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.     

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.     

A novel nucleocytoplasmic traffic GTPase identified as a functional target of the bipartite geminivirus nuclear shuttle protein

In contrast to the accumulated data on nuclear transport mechanisms of macromolecules, little is known concerning the regulated release of nuclear-exported complexes and their subsequent trans-cytoplasmic movement. The bipartite begomovirus nuclear shuttle protein (NSP) facilitates the nuclear export of viral DNA and cooperates with the movement protein (MP) to transport viral DNA across the plant cell wall. Here, we identified a cellular NSP-interacting GTPase (NIG) with biochemical properties consistent with a nucleocytoplasmic transport role. We show that NIG is a cytosolic GTP-binding protein that accumulates around the nuclear envelope and possesses intrinsic GTPase activity. NIG interacts with NSP in vitro and in vivo (under transient expression), and redirects the viral protein from the nucleus to the cytoplasm. We propose that NIG acts as a positive contributor to geminivirus infection by modulating NSP nucleocytoplasmic shuttling and hence facilitating MP–NSP interaction in the cortical cytoplasm. In support of this, overexpression of NIG in Arabidopsis enhances susceptibility to geminivirus infection. In addition to highlighting the relevance of NIG as a cellular co-factor for NSP function, our findings also have implications for general nucleocytoplasmic trafficking of cellular macromolecules.     

Cytosolic import factor- and Ran-independent nuclear transport of ribosomal protein L5

Ribosomal protein L5 is a shuttling protein that, in Xenopus oocytes, is involved in the nucleocytoplasmic transport of 5S rRNA. As demonstrated earlier, L5 contains three independent nuclear import signals (NLSs), which function in oocytes as well as in somatic cells. Upon physical separation, these NLSs differ in respect to their capacity to bind to nuclear import factors in vitro and to mediate the nuclear import of a heterologous RNP in vivo. As reported in this communication, analysis of the in vitro nuclear import activity of these three NLSs reveals that they also differ in respect to their requirements for cytosolic import factors and Ran. Nuclear import mediated by the N-terminal and the central NLS depends on cytosolic import factor(s) and Ran, whereas import via the C-terminal NLS occurs independently from these factors. Thus, the presence of multiple NLSs in ribosomal protein L5 appears to allow for efficient nuclear transport via utilisation of multiple, mechanistically different import pathways.     

GTP-dependent permeabilized neutrophil secretion requires a freely diffusible cytosolic protein

Guanosine triphosphate (GTP) has been implicated in the regulation of Ca(2+)-mediated secretion from neutrophils. We further examined the role of GTP in neutrophil secretion using streptolysin O permeabilized cells. We found that, in the presence of GTP, 1.0 microM free Ca(2+) causes maximum secretion-equivalent to that achieved with 100 microM free Ca(2+)-whereas GTPgammaS inhibits Ca(2+)-stimulated secretion. Interestingly, GTP by itself stimulates secretion. These results indicate the existence of a GTP-regulated mechanism of secretion in neutrophils that requires GTP hydrolysis to stimulate secretion in the presence and absence of Ca(2+). The stimulatory effect of GTP is only observed when GTP is present during permeabilization. Addition of GTP after permeabilization, when the cytosolic contents have leaked out from cells, gives no stimulatory response, implying that the GTP-dependent secretory apparatus requires at least one cytosolic protein. GTP-dependent secretion can be reconstituted with crude HL-60 and bovine liver cytosol. The reconstituting activity binds to GTP-agarose, suggesting that the cytosolic factor is a GTP-binding protein or forms a complex with a GTP-binding protein. However, it is not a member of the rho or rac families of GTPases. By gel filtration chromatography, the secretion-reconstituting activity eluted at 870 and 200 kDa, but in the presence of GTP, eluted at 120 kDa, indicating that it is part of a high-molecular-weight complex that dissociates in the presence of GTP. Retention of adenosine diphosphate-ribosylation factor (ARF) in permeabilized cells and insensitivity of the cytosolic reconstituting activity to brefeldin A led to our speculation that ARF6 may be the GTPase involved in GTP-dependent secretion, and that activity from a BFA-insensitive ARF6 guanine nucleotide exchange factor reconstitutes secretion.     

Mitogen activated protein kinase at the nuclear pore complex

Mitogen activated protein (MAP) kinases control eukaryotic proliferation, and import of kinases into the nucleus through the nuclear pore complex (NPC) can influence gene expression to affect cellular growth, cell viability and homeostatic function. The NPC is a critical regulatory checkpoint for nucleocytoplasmic traffic that regulates gene expression and cell growth, and MAP kinases may be physically associated with the NPC to modulate transport. In the present study, highly enriched NPC fractions were isolated and investigated for associated kinases and/or activity. Endogenous kinase activity was identified within the NPC fraction, which phosphorylated a 30 kD nuclear pore protein. Phosphomodification of this nucleoporin, here termed Nup30, was inhibited by apigenin and PD‐98059, two MAP kinase antagonists as well as with SB‐202190, a pharmacological blocker of p38. Furthermore, high throughput profiling of enriched NPCs revealed constitutive presence of all members of the MAP kinase family, extracellular regulated kinases (ERK), p38 and Jun N‐terminal kinase. The NPC thus contains a spectrum of associated MAP kinases that suggests an intimate role for ERK and p38 in regulation of nuclear pore function.     

A GTPase distinct from Ran is involved in nuclear protein import

Signal-dependent transport of proteins into the nucleus is a multi-step process mediated by nuclear pore complexes and cytosolic transport factors. One of the cytosolic factors, Ran, is the only GTPase that has a characterized role in the nuclear import pathway. We have used a mutant form of Ran with altered nucleotide binding specificity to investigate whether any other GTPases are involved in nuclear protein import. D125N Ran (XTP-Ran) binds specifically to xanthosine triphosphate (XTP) and has a greatly reduced affinity for GTP, so it is no longer sensitive to inhibition by nonhydrolyzable analogues of GTP such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). using in vitro transport assays, we have found that nuclear import supported by XTP-Ran is nevertheless inhibited by the addition of non-hydrolyzable GTP analogues. This in conjunction with the properties of the inhibitory effect indicates that at least one additional GTPase is involved in the import process. Initial characterization suggests that the inhibited GTPase plays a direct role in protein import and could be a component of the nuclear pore complex.     

Guanylate cyclase. Existence of different forms and their regulation by nucleotides in calf uterus.

The activity of calf uterus guanylate cyclase (EC 4.6.1.2) exists in at least two and most probably three distinct forms. The cytosolic enzyme exhibits hyperbolic substrate curves with respect to GTP and Mn2+, while the particulate cyclases (nuclear and microsomal)display sigmoidal (GTP) and hyperbolic (Mn2+) relationships. The Hill coefficient for the GTP dependence is 0.9 for the cytosolic, 1.5 for the nuclear, and 1.4 for the microsomal enzyme. The cytosolic enzyme has a Km for GTP of 70 muM while half maximal velocity occurs at 90 and 100 muM GTP for the nuclear and microsomal enzymes, respectively. The Ka for Mn2+ is 0.57, 0.71 or 0.75 mM for the cytosolic, nuclear, or microsomal enzyme, respectively.     

Nuclear proteasomal degradation of Saccharomyces cerevisiae inorganic pyrophosphatase Ipp1p,a nucleocytoplasmic protein whose stability depends on its subcellular localization

Inorganic pyrophosphate (PPi) is an abundant by-product of cellular metabolism. PPi-producing reactions take place in the nucleus concurrently with reactions that use PPi as a substrate. Saccharomyces cerevisiae possesses two soluble pyrophosphatases (sPPases): Ipp1p, an essential and allegedly cytosolic protein, and Ipp2p, a mitochondrial isoenzyme. However, no sPPase has yet been unambiguously described in the nucleus. In vivo studies with fluorescent fusions together with activity and immunodetection analyses demonstrated that Ipp1p is a nucleocytoplasmic protein. Mutagenesis analysis showed that this sPPase possesses a nuclear localization signal which participates in its nuclear targeting. Enforced nucleocytoplasmic targeting by fusion to heterologous nuclear import and export signals caused changes in polypeptide abundance and activity levels, indicating that Ipp1p is less stable in the nucleus that in the cytoplasm. Low nuclear levels of this sPPase are physiologically relevant and may be related to its catalytic activity, since cells expressing a functional nuclear-targeted chimaera showed impaired growth and reduced chronological lifespan, while a nuclear-targeted catalytically inactive protein was not degraded and accumulated in the nucleus. Moreover, nuclear proteasome inhibition stabilized Ipp1p whereas nuclear targeting promoted its ubiquitination and interaction with Ubp3p, a component of the ubiquitin-proteasome system. Overall, our results indicate that Ipp1p is nucleocytoplasmic, that its stability depends on its subcellular localization and that sPPase catalytic competence drives its nuclear degradation through the ubiquitin-proteasome system. This suggests a new scenario for PPi homeostasis where both nucleocytoplasmic transport and nuclear proteasome degradation of the sPPase should contribute to control nuclear levels of this ubiquitous metabolite.     

Phospholipase A2 inhibits nuclear nucleoside triphosphatase activity and mRNA export in isolated nuclei from rat liver

The present study is undertaken to investigate whether the phospholipase A(2) (PLA(2)) influences mRNA nucleocytoplasmic transport evaluated by nucleoside triphosphatase (NTPase) activity and mRNA export in isolated hepatic nuclear envelope. Isolated hepatic nuclei from rat liver were exposed to PLA(2) (10(-5) approximately 10(-2)/ml) with or without incorporation of nuclei with phosphatidylcholine (PC) liposome. Messenger RNA exports and NTPase activities of nuclear membrane were assayed using ATP and GTP as substrates. We found that the RNA efflux, evaluated by [3H] uridine, was potently decreased in a concentration-dependent manner, by incubation of hepatic nuclei with PLA(2), regardless using ATP or GTP as substrates. The PC content in nuclear membrane was also decreased by PLA(2)-treatment. The PC was incorporated into the nuclear membrane by addition of phospholipid liposomes into the incubation mixture. PC incorporation into the nuclear membrane did not alter mRNA export. However this resulted in a significant increase in mRNA export rate in PLA(2)-treated group. Messenger RNA export rate in PLA(2) (10(-3) unit/mL)- treated nuclear membrane was positively correlated with level of PC incorporation, both using ATP and GTP as substrates. The activity of nucleoside triphosphatase, a nuclear membrane-associated enzyme, showed parallel variations with mRNA transport. It is concluded that nuclear PLA(2) plays a regulatory role in RNA transport, which can be antagonized by exogenous PC. These might be pathophysiologically significance, although the mechanisms by which this effect takes place remain to be clarified.     

Mechanisms of receptor-mediated nuclear import and nuclear export

Nuclear transport of proteins and RNA occurs through the nuclear pore complex and is mediated by a superfamily of transport receptors known collectively as karyopherins. Karyopherins bind to their cargoes by recognition of specific nuclear localization signals or nuclear export signals. Transport through the nuclear pore complex is facilitated by transient interactions between the karyopherins and the nuclear pore complex. The interactions of karyopherins with their cargoes are regulated by the Ras-related GTPase Ran. Ran is assisted in this process by proteins that regulate its GTPase cycle and subcellular localization. In this review, we describe several of the major transport pathways that are conserved in higher and lower eukaryotes, with particular emphasis on the role of Ran. We highlight the latest advances in the structure and function of transport receptors and discuss recent examples of steroid hormone receptor import and regulation by signal transduction pathways. Understanding the molecular basis of nuclear transport may provide insight into human diseases by revealing how nucleocytoplasmic trafficking regulates protein activity.     

Facilitated nucleocytoplasmic shuttling of the Ran binding protein RanBP1

The Ran binding protein RanBP1 is localized to the cytosol of interphase cells. A leucine-rich nuclear export signal (NES) near the C terminus of RanBP1 is essential to maintain this distribution. We now show that RanBP1 accumulates in nuclei of cells treated with the export inhibitor, leptomycin B, and collapse of the nucleocytoplasmic Ran:GTP gradient leads to equilibration of RanBP1 across the nuclear envelope. Low temperature prevents nuclear accumulation of RanBP1, suggesting that import does not occur via simple diffusion. Glutathione S-transferase (GST)-RanBP1(1-161), which lacks the NES, accumulates in the nucleus after cytoplasmic microinjection. In permeabilized cells, nuclear accumulation of GST-RanBP1(1-161) requires nuclear Ran:GTP but is not inhibited by a dominant interfering G19V mutant of Ran. Nuclear accumulation is enhanced by addition of exogenous karyopherins/importins or RCC1, both of which also enhance nuclear Ran accumulation. Import correlates with Ran concentration. Remarkably, an E37K mutant of RanBP1 does not import into the nuclei under any conditions tested despite the fact that it can form a ternary complex with Ran and importin beta. These data indicate that RanBP1 translocates through the pores by an active, nonclassical mechanism and requires Ran:GTP for nuclear accumulation. Shuttling of RanBP1 may function to clear nuclear pores of Ran:GTP, to prevent premature release of import cargo from transport receptors.     

Nucleocytoplasmic trafficking of proteins: With or without Ran?

Proteins and RNAs move between the nucleus and cytoplasm by translocation through nuclear pore complexes in the nuclear envelope. To do this, they require specific targeting signals, energy, and a cellular apparatus that catalyzes their transport. Several of the factors involved in nucleocytoplasmic trafficking of proteins have been identified and characterized in some detail. The emerging picture for nuclear transport proposes a central role for the small GTPase Ran and proteins with which it interacts. In particular, asymmetric distribution of these proteins between nucleus and cytoplasm appears to be responsible for the vectorial nature of nucleocytoplasmic transport. Here, we summarize the role of Ran and Ran-binding proteins in nuclear trafficking of proteins with classical nuclear localisation signals. We also discuss examples of the growing number of alternative pathways that are involved in transport of proteins across the nuclear envelope. BioEssays 21:579–589, 1999. © 1999 John Wiley & Sons, Inc.     

Calcium influx mediated by nicotinic receptors and voltage sensitive calcium channels in SK-N-SH human neuroblastoma cells

When SK-N-SH human neuroblastoma cells were exposed to nicotine (NIC) or KCl they showed a dose-dependent transient increase (2- to 4-fold) in intracellular Ca2+ concentration ([Ca2+])i as detected by quin-2 fluorescence, with half maximal effects (EC50) observed at 13 microM and 26 mM, respectively. Tubocurarine and 1-isodihydrohistrionicotoxin potently blocked the NIC-evoked (IC50 congruent to 1 microM and 0.3 microM, respectively), but not the high [K+]o-evoked [Ca2+]i accumulation. The KCl-induced response was inhibited by verapamil and diltiazem (IC50 = 1.4 and 10.9 microM, respectively). Tetrodotoxin (3 microM) and tetraethylammonium (10 microM) had no effect on [Ca2+]i accumulation induced by either agent. Increases in [Ca2+]i could be evoked sequentially by NIC and KCl in the same cells suggesting independent mechanisms of Ca2+ entry. In a Ca2+-free medium, no response to either KCl or NIC was observed. However, when Ca2+ ions were restored, [Ca2+]i accumulation was enhanced to the same extent as cells suspended in a Ca2+-containing buffer. Long-term (18 hr) pretreatment of SK-N-SH cells with pertussis (100 ng/ml) or cholera toxins (10 nM) had no effect on NIC or KCl-induced [Ca2+]i accumulation. Together, these data demonstrate the presence of NIC receptors and voltage-sensitive Ca2+ channels on SK-N-SH neuroblastoma cells, through which [Ca2+]i may be modulated.     

Sphingosylphosphorylcholine induces Ca(2+)-sensitization of vascular smooth muscle contraction: possible involvement of rho-kinase

Sphingosylphosphorylcholine (SPC), a sphingolipid, concentration-dependently (1-50 microM) induced contraction and slight elevation of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in smooth muscle of the pig coronary artery, the result being a marked increase in the force/[Ca(2+)](i) ratio. In alpha-toxin- or beta-escin-permeabilized, but not Triton X-100-permeabilized, vascular strips, SPC induced contraction at constant [Ca(2+)](i) (pCa 6.3) in the absence of GTP, whereas a G-protein-coupled receptor agonist, histamine, required the presence of GTP to induce the contraction. The Rho-kinase blocker, Y-27632 (10 microM) abolished the SPC-induced Ca(2+)-sensitization, without affecting the Ca(2+)-induced contraction. These results suggest that SPC induces Ca(2+)-sensitization of force in vascular smooth muscle, presumably through the activation of Rho-kinase (or a related kinase).