs are embedded in the nuclear envelope (NE) in yeast, their known functions are quite distinct. Previous work demonstrated that NDC1 function is required for proper SPB duplication (Winey, M., M.A. Hoyt, C. Chan, L. Goetsch, D. Botstein, and B. Byers. 1993. J. Cell Biol. 122:743–751). Here, we show that Ndc1p is a membrane protein of the NE that localizes to both NPCs and SPBs. Indirect immunofluorescence microscopy shows that Ndc1p displays punctate, nuclear peripheral localization that colocalizes with a known NPC component, Nup49p. Additionally, distinct spots of Ndc1p localization colocalize with a known SPB component, Spc42p. Immunoelectron microscopy shows that Ndc1p localizes to the regions of NPCs and SPBs that interact with the NE. The NPCs in ndc1-1 mutant cells appear to function normally at the nonpermissive temperature. Finally, we have found that a deletion of POM152, which encodes an abundant but nonessential nucleoporin, suppresses the SPB duplication defect associated with a mutation in the NDC1 gene. We show that Ndc1p is a shared component of NPCs and SPBs and propose a shared function in the assembly of these organelles into the NE.     

Changes of N-methyl-D-aspartate activated channels of cerebellar granule cells with days in culture

N-Methyl-D-Aspartate (NMDA) activated channels were studied in enzymatically dissociated cerebellar granule cells primary cultures. Measurements of single channel currents were made on different days in culture. Changes in the electrophysiological behavior of NMDA-activated channels, which were dependent on the time in culture, were found. The variations of single channel maximum conductance during the developement of the cells in culture were detected. Three different characteristic periods could be distinguished: the first period (1-3 days) in which the conductance assumed a value of 15.5 pS; the second one (5-8 days) characterized by a value of 35.7 pS and the last one (9-11 days) in which the conductance reached values of 46.8 pS. Moreover mean open time of NMDA-activated channels was less than 1 msec during the first two days in culture and stabilized at 3 to 6 msec around the fifth day.     

The role of nuclear pores in gene regulation,development and disease

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.     

Phosphorylation-dependent mitotic SUMOylation drives nuclear envelope–chromatin interactions

In eukaryotes, chromatin binding to the inner nuclear membrane (INM) and nuclear pore complexes (NPCs) contributes to spatial organization of the genome and epigenetic programs important for gene expression. In mitosis, chromatin–nuclear envelope (NE) interactions are lost and then formed again as sister chromosomes segregate to postmitotic nuclei. Investigating these processes in S. cerevisiae, we identified temporally and spatially controlled phosphorylation-dependent SUMOylation events that positively regulate postmetaphase chromatin association with the NE. Our work establishes a phosphorylation-mediated targeting mechanism of the SUMO ligase Siz2 to the INM during mitosis, where Siz2 binds to and SUMOylates the VAP protein Scs2. The recruitment of Siz2 through Scs2 is further responsible for a wave of SUMOylation along the INM that supports the assembly and anchorage of subtelomeric chromatin at the INM and localization of an active gene (INO1) to NPCs during the later stages of mitosis and into G1-phase.     

Current concepts in nuclear pore electrophysiology

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.     

GABA increases both the conductance and mean open time of recombinant GABAA channels co-expressed with GABARAP

The single channel properties of recombinant gamma-aminobutyric acid type A (GABA(A))alphabetagamma receptors co-expressed with the trafficking protein GABARAP were investigated using membrane patches in the outside-out patch clamp configuration from transiently transfected L929 cells. In control cells expressing alphabetagamma receptors alone, GABA activated single channels whose main conductance was 30 picosiemens (pS) with a subconductance state of 20 pS, and increasing the GABA concentration did not alter their conductance. In contrast, when GABA(A) receptors were co-expressed with GABARAP, the GABA-activated single channels displayed multiple, high conductances (> or =40 pS), and GABA (> or =10 microM) was able to increase their conductance, up to a maximum of 60 pS. The mean open time of GABA-activated channels in control cells expressing alphabetagamma receptors alone was 2.3 +/- 0.1 ms for the main 30-pS channel and shorter for the subconductance state (20 pS, 0.8 +/- 0.1 ms). Similar values were measured for the 30- and 20-pS channels active in patches from cells co-expressing GABARAP. However higher conductance channels (> or =40 pS) remained open longer, irrespective of whether GABA or GABA plus diazepam activated them. Plotting mean open times against mean conductances revealed a linear relationship between these two parameters. Since high GABA concentrations increase both the maximum single channel conductance and mean open time of GABA(A) channels co-expressed with GABARAP, trafficking processes must influence ion channel properties. This suggests that the organization of extrasynaptic GABA(A) receptors may provide a range of distinct inhibitory currents in the brain and, further, provide differential drug responses.     

核孔复合体的结构及其功能

核孔复合体(Nuclear pore complexes, NPCs)镶嵌在核膜上,是细胞核与细胞质之间的唯一通道。冷冻电子X射线断层扫描将环状NPCs分为三个环,分别称为胞质环、内环和核质环,胞质环上附有胞质纤丝,核质环上附有核篮。由于物种不同,NPCs由30～50多种不同的核孔蛋白(nucleoporins, Nups)组成,但结构和功能高度保守。根据其结构、氨基酸序列,NPCs定位和功能,Nups被分为跨膜Nups、屏障Nups、骨架Nups、胞质纤丝Nups和核篮Nups。相互间作用稳定、紧密连接的数个Nups可组成亚复合体。为了应对不同生理需要,NPCs处于高度动态变化中,间期和有丝分裂期均可通过组装和去组装改变核孔数量和功能。NPCs的主要功能是调控核质转运,小分子物质可自由扩散,大分子物质则需在核转位信号和转运载体的介导下以主动运输的方式进行转运。除了核质转运这一主要功能外,Nups还能以一个独立于转运的方式影响基因组功能。通过影响染色质结构和影响转录调控元件对靶基因的访问,Nups促进或抑制转录。在酵母,Nups介导的基因调控主要由位于NPCs中的Nups执行;在多细胞生物,不仅NPCs中的Nups,核质内游离的Nups也具有基因调控功能。此外,Nups还能通过参与形成染色质边界和形成转录记忆对基因进行调控。在增殖细胞, Nups通过与DNA修复机器相互作用,参与DNA损伤修复,保护基因组完整性。有丝分裂时,Nups协助核膜解体和中心体迁移,并通过作用于着丝粒来控制有丝分裂组件的空间定位与活性,稳定它们与微管之间的相互作用,保证纺锤体正常组装和染色体准确分离。总之,NPCs与生物分子的核质转运、基因表达和细胞周期密切相关,它的结构和功能的稳定是真核细胞生长、增殖、分化等生命活动的基本保证。     

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 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.