Patch-clamp detection of macromolecular translocation along nuclear pores · 2003-04-07 · 335 Braz J Med Biol Res 31(3) 1998 Patch-clamping macromolecular translocation along nuclear

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Braz J Med Biol Res 31(3) 1998

Patch-clamping macromolecular translocation along nuclear poresBrazilian Journal of Medical and Biological Research (1998) 31: 333-354ISSN 0100-879X

Patch-clamp detection ofmacromolecular translocationalong nuclear pores

Departamento de Fisiologia, Faculdade de Medicina de Ribeirão Preto,Universidade de São Paulo, Ribeirão Preto, SP, Brasil

J.O. Bustamanteand W.A. Varanda

Abstract

The present paper reviews the application of patch-clamp principles tothe detection and measurement of macromolecular translocation alongthe nuclear pores. We demonstrate that the tight-seal ‘gigaseal’ be-tween the pipette tip and the nuclear membrane is possible in thepresence of fully operational nuclear pores. We show that the ability toform a gigaseal in nucleus-attached configurations does not mean thatonly the activity of channels from the outer membrane of the nuclearenvelope can be detected. Instead, we show that, in the presence offully operational nuclear pores, it is likely that the large-conductanceion channel activity recorded derives from the nuclear pores. Weconclude the technical section with the suggestion that the best way todemonstrate that the nuclear pores are responsible for ion channelactivity is by showing with fluorescence microscopy the nucleartranslocation of ions and small molecules and the exclusion of thesame from the cisterna enclosed by the two membranes of the enve-lope. Since transcription factors and mRNAs, two major groups ofnuclear macromolecules, use nuclear pores to enter and exit thenucleus and play essential roles in the control of gene activity andexpression, this review should be useful to cell and molecular biolo-gists interested in understanding how patch-clamp can be used toquantitate the translocation of such macromolecules into and out ofthe nucleus.

CorrespondenceJ.O. Bustamante

Departamento de Fisiologia

FMRP, USP

Av. Bandeirantes, 3900

14049-900 Ribeirão Preto, SP

Brasil

Fax: 55 (016) 633-0017

E-mail: [email protected] and

[email protected]

Presented at the XII Annual Meeting

of the Federação de Sociedades de

Biologia Experimental, Caxambu,

MG, Brasil, August 27-30, 1997.

J.O. Bustamante and W.A. Varanda

were recipients of grants from

MRC, Canada and FAPESP.

Received August 8, 1997

Accepted November 11, 1997

Key words• Nuclear pores• Nuclear envelope• Cell nucleus• Nucleocytoplasmic transport• Macromolecular transport• Gene activity• Gene expression• Transcriptional control• Ion channels• Patch-clamp• Measurement

Introduction

In the present review we attempt to ex-plain how the patch-clamp technique can beused to detect and measure macromoleculartranslocation (MMT) by using both qualita-tive and quantitative analysis of gated ionflow along the nuclear pore complex (NPC).In doing so, we hope that the concepts usedcan be extrapolated to other systems such asthose of channels from other organellar mem-branes. The mechanisms of NPC-mediated

MMT (NPC-MMT) have been mostly stud-ied by non-electrophysiologists. For this rea-son, we have made an effort to provide assis-tance to those less familiar with patch-clampconcepts and procedures. At the same time,because patch-clamp and other ion channelspecialists are not familiar with NPC-MMT,we pay attention to the essential non-electro-physiological ideas of NPC-MMT. Throughthis exercise, we hope to bring together thesedistant fields of investigations by facilitatingthe understanding by a wide audience of how

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patch-clamp recording of the large-conduc-tance ion channel activity at the nuclearenvelope (NE) represents gating of the NPCchannel (NPCC, the central NPC hole) andhow this measurement can be used to inves-tigate NPC-MMT. We hope that this balanc-ing-act, tight-rope approach, although trivialat times for one or another group of scientists(i.e., electrophysiologists or cell biologists),contributes to the development of new meth-odologies for the study of the mechanisms ofgene control by imported transcription fac-tors and of gene expression by the export ofmessenger RNAs (mRNAs). Both of theseprocesses are relevant to the maintenance ofnormal cell function and to the developmentof various cellular pathologies and pro-grammed cell death. Space limitations re-strict the number of publications cited in thepresent review. For this reason, we refer thereader to further reading on these special-ized areas.

Nuclear pore ion channel behavior

Ion channels and ion channel activity

Here we review some basic conceptson ion channels. The more demandingreader may consult other sources (e.g., 1).The simplest channel system is one thatswitches in a binary scheme between twostates: open and closed, with values of, say,1 and 0 (picoamperes, picosiemens, etc.).In the steady state, this simple two-statesystem exhibits open and closed probabili-

ties, po and pc, such that:

po + pc = 1 (Eq. 1)

Although a popular term, ion channelactivity has both qualitative and quantitativeconnotations. Channel activity can be takento be the set of all those parameters whichdetermine the observable or measured quan-tity which, in patch-clamp, is the time-de-pendent electrical current, i(t), carried out bythe ions moving along the channel. At eachpoint in time, the current will be determinedby the value of the conductance, γ:

i(V,t) = γ (V,t) V(t) (Eq. 2)

where the parentheses next to the ‘depend-ent’ variables indicate that the variable is afunction of the ‘independent’ variables (e.g.,V(t) indicates that V is a function of t).

In a first, gross approximation, if wethink of the channel as a hollow cylinder,then the channel conductance is related tothe cylinder geometry and electrolyte resis-tivity, ρ,

γ = A/(lρ) or γ = πr 2/(lρ) (Eq. 3)

where A is the cross-sectional area of thecylinder and r and l are the radius and lengthof the cylinder. Equation 3 summarizes therelationship between conductance, area,length and resistivity of the channel hole.The wider and shorter the channel, the largerthe conductance. But, what equation 3 doesnot describe is the gating mechanism, whichcould be represented as a multiplying termwhose values are either 0 or 1. We could use

Abbreviations and acronyms:AP-1: activator protein-1, a transcription factor complex

consisting of c-Fos and c-JunAP-1/c-Jun: the c-Jun component of AP-1ATP and GTP: adenosine and guanosine triphosphatecAMP: cyclic adenosine monophosphateCFTR: cystic fibrosis transmembrane conductance regulatorB-PE and FITC: B-phycoerythrin and fluorescein

isothiocyanate; two fluorescent probesINM and ONM: inner and outer nuclear membranes of the

nuclear envelopeIP3: inositol 1,4,5-trisphosphatemAb414: monoclonal antibody 414; raised against the

nuclear pore

MMT: macromolecular translocationNE: nuclear envelopeNF-κB: the transcription factor named nuclear factor kappa BNLS: nuclear localization signalNPC and NPCC: nuclear pore complex and its central

channel or holeNPC-MMT: NPC-mediated MMTPKA: cAMP-dependent protein kinaseRan/TC4: a small GTPase involved in NPC-MMTRNA and mRNA: ribonucleic acid and messenger RNASP1: a transcription factorTATA: a deoxynucleotide sequence used here in connection

to the transcription factor that binds to it: the TATA-bindingprotein

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Patch-clamping macromolecular translocation along nuclear pores

a time-dependent Dirac’s delta function, δ =δ(t), widely used to represent 0 everywhereexcept at a single point (which we envisionas that representing the proper gating condi-tions):

γ = δ(t) [A/(lρ)] or γ = δ(t) [πr 2/(lρ)] (Eq. 4)

In a random, stochastic system such as anion channel switching between the open andclosed states, the macroscopic behavior ofthe system is represented by time averagequantities which give a measure of how themacroscopic system (the collection of single,microscopic units) works. For example, thetime average channel conductance, <γ>would be given by:

<γ> = <δ(t)> [A/(lρ)] or<γ> = <δ(t)> [πr 2/(lρ)] (Eq. 5)

which can also be written as:

<γ> = po [A/(lρ)] or <γ> = po [πr 2/(lρ)] (Eq. 6)

Thus, the average ion current can be writtenas:

<i(V)> = <γ> V (Eq. 7)

where V = V(t) is the applied voltage, whichin patch-clamp is most of the time conve-niently set to be constant. If po, and V, as wellas channel geometry and resistivity of envi-ronment (i.e., ρ), do not change with time orvoltage, then:

<i(t)> = po γ V (Eq. 8)

Together, these microscopic and macro-scopic parameters give a measure of channel

activity, function and behavior. The morefrequently the channel is open (i.e., the higherthe po), and/or the greater the conductanceand voltage, γ and V, the greater the value ofthe average current, <i>. Here lies the powerof patch-clamp. While patch-clamp allowsprecise determination of the parameters thatdetermine <i> changes, macroscopic meas-urements (such as whole-nucleus voltage-clamp which detects whole nucleus ion cur-rent or fluorescence microscopy which de-tects integrated changes in probe concentra-tion) do not permit such determination be-cause a larger value of <i> can be obtainedwith either greater po, γ, or V, individually.

Figure 1 illustrates an equivalent circuitfor an organellar ion channel with negligiblereversal potential (Vrev, the value of voltageat which the ion current reverses direction).The channel components are delimited bythe rectangular box formed by the discontin-ued line. The channel is represented by aresistor or conductor (i.e., the vertical, solidline rectangular box represents the channelcylinder) plus a gating mechanism (a switch).The organelle electrical potential, Vorganelle,is represented by the pair of thin-and-thickhorizontal lines near the bottom. The voltagein the system is measured with reference tothe bath potential, Vreference or Vbath (repre-sented as the thick horizontal line at thebottom). The pipette potential, Vpipette, is ap-plied at one side of the channel. The meas-ured quantity, the pipette current, ipipette, isgiven by:

ipipette = γ (Vpipette - Vorganelle) (Eq. 9)

Variables:i and γ : current and conductance of a single ion channel;

microscopic variablesI and Γ : current and conductance of a channel population;

macroscopic variablesN: number of functional units in the ion channel populationpo and pc: open and close probabilities of an ion channelPo and Pc: open and close probabilities of the ion channel

populationτ : time constant of decayR, V and t : electrical resistance, voltage and timeVrev: reversal potential, voltage at which the ion current

changes direction

A, r, l and ρ: cross-sectional area, radius, length and resistivity of a cylinder-like channel

ncarrier and ninert: number of electrical charge carriers andinert particles inside the NPCC

vNPCC: NPCC volumevcarrier and vinert: volume occupied by the electrical charge

carriers and inert particles inside the NPCCρcharge: electrical charge density per unit timeXcarrier and Xinert: generic designation of the electrical

charge carriers and inert particles

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Essential features of the NPC

The NPC is the only direct pathway con-necting nucleus and cytoplasm (e.g., 2-6).Under electron microscopy, the NPC is oneof the most conspicuous features of the NE(e.g., 7,8). The NPC stands out as the mostprominent structure in topological imagingof the NE with scanning electron micros-copy (e.g., 9) and atomic force microscopy(e.g., 10). This supramolecular structure con-sists of many proteins which together have amass estimated at about 124 MDa (megadal-tons, e.g., 8). Figure 2 shows a 3-D recon-struction model generated with electron mi-croscopy data (from Ref. 8). The figure dis-plays the putative plug in the center in afuzzy, transparent fashion to indicate thatthe concept of the plug is not clear (8).Cytoplasmic filaments (vertical rod-like fila-ments) as well as nucleoplasmic ones (con-vergent at the bottom) are shown. The NPCspans the two membranes of the NE: theouter and the inner nuclear membranes (ONMand INM, respectively). This feature is es-

sential to the electrophysiological discus-sion that will follow. The space delimited byONM and INM is a cisterna used by manyprocesses to store proteins, molecules, etc. Itis there that Ca2+ is thought to mediate NPC-MMT (e.g., 11,12). Note that the outer mem-brane of the NE is contiguous with the roughendoplasmic reticulum. Therefore, the ONMand rough endoplasmic reticulum, althoughdistinct structures, share many properties.This contiguity may cause arguments whenit comes to identifying the source of ionchannel activity. Therefore, it is not suffi-cient to simply place the patch-clamp pipetteat the outside of the nucleus but one is re-quired to prove that the activity is not de-rived from the rough endoplasmic reticu-lum. In a recent paper, it was shown that thelarge-conductance ion channel activity re-corded in cardiac myocyte nucleus-attachedpatches does not derive from the endoplas-mic reticulum (13). That this was the casewas demonstrated by the inefficacy of vari-ous maneuvers known to affect protein-con-ducting channels of the endoplasmic reticu-lum (14).

The patent diameter of the NPC

Many electron and fluorescence micros-copy studies indicate that small molecules,ions and particles placed on one side of theNE reach the other side (e.g., 7,15,16). Theseobservations have been interpreted to meanthat the NPCs have a ‘patent’ diameter (asort of effective cross-section for the electro-lyte-filled hole) of about 10 nm. The value ofthe patent diameter can vary from a few toseveral tens of nanometers according to thecell type and cycle (e.g., 7,16). This largepatent diameter leads to the inescapable con-clusion that monoatomic ions flow freelyalong the NPCs and, consequently, that anyobserved heterogeneous concentration dis-tribution of small ions and molecules mustresult from ‘cytoplasmic exclusion’ (17)caused by the compartmentalized distribu-

Figure 1 - Equivalent electricalcircuit for an organelle ion chan-nel. The model is drawn for anorganelle with a simple mem-brane (vis à vis a dual membranelike the nuclear envelope). Thechannel itself is delimited by thediscontinued line. The channelis represented by a resistor orconductor (i.e., the rectangularbox represents the channel cyl-inder) plus a gating mechanism(a switch). Allowance has beenmade for organelle electrical po-tential, Vorganelle (represented bythe pair of thin-and-thick horizon-tal lines near the bottom). Thevoltage in the system is meas-ured with reference to the bathpotential, Vreference (representedas the thick horizontal line at thebottom). The pipette potential,Vpipette, is applied to one side ofthe channel and the resultingcurrent, ipipette, measured. Thereversal potential, Vrev, for theion current has been assumednegligible (see 31).

ipip

ett

e

Vorganelle

Vreference

Channelgatingmechanism

Channelconductor

Channel

Vpipette

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tion of water- and molecular-binding sites(e.g., 7,15,18) from another (or more) non-NE mechanism(s), or from technical arti-facts. The ‘cytoplasmic exclusion’ principlestates that because the state of water is dif-ferent in the different cellular compartments,the solubility, and thus the concentration, ofa solute is compartmentalized (in our case,between the nucleus and cytoplasm). Thisexclusion principle resembles the ‘associa-tion-induction hypothesis’ introduced at thesame time to explain the resting potential ofcells without the need for a plasmalemmalNa-K pump (e.g., 19,20). A discussion ofnuclear resting potentials is outside the scopeof this review.

Agreement between patch-clamp andnon-electrophysiological data

The patent diameter view of unrestrictedflow of ions along the NPCC is in apparentcontradiction with the electrophysiologicaldata indicating that the NPC behaves as anion channel (e.g., 13,21-24). However, thisdiscrepancy is eliminated when both qualita-tive and quantitative analyses are performed(15). Qualitatively, at the present time onlythe patch-clamp is capable of detecting livesingle NPC conformational changes throughthe measurement of NPCC ion-conductiveproperties. Not even the most sophisticatedelectron or light microscope techniques arecapable of giving a measurement of the livefunction of a single NPC and, although atomicforce microscopy and other scanning probemicroscopies hold this promise, reports haveyet to appear with the time-resolution re-quired. All we can say with any kind ofmicroscopy is that the particles have movedfrom one side to the other within an approxi-mate time interval of minutes, whereas withthe patch-clamp we can have time resolutiondown to the millisecond range or even less.Thus, we can say that 10-kDa dextran mol-ecules have passed from the outside of thenucleus to the inside within, say, 15 min, but,

in physical terms, this does not mean that theNPCC is permanently open as commonlyconcluded. What patch-clamp measurementsshow is that the NPC gates behave like anyother gate controlling the traffic of largequantities. Quantitatively, one may neglectparticle-particle and particle-NPCC wall in-teractions, and simply use the geometricalparameters reported for the NPCC to esti-mate the single channel conductance of theNPCC, γNPCC. Table 1 gives the values ofγNPCC assuming that the hydrated radius, aion,of the current carrying ions (e.g., K+) is 0.2nm (15), and that the NPC has a hole of 3-10-nm radius, rNPCC, 25-100-nm length, lNPCC,(to accommodate published data; see 8,15)and filled with an electric conductor (theelectrolyte) of specific resistivity, ρ, of 100-250 Ω.cm (around that of cytoplasm andnucleoplasm, plus or minus small inert con-taminants, not medium-sized or larger mac-romolecules). The values are calculated whenthe resistance of each NPCC, RNPCC, is ap-proximated by the following equation (15):

RNPCC = ρ [lNPCC + (1.64 rNPCC)]/ π(rNPCC)2

[1-(aion/rNPCC)]6 (Eq. 10)

Table 1 shows examples of RNPCC valuescalculated with equation 10 and covers pub-lished values (25-29). In consideration of theNPC variation with cell type and cell cyclephase as well as the measurement errors(instrument, sample preparation, etc.), wecan say that there is good agreement be-tween electrophysiological and non-electro-

Figure 2 - 3D-Reconstructionmodel of the nuclear pore com-plex (NPC) generated from elec-tron microscopy data. Cyto- andnucleoplasmic filaments areshown pointing to their respec-tive compartments. The outerand inner nuclear membranes(ONM and INM, respectively) ofthe nuclear envelope (NE) cre-ate a space which serves as cis-ternae for the storage of impor-tant factors, one of which isCa2+. Note that the putativecentral plug is shaded to indi-cate the uncertain nature of theconcept. (From Ref. 5, with per-mission).

Cytoplasm

ONM

INM

Nucleoplasm

Cisternae

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physiological data. For example, avian eryth-rocytes display a channel activity with acationic channel conductance of 800 pS (23),coconut endosperm cells with one of about1,000 pS (30), and murine cardiac myocytesshow conductance values between 106 and532 pS (22-36oC), with higher values corre-sponding to temperatures closer to that ofthis warm-blooded species (31). This tem-perature dependence of γNPCC demonstratesthe complexity of the biochemical processesinvolved in NPCC gating (e.g., ATPases andGTPases). Reports of nuclear ion channelswhich could be attributed to NPCCs havebeen published (e.g., 23,24,30,32). How-ever, these did not include demonstrationsthat the NPCCs were either functional orpermanently closed, blocked or plugged.Other ion channels which are putatively not-NPCCs have also appeared. These concludethat the recorded channel activity can beattributed to Ca2+-activated K+ channels (33;see also 34), IP3-sensitive Ca2+-channels (35-37; see also 34) and even now the Cl--channel known as the cystic fibrosis trans-membrane conductance regulator or CFTR(38). Despite the assertion by the authorsthat these channels were not NPCCs, noproof was given that the NPCs were closedor plugged. We will discuss the importanceof providing such proof in the followingparagraphs (see section Identification ofNPCs as a source of ion channel activity).

Patch-clamp principles

The patch-clamp demonstration that theNPCC opens probabilistically (13) is con-sistent with non-electrophysiological obser-vations. Briefly, the NPCCs switch betweenopen and closed states with open and closeprobabilities (po and pc, respectively). Theseprobabilities are regulated by cytosolic andnucleosolic factors such as GTPases, andlocalization signal receptors (e.g., 13,25,26).Therefore, despite the large diameter of theNPCC, the time average of its opening is lessthan the geometrical diameter of the NPChole (what we are calling here the NPCC)unless, of course, po is 1. As a corollary,conditions that favor the closed state willreduce the average nucleocytoplasmic ionflow. The more the NPCC dwells in eitherthe closed or the plugged state (where ionflow/current is zero), the lower the po and thesmaller the macroscopic ion current. A re-duced macroscopic NE ion current favorsthe build-up of nucleocytoplasmic gradientsof small ions, molecules and particles. Suchgeneration of nucleocytoplasmic gradients(e.g., during Ca2+ transients, see 39) lendthemselves to be misinterpreted as a reduceddiameter of the NPCC but, instead, they area reflection of the lower than unity openprobability (i.e., po<1).

Early patch-clamp investigations sug-gested that ion channel activity recordedfrom the NE was attributable to NPCs (e.g.,23,24,30-32,40). In pure saline solutionscontaining high [K+], these NPCC candi-dates have a linear current-voltage relation-ship and, therefore, they have constant singlechannel conductance, γNPCC (e.g., 23,24,30-32,40). To date, only the large-conductancecardiac channel activity has been directlydemonstrated to derive from NPCs (13; seebelow). Like some of the NE channels inother nuclear preparations, these cardiacchannels show a preference for cations (40).Cardiac myocyte NPCCs display two modesof operation labeled ‘stationary’ and ‘inacti-

Table 1 - NPCC conductance, γNPCC, calculated from equation 10 with the ion-conductive hole parameters: length (lNPCC), radius (rNPCC) and resistivity (ρ).

Parameter γNPCC RNPCC ρ lNPCC rNPCC aionchanged (pS) (GΩ) (Ω.cm) (nm) (nm) (nm)

lNPCC 451 2.2 100 100 4.5 0.2lNPCC 844 1.2 100 50 4.5 0.2lNPCC 1496 0.7 100 25 4.5 0.2rNPCC 1852 0.5 100 25 5 0.2rNPCC 1171 0.9 100 25 4 0.2rNPCC 625 1.6 100 25 3 0.2ρ 416 2.4 150 25 3 0.2ρ 312 3.2 200 25 3 0.2ρ 250 4.0 250 25 3 0.2

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vating’ (31). This behavior has been ob-served also in hepatocytes (41). In the ‘sta-tionary’ or steady-state mode, the NPCCsopen and close with a binomial behaviorsimilar to an electron spin ensemble. That is,not only do all the NPCCs appear to beidentical but they also switch between theopen and closed states with open probabili-ties which are well fitted by a binomial mo-del:

Po(n) = N! pon (1-po)N-n/[n! (N-n)] (Eq. 11)

where Po(n) is the probability of finding nchannels simultaneously open, po is, as be-fore, the single channel open probability,and N is the total number of ion-conductingchannels in the patch (31). Note that equa-tion 2 does not contain reference to time.This is because the system is in steady stateand its stochastic characteristics are the sameat any given time. Also note that, as indi-cated at the beginning of this review, for aperfect binomial system, po + pc = 1. In the‘inactivating’ mode of operation, NPCCsrespond to voltage stimulation (equivalent toelectrochemical gradient stimulation; see Ref.42) with an exponential decay in Po(n). Thatis, Po(n) is time-dependent.

Po(n,t) = Po(n,0) e-t/τ (Eq. 12)

with t being time - the independent variable,τ the decay time constant, and Po(n,0) theinitial value of Po. Note that Po does notnecessarily follow the binomial behaviordescribed in equation 10. The term inactivat-ing was assigned to this mode of operationbecause there are instances where the chan-nels can no longer open upon successiveapplication of voltage pulses but require atime for recovery (31). This time, althoughnot always the same, is equivalent to therefractory period of nerves associated withsodium channel inactivation (see 42). Thelack of constancy of the refractory periodmay be indicative of the much greater com-plexity of the NPCC mechanism of opera-tion. For example, GTPase may play an im-

portant role in the inactivating machinery(see 40). Similarly to classical inactivation,the time constants for this putative inactiva-tion are reduced with voltage (see 42). Atvoltages between -10 mV and +10 mV, car-diac NPCCs showed no measurable inacti-vation: they were open all the time (31). Thissuggests that, at least in quiescent, non-stim-ulated cardiac myocytes (where the reversalpotential is negligible), the NPCCs offer littleopposition to ion flow. However, when thevoltage gradient across the NE increased(i.e., increase in electrochemical gradientacross the NE), the probability of findingopen channels decreased. This dependenceof NPCC gating on voltage (electrochemicalgradient) is consistent with the fluorescencemicroscopy observation that the NE becomesa Ca2+-barrier when the cytosolic [Ca2+] in-creases over 300 nM (39). This can also bedue to Ca2+-induced Ca2+ release from theNE cisternae which, in turn, leads to NPCCplugging by macromolecules (see below).Since live cardiac myocytes in situ are neverat rest, it is likely that NPCCs play a signifi-cant role in determining the signals that crossto one or the other side of the NE.

Putative NPCC substates of 25-50 pSwere reported for cardiac myocytes (31,40,43). However, it is not clear whetherthese may represent NPCs whose holes havebeen partially occluded by medium-sizedmolecules (see below). Cardiac myocyteNPCC activity was enhanced by cAMP-de-pendent protein kinase (PKA) and reducedby a PKA inhibitor (31). It was also shownthat their activity could be suppressed withGTP-γ-S, a non-hydrolyzing analog of GTP,suggesting a direct involvement of GTPasein NPCC gating (40). It is unclear how thisphenomenon correlates with the essentialrole that the small GTPase Ran/TC4 plays inMMT (e.g., 2-6,44). The current availabilityof methods for the isolation of NPCs (e.g.,45) should make it possible in the future todissect the single NPCC gating characteris-tics in artificial lipid bilayer systems.

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Basic concepts in the identification of NPCsas a source of ion channel activity

The idea that NE ion channel activityderives from NPCCs is very appealing fromthe electrophysiological point of view and,indeed, efforts have been made towards dem-onstrating that this activity derives fromNPCCs (e.g., Innocenti and Mazzanti (46)used osmotic shock). However, patch-clampinvestigations complemented with laser-scan-ning confocal fluorescence microscopy,transmission electron microscopy, field-emis-sion scanning electron microscopy, andatomic force microscopy showed a directcorrelation between the large-conductanceion channel activity recorded from cardiacmyocytes and myocyte NPCs (13). Both inthe classical microelectrode studies ofLoewenstein and coworkers (reviewed in47) and in the first patch-clamp investiga-tions of NE ion channel activity (23,24), itwas proposed that the NPC was the structureunderlying the recorded activity. However,this proposition was hindered by patch-clampinvestigations indicating that ion channelsdifferent from NPCCs also exist in either ofthe two membranes delimiting the NE. Suchis the case for standard and CFTR chloridechannels (38,48), for Ca2+-sensitive K+ chan-nels (33), for Ca2+- and Zn2+-channels (49,see 34), IP3-sensitive Ca2+-channels (34-37),etc. (50). That non-NPCCs do exist at the NEis further suggested by the demonstrationthat ion channel-forming proteins have beenisolated from NE preparations (e.g., 34,51).Indeed, even a chloride ion channel proteinwhich localizes primarily in the nucleus hasbeen cloned and characterized (e.g., 52).Despite the non-NPCCs identified or yet tobe identified at the NE, the NPCCs continueto stand out as the sole structures providingthe direct pathway for the exchange of mate-rial between the nuclear and cytoplasmiccompartments. For this reason, when thepatch-clamp technique is used in the nucleus-attached mode (i.e., the pipette tip forming a

gigaseal with the outer membrane of theNE), and when it is demonstrated that smalland medium-sized (e.g., <10 kDa) moleculesenter the nucleus, then it must be unavoid-ably concluded that the NPCCs are effectiveelectrical shunts for any non-NPCCs be-tween the recording patch-clamp pipette andbath electrodes (note that all non-NPPCs arelocated on either membrane of the NEwhereas the NPCCs join the two membranes).Therefore, from an electrophysiological pointof view, NPCCs should be the natural candi-dates for the source of large conductancechannel activity. Figure 3 aims at explainingthese concepts with the equivalent circuit.For the sake of clarity, we will only discussthe nucleus-attached patch-clamp mode ofrecording. Figure 3 summarizes the threegroups of channels found in the NE prepara-tion. They are the NPCCs and the outer andthe inner nuclear membrane channels(ONMC, and INMC, respectively). Innucleus-attached patches, the ONM and theINM can be thought of as two membranes inseries. Since the pipette tip only touches theONM, only the pipette voltage (Vpipette) isapplied to the NPCCs and the ONMCs. Thevoltage of the NE cisternae (VNE-cisternae) doesnot change appreciably and, consequently,there is no voltage drop across the INM andthe INMCs. The NPCCs and ONMCs form asystem of parallel conductors through whichthe measured pipette current (ipipette) is dis-tributed into essentially two branches: theNPCC current (iNPCC) and the ONMC cur-rent (iONMC). That is: ipipette = iNPCC + iONMC.Each current component is determined bythe value of the conductance of the corre-sponding channel and the voltage applied tothe channel. In cardiac myocytes, Vnucleus isnegligible in buffered 150 mM KCl (31).That is, the nuclear interior is short-circuitedto the bath. Thus, Vnucleus equals the refer-ence or bath voltage (Vreference). For this rea-son, the voltage sensed by the NPCCs andthe ONMCs equals Vpipette.

Since the density of NPCs is relatively

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high (e.g., 7,16), the patch-clamp pipettealways contains a few or more NPCCs. Fur-thermore, because inside the patch there aremany NPCCs with γNPCC of several hundredsof pS, it is clear that NPCCs are the majorcontributors to the recorded ion channel activ-ity. Therefore, the only available explana-tion against the NPCCs being responsiblefor the observed ion channel activity is thatthe NPCCs are permanently closed (orplugged) due to the conditions used in patch-clamp studies (e.g., saline not supplementedwith MMT substrates). This counter-argu-ment remains to be proven. Furthermore,fluorescence microscopy data clearly dem-onstrate that under saline conditions thatprevent MMT, the small monoatomic ionsand molecules diffuse freely between thenucleus and cytosol (e.g., 53). That is, evenin the most unfavorable conditions for MMT,the NPCs still allow ion transport. There-fore, a corollary of this discussion is that anyreport claiming non-NPCC activity shouldbe accompanied by a direct demonstration

that the NPCCs are either closed, plugged orboth.

Peripheral channels of the NPC

Only one computer-assisted 3-D recon-struction work, based on electron micros-copy data, shows that the eight-fold geom-etry of the NPC contains eight parallel path-ways for the transport of ions which areindependent of the large central channel ofthe NPC (54). If these pathways behavedlike ion channels, then one should see tens tohundreds of channels in a single nucleus-attached patch because the NPC density perunit area ranges from a few NPCs to tens ofNPCs per square micrometer or several chan-nels with identical substates. However, thisobservation has never been reported and the3-D reconstruction work has not been dupli-cated. Nevertheless, it should be noted thatefforts to find a large-conductance ion chan-nel which shows eight substates have beenrecently published (41). Therefore, the only

Figure 3 - Equivalent circuit for the ion channels at the nuclear envelope (NE). A, Schematics of the nucleus-attached patch-clamp pipette system. B, Equivalent circuit. Three groups of channels are represented: NPCchannels (NPCC), outer nuclear membrane channels (ONMC), and inner nuclear membrane channels (INMC).Under nucleus-attached patch-clamp configuration, the NPCCs and ONMCs form a system of parallel conductorsthrough which the measured pipette current (ipipette) is distributed: the NPCC current (iNPCC) and the ONMCcurrent (iONMC). There is no contribution of INMC current (iINMC). That is: ipipette = iNPCC + iONMC. Each currentcomponent is determined by the value of the conductance of the corresponding channel and the voltage applied tothe channel. The applied voltage for NPCCs equals the difference between the pipette and nucleus voltages(Vpipette - Vnucleus). The applied voltage for ONMCs and INMCs is (Vpipette - VNE-cisternae) and (VNE-cisternae -Vnucleus), respectively, where VNE-cisternae is the voltage at the NE cisternae. That large inert particles (e.g., colloidalgold and spherical dendrimers) go into the nuclear interior but not into the NE cisternae which indicates that theONMC conductance is much smaller than γNPCC. Vrev is assumed negligible.

A BPatch-clampamplifier

ipipette

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remaining possibility is that these pathwaysare not ion channels in the electrophysi-ological sense but, instead, permanently openperipheral holes as reported in the electronmicroscopy studies (54). However, if thiswere the case then the baseline current inpatch-clamp recordings should be muchgreater than the zero value reported for allpatch-clamp work. A simple computationdemonstrates this fact:

ipipette = iNPCC + iperipheral = γNPCC Vpipette +8γperipheral Vpipette (Eq. 13)

where the measured pipette current equalsthe sum of the current through the NPCC andthe current through the peripheral channels(indicated by the subscripts). γNPCC and γpe-

ripheral are the conductance values for theNPCC and one peripheral channel, respec-tively. Figure 4 shows the equivalent circuitfor this concept. The measured pipette cur-rent, ipipette, is the sum of the currents flowingthrough the central and peripheral channels.That is, ipipette = iNPCC + Niperipheral, where N isthe number of peripheral channels (8 in mostcases, see Ref. 8). The putative peripheralchannels should cause a baseline current tobe recorded. However, patch-clamp investi-

gations have reported no such current, sug-gesting that the peripheral channels do nothave access to the cytosol.

Technical issues

A question commonly asked is whether itis possible to attain a gigaseal in prepara-tions that have functional NPCs. In otherwords, do open NPCCs prevent gigaseal for-mation? The answer is no, they do not. Anessential condition that must be technicallyattained prior to patch-clamp recording isthe formation of the tight seal between thepipette tip and the membrane surface (in ourcase, the outer NE membrane). This seal has(or must have for a successful recording tobe made) an electrical resistance, Rseal, greaterthan 1 GΩ and, for this reason, it is called thegigaseal. The gigaseal is assessed indirectlyby reading the current flowing through thepipette, ipipette, elicited with small currentpulses (resulting from the application of smallvoltage pulses to the pipette electrode). If themembrane contains many open NPCCs, it islikely that the investigator may think that agigaseal has not been reached. However, thisinterpretation is erroneous. Say that underthe pipette tip there are 10 permanently openNPCCs and that their single channel conduc-tance, γNPCC, is 500 pS. This would cause thepipette tip to see a patch conductance notlower than 5,000 pS or 5 nS. This is equiva-lent to 0.2 GΩ. The investigator could mis-takenly take this to suggest that a gigasealhas not been formed. However, the fact isthat the seal resistance between the rim ofthe pipette tip and the outer NE membrane,Rseal, cannot be measured directly with thepatch-clamp technique. All that can be saidis that the measured resistance, Rmeasured, isnot greater than that of either the NE patchdirectly under the pipette tip, Rpatch, or theseal resistance, Rseal. The relationship amongthese three quantities is:

1/Rmeasured = (1/Rpatch) + (1/Rseal) (Eq. 14)

Figure 4 - Equivalent circuit forthe proposed NPC systemformed by the central and pe-ripheral channels. The measuredpipette current, ipipette, is splitinto the current for the centraland peripheral channels (iNPCCand iperipheral, respectively). Thatis, ipipette = iNPCC + Niperipheral,where N is the number of pe-ripheral channels (8 most of thetimes, see Ref. 8). The peripher-al channels should cause abaseline current to be recordedin nucleus-attached experi-ments. However, this is yet tobe reported, suggesting that theperipheral channels have no ac-cess to the cytosol (i.e., the sidewhere the pipette is attached).Vrev is assumed to be negligible.

Per

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Note that the contribution of other resis-tances (e.g., pipette, bath) are negligibleand, thus, ignored in this discussion. There-fore, one can have a gigaseal and yet have apatch of NE containing many NPCs. Thevalue of the gigaseal is revealed during mo-ments of simultaneous NPCC closure ob-served during prolonged periods of record-ing (up to 72 h), when the NPCs seem toexhaust the substrates for their gating mech-anism (Bustamante JO, unpublished obser-vations). Thus, the initial current recordedfrom a nucleus-attached patch may suggestthat a gigaseal has not been reached and,consequently, the experimenter may discardthe preparation. However, after many min-utes of observation one may capture a si-multaneous closure of the channels(Bustamante JO, unpublished results).Clearly, if the gigaseal had not been at-tained, this could be considered a poor pi-pette-membrane seal. To further confirmthat the tight seal is retained, one may plugthe NPCCs by adding diluted amounts ofnuclear proteins (those which have nuclear-targeting sequences) without transport sub-strates (i.e., all those substances that par-ticipate in the successful NPC-MMT: ATP,GTP, cytosolic and nucleosolic receptors,etc.). In this manner, it is possible to reducethe number of unplugged NPCCs and,therefore, to clearly differentiate and countthe NPCCs in the NE patch (BustamanteJO, unpublished results). It is also possibleto obtain a gigaseal between the pipette tipand the NE but not see ion channel activity(Bustamante JO, unpublished observa-tions). This, however, cannot be taken asindicative that the NPCs are not present orthat they are not functioning, for if given asufficient amount of time (15-60 min, de-pending on the availability of NPC-MMTsubstrates), NPCC activity will appear as aconsequence of macromolecular unplug-ging. As discussed below, during transloca-tion, macromolecules plug the NPCC for aperiod of time that will depend on the ex-

perimental conditions. Since most patch-clamp studies are carried out in pure salinesolutions, macromolecular transport has theleast favorable conditions. Therefore, thoseNPCCs plugged during nucleus isolationwill remain that way for a very long time orforever, depending on the specific method-ologies used. The longer the isolated nucleiare exposed to saline, the less transport sub-strates are left in the preparation and thegreater the number of plugged channels(Bustamante JO, unpublished observa-tions). This observation can be used to theinvestigator’s advantage because it providesa means of controlling the number of un-plugged, ion-conducting NPCCs. This is ad-vantageous because for the analysis ofsingle channel activity it is best to have aslow a number of gating channels as pos-sible (in the best case, only one). Therefore,with some fine tuning, a method can bedeveloped that provides such a favorablesituation for the statistical analysis of singlechannel activity (e.g., 13,31,40).

Nucleocytoplasmic gradients

We indicated above that electrophysi-ological and non-electrophysiological dataare not adversaries (see also Table 1) but,does NPCC activity explain nucleocytoplas-mic gradients of small ions and other mol-ecules? On the basis of NPCC activity alone(i.e., po, γNPCC and N), the amount ofmonoatomic ion flow exchanged per cardiacmyocyte patch was estimated at leading to aconcentration change of 1 mM/s (see Ap-pendix in Ref. 31). Briefly, the maximal rateof change in ion concentration caused by theNPCC current in an NE patch is calculatedby dividing the ionic current by the nuclearvolume (e.g., about 500 µm3 for a smallspherical nucleus of 5 µm radius, rnucleus).For a patch with an average of 0.5 pA in 1 s(Figure 1 in Ref. 31), the change in ionconcentration per unit time was calculated at1 mM/s. Clearly, for the whole NE, the value

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would be several-fold greater. For example,for a pipette tip of 0.5 µm radius, rpipette, theminimal NE patch area (corresponding to aflat plane and not to an Ω-shaped surfacebecause no suction is applied) would be 0.8µm2 orπ (rpipette)2. The whole NE surface forthe 5-µm radius nucleus in our example hasan area of 314 µm2 or 4π (rnucleus)2. There-fore, the estimated concentration change in 1s for the whole NE is 393 mM. This estimatedemonstrates that NPCC behavior does notoppose the concept of diffusion, althoughthe latter is usually assumed to indicate thatthe NPCs do not switch between open andclosed states. Table 2 shows calculations forvarious changes in the parameters of thenucleus and NPCC.

NPC-mediated macromoleculartranslocation

Although it has been demonstrated byfluorescence microscopy that nucleocyto-plasmic ion flow is maintained even in iso-lated nuclei under pure saline conditions(e.g., 53), one arguable flaw in most patch-clamp studies is the lack of simultaneoustests for the basic transport properties ofNPCs. In those studies favoring NPCs as thesource for channel activity, there is no visualdemonstration of translocation. Whereas inthose assuming that the activity derives fromstructures different from NPCs, there is ab-sence of visual demonstration that the NPCsare closed. To resolve this problem, one can

Table 2 - Computed changes in nuclear ion concentration ∆Cion in 1 s, ∆Cion/∆t, caused by a 10 mV electrochemical gradient applied to the wholenuclear envelope (i.e., a voltage drop between the cytoplasmic and the nucleoplasmic layers in the immediate vicinity of the NE). Definitions:γNPCC is the ion conductance of a single NPC channel or NPCC; VNE is the voltage applied to the NE; popen is the probability of opening of a singleNPCC; <Flow Rate> is the mean rate of flow measured in terms of charges; DNPC is the NPC surface density; rN is the radius of the nucleus; ANE

is the total NE area; NNPC is the total number of NPCs in the nucleus; VN is the volume of the nucleus; ∆mion /∆t is the rate of change in the massof the total number of ions. Numbers are represented in the computer version of scientific notation, with x10n being indicated as En (e.g., x10-17

is given as E-17).

NPCC Nucleus

Parameter γNPCC VNE popen <Flow rate> DNPC rN ANE NNPC VN ∆mion /∆t ∆Cion/∆tchanged (pS) (mV) (NPC/µm2) (µm) (µm2) (Total) (mol/s) (mM/s)

(pA) (mol/s) (µm3) (l)

popen 500 10 1.00 5.00 5E-17 10 5 314.2 3142 523.6 5E-13 1.5E-13 300

popen 500 10 0.50 2.50 2.5E-17 10 5 314.2 3142 523.6 5E-13 7.9E-14 150

popen 500 10 0.25 1.25 1.3E-17 10 5 314.2 3142 523.6 5E-13 4.0E-14 75

popen 500 10 0.10 0.50 5.0E-18 10 5 314.2 3142 523.6 5E-13 1.6E-14 30

popen 500 10 0.05 0.25 2.5E-18 10 5 314.2 3142 523.6 5E-13 7.9E-15 15

popen 500 10 0.01 0.05 5.0E-19 10 5 314.2 3142 523.6 5E-13 1.8E-15 3

γNPCC 400 10 0.50 2.00 2.0E-17 10 5 314.2 3142 523.6 5E-13 6.3E-14 120

γNPCC 300 10 0.50 1.50 1.5E-17 10 5 314.2 3142 523.6 5E-13 4.7E-14 90

γNPCC 200 10 0.50 1.00 1.0E-17 10 5 314.2 3142 523.6 5E-13 3.1E-14 60

γNPCC 100 10 0.50 0.50 5.0E-18 10 5 314.2 3142 523.6 5E-13 1.6E-14 30

γNPCC 50 10 0.50 0.25 2.5E-18 10 5 314.2 3142 523.6 5E-13 7.9E-15 15

γNPCC 25 10 0.50 0.13 1.3E-18 10 5 314.2 3142 523.6 5E-13 3.9E-15 8

DNPC 500 10 0.50 2.50 2.5E-17 5 5 314.2 1571 523.6 5E-13 3.9E-14 75

DNPC 500 10 0.50 2.50 2.5E-17 3 5 314.2 942 523.6 5E-13 2.4E-14 45

DNPC 500 10 0.50 2.50 2.5E-17 1 5 314.2 314 523.6 5E-13 7.9E-15 15

rN 500 10 0.50 2.50 2.5E-17 10 10 1257 12566 4188.8 4E-12 3.1E-13 75

rN 500 10 0.50 2.50 2.5E-17 10 20 5027 50266 33510 3E-11 1.3E-12 38

rN 500 10 0.50 2.50 2.5E-17 10 30 11310 113098 113098 1E-10 2.8E-12 25

rN 500 10 0.50 2.50 2.5E-17 10 40 20106 201062 268083 3E-10 5.0E-12 19

rN 500 10 0.50 2.50 2.5E-17 10 50 31416 314160 523600 5E-10 7.9E-12 15

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test the NPC ion transport properties withfluorescent probes (e.g., 53). Either 4-kDaFITC-dextran (Molecular Probes, Eugene,OR) or 5.4-nm FITC-labeled StarburstTM

dendrimers (Polysciences, Warrington, PA)can be used (Bustamante JO, unpublishedresults). Starburst dendrimers appear a bet-ter choice as they have an almost perfectspherical shape (see Ref. 55). Laser-scan-ning confocal fluorescent microscopy meas-urements performed during electrophysi-ological investigations show that both 4-kDaFITC-dextran and 5.4-nm FITC-dendrimerstranslocate under simple, high-K saline con-ditions (13; Bustamante JO, Geibel JP,Liepins A, Hanover JA and McDonnell TJ,and also Bustamante JO, unpublished re-sults). This observation is consistent withthe demonstration that ≤10-kDa FITC-dex-tran molecules translocate across the NEunder simple saline conditions that normallyprevent MMT (53). These results demon-strate that the nuclei utilized in those electro-physiological studies conformed to the preva-lent concept that NPCs allow translocationof not only monoatomic ions but also ofsmall-to-medium-sized molecules and par-ticles (53). More importantly, the nuclei uti-lized in one electrophysiological laboratoryhave been shown to retain their macromo-lecular transport capacity (13; BustamanteJO, Geibel JP, Liepins A, Hanover JA andMcDonnell TJ, unpublished results). Thiscapacity was demonstrated by constructing anuclear protein probe with the fluorescentB-phycoerythrin (B-PE, 240 kDa) conjugatedto the nuclear localization signal (NLS) ofthe SV40 large T antigen (13). Readers inter-ested in the details of the mechanisms re-quired for NPC-MMT are encouraged toconsult excellent reviews which have ap-peared elsewhere (e.g., 2-6,45). For this rea-son, we focus here on the highlights of MMT.Briefly, various mechanisms have been iden-tified for several classes of molecules. Forproteins, the molecules carrying the NLSmust first bind to a cytosolic receptor, then

bind to a docking site at the NPC, and thentranslocate in an ATP-dependent process.The process depends on other factors, in-cluding Ran/TC4 and phosphorylation ofsome of the substrates involved. Note thatthe conjugation of B-PE to the NLS wasrequired to make the complex recognizableby the NPC-mediated mechanism for MMT(13). Without conjugation to an NLS, B-PEwill not enter the nucleus. Thus, the cardiacmyocyte investigations demonstrated that ionchannel activity was recorded in a prepara-tion which retained the NPC capacity for ionand macromolecule transport. An advantageof conjugating a large fluorescent moleculeto an NLS rather than conjugating a nuclearprotein to a fluorescent probe such as FITCis that the large fluorescent probe will notenter the nucleus if not conjugated to theNLS whereas if the FITC conjugation of thenuclear protein is not strong enough, thenthe probe may enter the nucleus and give afalse reading. Recent observations on changesof fluorescence response of fluorochromescaused by nuclear compartmentalization areimportant for absolute value determinationsbut not for relative, qualitative or semi-quan-titative observations (56).

The identification of NPCs as asource of ion channel activity

Direct demonstration that the NPCs arethe structure responsible for the open-closegating of large-conductance NE ion chan-nels was given in isolated cardiac myocytenuclei (nucleus-attached mode) by differenttests (13). One of these showed the directblockade of channel activity by the NPCmonoclonal antibody mAb414 (known toblock NPC-mediated macromolecular trans-port). Only this antibody, out of a series ofNPC and NLS antibodies, did block the large-conductance ion channel activity (13). Thelack of action was also seen with other non-NPC commercial antibodies (13). These non-NPC antibodies included anti-FITC antibody,

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anti-myosin, and the mAb414 isotype con-trol (13). The blocking effect of mAb414 onchannel activity was independently con-firmed (57). The effect of mAb414 on NPCCactivity parallels that seen in the mitochon-drial multiple conductance channel upon theapplication of an antibody to a protein im-port component of the mitochondrial innermembrane (58). That mAb414 did not plugthe NPCC, but instead blocked it, was shownby the unidirectional blockade (i.e., block-ade seen with only voltage of one sign) seenin some preparations (13). The reader shouldnote that this unidirectional blockade is morethan a simple voltage-dependent blockadebecause 100% block occurred with only onevoltage polarity. Taken together, the resultssupport the conclusion that the NPC, likeother organellar and junctional ion channels,has an intrinsic ion channel behavior withhigh conductance at high [K+]. That a su-pramolecular structure may have an intrinsicion channel activity (switching between theopen and closed states) and at the same timemay account for a large ion permeability isexemplified by the many reports on organellarion channels. For example, at these high [K+]values, both the ryanodine receptor Ca2+

channel (reviewed in Ref. 59) and the mito-chondrial multiple conductance channel (e.g.,60) have a single channel conductance of theorder of 103 pS. In analogy to other ionchannels, NPCC activity depends on the volt-age across the NE (and thus, the electro-chemical potential). Note, however, thatwhile the open probability decays faster(smaller time constants) at larger electro-chemical potentials, the single channel con-ductance remains constant.

Electrophysiological argumentsfavoring NPCs as ion channels

In addition to these supportive data, onecan use electrical circuit theory alone to de-duce that in nucleus-attached patches of car-diac myocytes (with negligible resting poten-

tial, Vnucleus) the NPCCs are the source for therecorded electrical activity. First, the hole ofeach NPC (i.e., the NPCC) allows a significantamount of ion flow and is solely responsiblefor the nucleocytoplasmic exchange of elec-trolytes (e.g., 8). Second, NPCCs have a widediameter and are present in large numbers perunit area (e.g., 16). In cardiac myocytes, thenumber of NPCs per µm2 was estimated at 12± 4 (see Ref. 13). Third, the NE outside thearea covered by the pipette tip has negligibleelectrical resistance because it contains manyion-conducting NPCs. Therefore, the poten-tial inside the nucleus is pretty much the sameas that of the bath. Consequently, the potentialsensed by the NPCs under the recording patch-clamp pipette is given by the potential differ-ence between the bath and pipette electrodes.Fourth, in nucleus-attached patches, where thepipette is sealed to the outer membrane of theNE, only non-NPC channels on the outer NEmembrane are directly affected by the changesin the pipette voltage (as typically occurs in thepatch-clamp technique). Fifth, any ion currentcontribution from non-NPCCs (Iother) at theONM flows in parallel to current contributedby the NPCCs (INPCC), i.e.,

Imeasured = ∑INPCC + ∑Iother (Eq. 15)

where ∑ represents the sum total of thecurrent contributed by all the channels of thecorresponding class. Therefore, for the con-tribution of non-NPCCs to overcome that ofNPCCs it would be necessary to have ahigher density of channels per unit area and/or a higher single channel conductance. Asmentioned above, to date, this has not beenshown. On the contrary, NPCCs have beenshown to allow ion and small molecule trans-location under pure saline conditions (53).Dextran molecules go into the nucleus butnot into the NE (e.g., 53).

Macromolecules nullify singleNPC channel conductance

Nuclear proteins, mRNA, native and for-

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eign nucleic acids and other macromoleculesutilize the NPCs to cross the NE (e.g., 2-6).Since these macromolecules are poor elec-trical charge carriers and since they takeover the volume previously occupied by themonoatomic ions (the good electrical chargecarriers), one would expect NPC-MMT toreduce the ion conductance of a single NPCC,γNPCC. To date, only one laboratory has re-ported the effects of nucleocytoplasmic MMTon NPCC activity (e.g., 13,61,62). Nuclear-targeted B-PE silenced the NPCC activity(13). However, the channel activity was pro-gressively recovered with time, in a time-course that resembled that required for thecomplete nuclear transport of the nuclear-targeted B-PE probe in fluorescence micros-copy experiments. It must, therefore, be con-cluded that while the channel activity wassilent, the B-PE macromolecules were con-tinuously transported into the nucleus. Ex-periments with transcription factors (an im-portant group of naturally occurring nuclear-targeted macromolecules involved in theregulation of gene activity) also transientlysilenced ion channel activity (13,61,62).These experiments demonstrated that whentranscription factors (AP-1/c-Jun, NF-κB,SP1 and TATA-binding protein) and othernuclear-targeted molecules (e.g., B-PE con-jugated to NLS) with a molecular mass ofabout 40 kDa or higher were added, the ionchannel activity was silenced for a period oftime ranging from a few seconds (with sub-strates) to hours (without substrates). Bothcases correlate with the electron microscopyobservation that, without effective NPC-MMT substrate (e.g., Ca2+, ATP, GTP, Ran/TC4, cytosolic and nucleosolic receptors,etc.), the NPC is plugged (e.g., 8). Sincetranscription factors enter the nucleus exclu-sively through the NPCC, the conclusionfollowed that the recorded ion channel activ-ity in these MMT-competent nuclei derivedfrom gated ion flow along the NPC and notfrom an indirect action of the factors (13).When comparing the translocation times in

these nuclei with the times in situ, the readershould be aware that the electrophysiologi-cal experiments were conducted under con-ditions which are far from optimal for nor-mal macromolecular transport (e.g., 2-6).For example, cytosolic factors such as Ran/TC4 and NLS receptors were not added.Furthermore, the rate of nuclear cytoplasmicprotein transport has been shown to be deter-mined by the protein phosphorylation (re-viewed in Ref. 63). Therefore, it is likely thatprotein kinases and phosphatases play a rolein nuclear transport by controlling the phos-phorylation levels of both transport substratesand NPCs (see 63). Despite these limitationsof the patch-clamp experiment, we considerthese patch-clamp conditions (with less thanoptimal content of NPC-MMT substrates) tofacilitate the isolation of single ion channelactivity because under normal conditionsthe translocation should take place at a fasterpace and might make it difficult to isolate theNPCC behavior. Furthermore, despite thesenon-physiological conditions, the prepara-tions demonstrated an extraordinary stabil-ity, yet to be reported elsewhere (>72 h; 62;Bustamante JO, unpublished results). There-fore, we would like to note the remarkable(albeit difficult) success in patch-clampingthe nucleus in situ (e.g., 64). This achieve-ment should facilitate patch-clamp studiesunder more realistic conditions and makeeasier the comparison of patch-clamp resultswith those obtained in non-electrophysiologi-cal experiments (e.g., with permeabilizedcells, see Ref. 11). Note, however, that fill-ing the pipette with cytosol causes someelectrical problems in patch-clamp record-ing as the pipette resistance increases(Bustamante JO, unpublished results).

The observation that macromolecules si-lence other large ion channels is not new.Indeed, the concept of a plug channel statedistinct from the open, closed and inacti-vated states was proposed for other organellarpreparations. For example, in mitochondrialouter membranes, a mitochondrial address-

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ing peptide silences a large conductance cat-ionic channel (e.g., see 65 and referencestherein). This channel, known as the pep-tide-sensitive channel, is detected in mutantporin-less preparations (e.g., 66). The openprobability of the voltage-dependent anionchannel (VDAC) of the outer mitochondrialmembrane (porin type-1; which is also ex-pressed in sarcoplasmic reticulum; see Ref.67) was eliminated with a soluble 54-kDamitochondrial protein (reviewed in Ref. 68).Mitochondrion-targeting peptides also plugthe multiple conductance mitochondrial in-ner membrane channel whose properties re-semble those of the peptide-sensitive mito-chondrial channel (e.g., 58,60,69). Finally,protein translocation through the 4-6-nm di-ameter aqueous pore of the endoplasmicreticulum, the translocon, eliminated chan-nel activity (e.g., 14,70). Independent fluo-rescence accessibility studies show that theaqueous pore in a functioning translocon is4-6 nm in diameter, making it the largestmembrane channel, after that of the NPC,that maintains a permeability barrier (71).

NPC channel activity, MTT,and Ca2+ in NE cisternae

It has recently been recognized that [Ca2+]in the NE cisternae ([Ca2+]NE) plays a majorrole in the NPC-mediated translocation ofmacromolecules (11) and medium-sized (>10kDa) molecules (53,72, reviewed in 12).Briefly, when [Ca2+]NE is low, the transloca-tion of macromolecules and even of medi-um-sized molecules (>10 kDa) is not pos-sible. It should, therefore, be expected that incells where there is a great deal of Ca2+

release from the NE cisternae, the transportof macromolecules will be adversely affected.The impaired transport machinery, in turn,should cause the macromolecules to becaught in the middle of the NPC transloca-tion hole (the NPCC). Therefore, like a plug,they should obstruct the pathway for ionflow and, consequently, result in an impaired

translocation of small molecules andmonoatomic ions. The period of silence mustbe determined by many factors. When theconcentration of the macromolecules is suf-ficiently low, it may be possible to detectsingle translocations, whereas when it is rela-tively high, the many molecules may line upin single file to give the equivalent effect oftranslocation of a single long molecule.Translocation will stop when one of therequired NPC-MMT substrates is exhausted(e.g., Ca2+, ATP, GTP, Ran/TC4, cytosolicand nucleosolic receptors, etc.), even if amacromolecule containing the signal is stillpresent and ready for transport. That is, MMTcannot be expected to follow the customarydiffusion laws. Therefore, rather than a pro-cess described by a single exponential change,a multi-exponential process should be ex-pected due to the biochemical reactions in-volved (signal recognition, docking, etc.; see,e.g., 2-6). Thus, theoretically, each reaction(e.g., signal recognition) should correspondto an exponential function. Structural sup-port for the electrophysiological idea of thetranslocating macromolecule behaving likea plug comes from the consistent and repro-ducible electron microscopy observation thatwhen transport substrates are supplied, mostNPCs are unplugged whereas when the sub-strates are absent, most of the NPCs areplugged (e.g., 8). Figure 2 shows the putativeplug in the 3-D reconstruction NPC model ofPante and Aebi (8). The fuzzy, uncertainconcept of the plug is indicated by the shadedcoloring of the figure. That plugging mate-rial may account for a high NE resistancewas put forward by Loewenstein and co-workers (22) more than three decades ago.Since both transcription factors and mRNAuse the NPCs for their translocation, it canbe concluded that the cellular rhythm affect-ing [Ca2+]NE should be reflected on the levelsof gene activity and expression.

A plethora of reports with fluorescenceand luminescence microscopy support theconcept of the NE as a barrier (e.g., 73-78;

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see 79 and 80). As discussed in previousparagraphs, each time the NPCC opens, itallows a great deal of ions to be translocated.Therefore, reports showing steady-statenucleocytoplasmic gradients may be ex-plained by an NE that has many of its NPCCsplugged by translocating molecules or by theclassical view of binding sites or water states(e.g., 15,18-20). Luminescence experimentswith nucleoplasmin (a nuclear protein) fusedto aequorin demonstrated the Ca2+ barriernature of the NE (73,74). However, whenaequorin was fused to the nuclear localiza-tion signal of the glucocorticoid receptor,the nature of the NE as a barrier was notdetected (81). This result may be related tosimpler transport mechanisms for the recep-tor used for nuclear localization (e.g., 82). Inother words, the selection of the protein to beused for nuclear targeting is critical to theinterpretation of the results because themechanisms of nuclear transport for pro-teins (e.g., nucleoplasmin) are not the sameas those for steroid receptors (e.g., glucocor-ticoid receptor). The experimental data indi-cate the importance of macromolecular trans-port for the maintenance or development ofnucleocytoplasmic gradients. This relation-ship between macromolecular transport andthe ATP-independent flow of small mol-ecules, particles and ions, should be relevantto the interpretation of experimental results.For example, when evaluating data fromsimultaneous fluorescence and ‘whole-cell’patch-clamp recordings, the investigatorshould be aware that the pipette may diluteaway transport substrates without whichmacromolecular transport would be impos-sible. This condition would lead to the NPCCbeing plugged by a macromolecule and, con-sequently, would result in an envelope witha full ion-barrier character. The reader shouldnote that, due to the complexity of require-ments for NPC-MMT, having a macromol-ecule with a nuclear localization signal plusATP and Ca2+ is not in itself sufficient tosecure translocation.

Changes in NPC topography concomi-tant with changes in NPCC activity wereconfirmed by atomic force microscopy (62).In these investigations, the universal tran-scription factor TATA-binding protein (withboth structural and functional roles) facili-tated transport and unplugging of the chan-nels after the typical transient reduction insingle NPCC conductance (62). The atomicforce microscopy images suggested that theproteins docked and accumulated at the NPCcytoplasmic side (62). Part of the mecha-nisms of TATA-NPC interaction was attrib-uted to an oligomerization effect mediatedby protein glycosylation (62; see also 83 andreferences therein).

Relevance to studies of genecontrol and expression

The demonstration that the NPC has anintrinsic ion channel activity supports the useof the patch-clamp technique for the quantifi-cation of macromolecular transport along asingle functional NPC. To date, no other tech-nique has this capability. In analogy to theprinciples of the Coulter cell counters, basicelectrophysiological principles show that, whencompletely filled with electrolytes, the NPCCionic conductance is maximal (13,84). Theyalso show that, each time a macromolecule istranslocated, the NPCC conductance goes tozero because the macromolecule displaces allthe electrical charge carriers (13,84). This phe-nomenon is not exclusive to the NPC but isshared by channels of mitochondria (e.g.,58,60,69) and endoplasmic reticulum (e.g.,14,70).

Perhaps the most dramatic example ofthis channel plugging phenomenon is thewell-known NPC-mediated translocation ofmRNA and ribonucleoprotein particles (e.g.,85) as well as foreign nucleic acids (e.g.,86,87). Figure 5 shows the basic mechanismfor the translocation of the ribonucleopro-tein particle (Figure 2 of reference 85; repro-duced with permission). Not only do these

350



particles have wider diameter than the NPCCbut their mechanism of translocation involvestheir transient transformation into filamentsduring their ‘snaking’ through the NPCCs(e.g., 86). Clearly, for smaller moleculeswhich are poor charge carriers and do notplug the NPCC completely, the situation isintermediary and requires a more complexdescription. Indeed, experiments with 5.4-nm FITC-labeled dendrimers showed thatthe probes translocate to the nucleus andduring their translocation the binomial be-havior of the NPCC is transformed into low-frequency bursts (Bustamante JO, unpub-lished results). This subject is outside thescope of this review but, in principle, onecan reason that for smaller molecules withpoor charge carrier properties, the effectwould be directly proportional to the densityof the molecules inside the channel. Thehigher their density, the higher the resistivityof the electrolyte filling the NPPC and, there-fore, the smaller the single channel conduc-tance, γNPCC. A semi-quantitative analysisdemonstrates the point. Under pure salinecondition, when the NPC hole is completelyfilled with the electrolyte, the electrical chargedensity per unit time (ρcharge) is:

ρcharge = ncarrier/vNPCC (Eq. 16)

where ncarrier is the number of good electricalcharge carriers inside the NPC hole and vNPCC,the volume of the hole. When macromol-ecules and other large, inert, non-current-carrying particles enter the hole, they dis-place the charge carriers, thus reducing ncarrier

and thus, ρcharge.

vcarrier = vNPCC - vinert (Eq. 17)

where vcarrier and vinert are the volume occu-pied by carrier and inert particles, respec-tively.

ncarrier = vcarrier [Xcarrier] = (vNPCC - vinert) [Xcarrier](Eq. 18)

where [Xcarrier] is the concentration of elec-

Figure 5 - NPC-mediated ribo-nucleoprotein particle transloca-tion. The particles change theirgeometry to traverse the NPCC.(From Ref. 82, with permission).

351



trical charge carriers. Thus,

ρcharge = (vNPCC - vinert) [Xcarrier]/vNPCC =[1 - (vinert/vNPCC)] [Xcarrier] (Eq. 19)

vinert can be calculated from the concentra-tion of electrically inert particles, [Xinert], andtheir individual (or average) volume, vparticle:

vinert = ninert vparticle = vNPCC [Xinert] vparticle

(Eq. 20)

Therefore,

ρcharge = (1 - vparticle [Xinert]) [Xcarrier](Eq. 21)

This relationship may be expanded to incor-porate the presence of multiple charge carri-ers (e.g., K+, Na+ and Ca2+) and multiplepoor or inert carriers (e.g., transcription fac-tors and nuclear proteins). The above analy-sis does not take into account interactionsamong particles or between the particles andthe NPCC inner wall lining, which affectcarrier mobility. In the real world, these are asignificant factor opposed to free ion flow.

As we come to the end of this review, wewould like to summarize by saying that bothdata and theory about the cell nucleus andother cellular organelles show that the pe-riod of channel silence can be correlatedwith the translocation time of macromol-ecules. This principle, denoted the ‘macro-molecule-conducting ion channel paradigm’in the study of NPC ion channel behavior(13), is similar to that of Coulter cell counters.The principle has been applied to polymercounting along ion channels (84) and to the

determination of the size of nucleic acidswith 2.6-nm diameter channels (88). The useof molecules which are not electrical chargecarriers for the measurement of ion channeldiameter has been applied to the determina-tion of the diameter (ca. 3 nm) of a porinchannel (89). The observation that the patch-clamp technique can be used to measure andstudy NPC-MMT for as long as 72 h (62;Bustamante JO, unpublished observations)implies that the mechanisms of transcriptionfactor and mRNA translocation can be stud-ied by this electrophysiological technique.Therefore, the possibility of using the patch-clamp technique for the identification of theNPC mechanisms underlying transcriptionfactor control of gene activity and of mRNA-mediated gene expression offers a uniqueadvantage over other currently available tech-niques in that it facilitates quantitative stud-ies of macromolecular transport at the singleNPC level. By complementing this techniquewith low light-level fluorescence micros-copy, it should be possible to resolve manyof the current enigmas pervading the field.

Further information related to this sec-tion can be found at the following internetsite: http://www.geocities.com/TheTropics/6066/index.html.

Acknowledgments

The authors acknowledge the construc-tive criticisms of Alan Finkelstein, MichaelV.L. Bennett, Peter Satir and Renato Rozental(Albert Einstein College of Medicine).

352



References

1. Sakmann B & Neher E (1995). Single-Channel Recording. Plenum Press, NewYork.

2. Gorlich D (1997). Nuclear protein import.Current Opinion in Cell Biology, 9: 412-419.

3. Lee MS & Silver PA (1997). RNA move-ment between the nucleus and the cyto-plasm. Current Opinion in Genetics andDevelopment, 7: 212-219.

4. Moroianu J (1997). Molecular mecha-nisms of nuclear protein transport. CriticalReviews in Eukaryotic Gene Expression,7: 61-72.

5. Nakielny S & Dreyfuss G (1997). Nuclearexport of proteins and RNAs. CurrentOpinion in Cell Biology, 9: 420-429.

6. Nigg EA (1997). Nucleocytoplasmic trans-port: signals, mechanisms and regulation.Nature, 386: 779-787.

7. Maul G (1977). The nuclear and the cyto-plasmic pore complex: structure, dynam-ics, distribution, and evolution. Interna-tional Reviews of Cytology, S6: 75-186.

8. Pante N & Aebi U (1996). Molecular dis-section of the nuclear pore complex. Criti-cal Reviews in Biochemistry and Molecu-lar Biology, 31: 153-199.

9. Rutherford SA, Goldberg MW & Allen TD(1997). Three-dimensional visualization ofthe route of protein import: the role ofnuclear pore complex substructures. Ex-perimental Cell Research, 232: 146-160.

10. Oberleithner H, Brinckmann E, Schwab A& Kröhne G (1994). Imaging nuclear poresof aldosterone-sensitive kidney cells byatomic force microscopy. Proceedings ofthe National Academy of Sciences, USA,91: 9784-9788.

11. Greber UF & Gerace L (1995). Depletionof calcium from the lumen of endoplas-mic reticulum reversibly inhibits passivediffusion and signal-mediated transportinto the nucleus. Journal of Cell Biology,128: 5-14.

12. Perez-Terzic C, Jaconi M & Clapham DE(1997). Nuclear calcium and the regula-tion of the nuclear pore complex. Bioes-says, 19: 787-792.

13. Bustamante JO, Hanover JA & Liepins A(1995). The ion channel behavior of thenuclear pore complex. Journal of Mem-brane Biology, 146: 239-251.

14. Simon SM (1995). Protein-conductingchannels for the translocation of proteinsinto and across membranes. Cold SpringHarbor Symposia on Quantitative Biology,60: 57-69.

15. Paine PL & Horowitz SB (1980). The

movement of material between nucleusand cytoplasm. Cell Biology, 4: 299-338.

16. Miller M, Park MK & Hanover JA (1991).Nuclear pore complex: structure, function,and regulation. Physiological Reviews, 71:909-949.

17. Horowitz SB & Paine PL (1976). Cytoplas-mic exclusion as a basis for asymmetricnucleocytoplasmic solute distributions.Nature, 260: 151-153.

18. Paine PL (1993). Nuclear protein accumu-lation by facilitated transport and intra-nuclear binding. Trends in Cell Biology, 3:325-329.

19. Ling GN (1977). Potassium accumulationin frog muscle: the association-inductionhypothesis versus the membrane theory.Science, 198: 1281-1284.

20. Ling GN (1994). The new cell physiology:an outline, presented against its full his-torical background, beginning from thebeginning. Physiological Chemistry andPhysics and Medical NMR, 26: 121-203.

21. Loewenstein WR & Kanno Y (1962). Someelectrical properties of the membrane of acell nucleus. Nature, 195: 462-464.

22. Wiener J, Spiro D & Loewenstein WR(1965). Ultrastructure and permeability ofnuclear membranes. Journal of Cell Biol-ogy, 27: 107-117.

23. Matzke AJM, Weiger TM & Matzke MA(1990). Detection of a large cation-selec-tive channel in nuclear envelopes of avianerythrocytes. FEBS Letters, 271: 161-164.

24. Mazzanti M, DeFelice LJ, Cohen J &Malter H (1990). Ion channels in the nuclearenvelope. Nature, 343: 764-767.

25. Bustamante JO (1994). Nuclear electro-physiology. Journal of Membrane Biology,138: 105-112.

26. Bustamante JO, Liepins A & Hanover JA(1994). Nuclear pore complex ion chan-nels. Molecular Membrane Biology, 11:141-150.

27. Dale B, DeFelice LJ, Kyozuka K, Santella L& Tosti E (1994). Voltage clamp of thenuclear envelope. Proceedings of theRoyal Society of London, Series B: Bio-logical Sciences, 255: 119-124.

28. DeFelice LJ & Mazzanti M (1995). Bio-physics of the nuclear envelope. In:Sperelakis N (Editor), Cell Physiology. Ac-ademic Press, New York.

29. Stehno-Bittel L, Perez-Terzic C, LuckhoffA & Clapham DE (1996). Nuclear ion chan-nels and regulation of the nuclear pore.Society of General Physiologists Series,51: 195-207.

30. Matzke AJM, Behensky C, Weiger T &

Matzke MA (1992). A large conductanceion channel in the nuclear envelope of ahigher plant cell. FEBS Letters, 302: 81-85.

31. Bustamante JO (1992). Nuclear ion chan-nels in cardiac myocytes. Pflügers Ar-chives, European Journal of Physiology,421: 473-485.

32. Mazzanti M, DeFelice LJ & Smith EF(1991). Ion channels in murine nuclei dur-ing early development and in fully differ-entiated adult cells. Journal of MembraneBiology, 121: 189-198.

33. Maruyama Y, Shimada H & Taniguchi J(1995). Ca2+-activated K+-channels in thenuclear envelope isolated from single pan-creatic acinar cells. Pflügers Archives, Eu-ropean Journal of Physiology, 430: 148-150.

34. Guihard G, Proteau S & Rousseau E(1997). Does the nuclear envelope con-tain two types of ligand-gated Ca2+ re-lease channels? FEBS Letters, 414: 89-94.

35. Mak DO & Foskett JK (1994). Single-chan-nel inositol 1,4,5-trisphosphate receptorcurrents revealed by patch clamp of iso-lated Xenopus oocyte nuclei. Journal ofBiological Chemistry, 269: 29375-29378.

36. Mak DO & Foskett JK (1997). Single-chan-nel kinetics, inactivation, and spatial distri-bution of inositol trisphosphate (IP3) re-ceptors in Xenopus oocyte nucleus. Jour-nal of General Physiology, 109: 571-587.

37. Stehno-Bittel L, Luckhoff A & ClaphamDE (1995). Calcium release from thenucleus by InsP3 receptor channels. Neu-ron, 14: 163-167.

38. Pasyk EA & Foskett JK (1997). Cystic fi-brosis transmembrane conductance regu-lator-associated ATP and adenosine 3'-phosphate 5'-phosphosulfate channels inendoplasmic reticulum and plasma mem-branes. Journal of Biological Chemistry,272: 7746-7751.

39. Al-Mohanna FA, Caddy KW & BolsoverSR (1994). The nucleus is insulated fromlarge cytosolic calcium ion changes. Na-ture, 367: 745-750.

40. Bustamante JO (1993). Restricted ionflow at the nuclear envelope of cardiacmyocytes. Biophysical Journal, 64: 1735-1749.

41. Assandri R & Mazzanti M (1997). Ionicpermeability on isolated mouse liver nu-clei: influence of ATP and Ca2+. Journal ofMembrane Biology, 157: 301-309.

42. Hille B (1992). Na and K channels of axons.In: Hille B (Editor), Ionic Channels of Excit-able Membranes. Chapter 3. 2nd edn.

353



Sinauer, Sunderland, MA.43. Bustamante JO (1994). Open states of

nuclear envelope ion channels in cardiacmyocytes. Journal of Membrane Biology,138: 77-89.

44. Moore MS & Blobel G (1993). The GTP-binding protein Ran/TC4 is required forprotein import into the nucleus. Nature,365: 661-663.

45. Rout MP & Blobel G (1993). Isolation ofthe yeast nuclear pore complex. Journalof Cell Biology, 123: 771-783.

46. Innocenti B & Mazzanti M (1993). Identifi-cation of a nucleo-cytoplasmic ionic path-way by osmotic shock in isolated mouseliver nuclei. Journal of Membrane Biol-ogy, 131: 137-142.

47. Loewenstein WR, Kanno Y & Ito S (1966).Permeability of the nuclear membrane.Annals of the New York Academy of Sci-ences, 137: 708-716.

48. Tabares L, Mazzanti M & Clapham DE(1991). Chloride channels in the nuclearmembrane. Journal of Membrane Biol-ogy, 123: 49-54.

49. Longin AS, Mezin P, Favier A & Verdetti J(1997). Presence of zinc and calciumpermeant channels in the inner mem-brane of the nuclear envelope. Biochemi-cal and Biophysical Research Communi-cations, 235: 236-241.

50. Draguhn A, Börner G, Beckmann R,Buchner K, Heinemann U & Hucho F(1997). Large-conductance cation chan-nels in the envelope of nuclei from ratcerebral cortex. Journal of Membrane Bi-ology, 158: 159-166.

51. Rousseau E, Michaud C, Lefebvre D,Proteau S & Decrouy A (1996). Reconsti-tution of ionic channels from inner andouter membranes of mammalian cardiacnuclei. Biophysical Journal, 70: 703-714.

52. Valenzuela SM, Martin DK, Por SB,Robbins JM, Warton K, Bootcov MR,Schofield PR, Campbell TJ & Breit SN(1997). Molecular cloning and expressionof a chloride ion channel of cell nuclei.Journal of Biological Chemistry, 272:12575-12582.

53. Stehno-Bittel L, Perez-Terzic C & ClaphamDE (1995). Diffusion across the nuclearenvelope inhibited by depletion of nuclearCa2+ store. Science, 270: 1835-1838.

54. Hinshaw JE, Carragher BO & Milligan RA(1992). Architecture and design of thenuclear pore complex. Cell, 69: 1133-1141.

55. Tomalia DA (1995). Dendrimer molecules.Scientific American, 272: 62-65.

56. Perez-Terzic C, Stehno-Bittel L & ClaphamDE (1997). Nucleoplasmic and cytoplas-

mic differences in the fluorescence prop-erties of the calcium indicator Fluo-3. CellCalcium, 21: 275-282.

57. Prat AG & Cantiello H (1996). Nuclear ionchannel activity is regulated by actin fila-ments. American Journal of Physiology,270: C1532-C1543.

58. Lohret TA, Jensen RE & Kinnally KW(1997). Tim23, a protein import compo-nent of the mitochondrial inner mem-brane, is required for normal activity ofthe multiple conductance channel, MCC.Journal of Cell Biology, 137: 377-386.

59. Coronado R, Morrissette J, Sukhareva M& Vaughan DM (1994). Structure and func-tion of ryanodine receptors. AmericanJournal of Physiology, 266: C1485-C1504.

60. Lohret TA & Kinnally KW (1995). Target-ing peptides transiently block a mitochon-drial channel. Journal of Biological Chem-istry, 270: 15950-15953.

61. Bustamante JO, Oberleithner H, HanoverJA & Liepins A (1995). Patch clamp detec-tion of transcription factor translocationalong the nuclear pore complex channel.Journal of Membrane Biology, 146: 253-261.

62. Bustamante JO, Liepins A, PrendergastRA, Hanover JA & Oberleithner H (1995).Patch clamp and atomic force microscopydemonstrate TATA-binding protein (TBP)interactions with the nuclear pore com-plex. Journal of Membrane Biology, 146:263-272.

63. Jans DA & Hubner S (1996). Regulation ofprotein transport to the nucleus: centralrole of phosphorylation. Physiological Re-views, 76: 651-685.

64. Mazzanti M, Innocenti B & Rigatelli M(1994). ATP-dependent ionic permeabilityon nuclear envelope in in situ nuclei ofXenopus oocytes. FASEB Journal, 8: 231-236.

65. Henry JP, Juin P, Vallette F & Thieffry M(1996). Characterization and function ofthe mitochondrial outer membrane pep-tide-sensitive channel. Journal of Bioen-ergetics and Biomembranes, 28: 101-108.

66. Pelleschi M, Henry JP & Thieffry M(1997). Inactivation of the peptide-sensi-tive channel from the yeast mitochondrialouter membrane: properties, sensitivityto trypsin and modulation by a basic pep-tide. Journal of Membrane Biology, 156:37-44.

67. Jurgens L, Kleineke J, Brdiczka D, ThinnesFP & Hilschmann N (1995). Localization oftype-1 porin channel (VDAC) in the sarco-plasmatic reticulum. Biological ChemistryHoppe-Seyler, 376: 685-689.

68. Colombini M, Blachly-Dyson E & Forte M

(1996). VDAC, a channel in the outer mi-tochondrial membrane. Ion Channels, 4:169-202.

69. Jensen RE & Kinnally KW (1997). The mi-tochondrial protein import pathway: areprecursors imported through membranechannels? Journal of Bioenergetics andBiomembranes, 29: 3-10.

70. Crowley KS, Liao S, Worrell VE, ReinhartGD & Johnson AE (1994). Secretory pro-teins move through the endoplasmicreticulum membrane via an aqueous,gated pore. Cell, 78: 461-471.

71. Hamman BD, Chen J-C, Johnson EE &Johnson AE (1997). The aqueous porethrough the translocon has a diameter of40-60 Å during cotranslational proteintranslocation at the ER membrane. Cell,89: 535-544.

72. Perez-Terzic C, Pyle J, Jaconi M, Stehno-Bittel L & Clapham DE (1996). Conforma-tional states of the nuclear pore complexinduced by depletion of nuclear Ca2+

stores. Science, 273: 1875-1877.73. Badminton MN, Kendall JM, Sala-Newby

G & Campbell AK (1995). Nucleoplasmin-targeted aequorin provides evidence for anuclear calcium barrier. Experimental CellResearch, 216: 236-243.

74. Badminton MN, Campbell AK & RemboldCM (1996). Differential regulation ofnuclear and cytosolic Ca2+ in HeLa cells.Journal of Biological Chemistry, 271:31210-31214.

75. Dupont G, Pontes J & Goldbeter A (1996).Modeling spiral Ca2+ waves in single car-diac cells: role of the spatial heterogene-ity created by the nucleus. American Jour-nal of Physiology, 271: C1390-C1399.

76. Gerasimenko OV, Gerasimenko JV,Petersen OH & Tepikin AV (1996). Shortpulses of acetylcholine stimulation inducecytosolic Ca2+ signals that are excludedfrom the nuclear region in pancreatic aci-nar cells. Pflügers Archives, EuropeanJournal of Physiology, 432: 1055-1061.

77. Kong SK, Tsang D, Leung KN & Lee CY(1996). Nuclear envelope acts as a cal-cium barrier in C6 glioma cells. Biochemi-cal and Biophysical Research Communi-cations, 218: 595-600.

78. Seksek O & Bolard J (1996). Nuclear pHgradient in mammalian cells revealed bylaser microspectrofluorimetry. Journal ofCell Science, 109: 257-262.

79. Santella L (1996). The cell nucleus: anEldorado to future calcium research? Jour-nal of Membrane Biology, 153: 83-92.

80. Bachs O, Carafoli E, Nicotera R & SantellaL (Organizers) (1997). First European Con-ference on Calcium Signalling in the Cell

354



Nucleus. European Commission DG XII,Biotechnology Programme, October 4-8,Baia Paraelios, Calabria, Italy 4-8 October,1997.

81. Brini M, Marsault R, Bastianutto C, PozzanT & Rizzuto R (1994). Nuclear targeting ofaequorin. A new approach for measuringnuclear Ca2+ concentration in intact cells.Cell Calcium, 16: 259-268.

82. Miyashita Y, Miller M, Yen PM, HarmonJM, Hanover JA & Simons Jr SS (1993).Glucocorticoid receptor binding to rat livernuclei occurs without nuclear transport.Journal of Steroid Biochemistry and Mo-lecular Biology, 46: 309-320.

83. Haltiwanger RS, Busby S, Grove K, Li S,Mason D, Medina L, Moloney D,

Philipsberg G & Scartozzi R (1997). O-glycosylation of nuclear and cytoplasmicproteins: regulation analogous to phos-phorylation? Biochemical and BiophysicalResearch Communications, 231: 237-242.

84. Bezrukov SM, Vodyanoy I & Parsegian VA(1994). Counting polymers movingthrough a single ion channel. Nature, 370:279-281.

85. Daneholt B (1997). A look at messengerRNP moving through the nuclear pore.Cell, 88: 585-588.

86. Citovsky V & Zambryski P (1993). Trans-port of nucleic acids through membranechannels: snaking through small holes.Annual Review of Microbiology, 47: 167-197.

87. Dean DA (1997). Import of plasmid DNAinto the nucleus is sequence specific. Ex-perimental Cell Research, 230: 293-302.

88. Kasianowicz JJ, Brandin E, Branton D &Deamer DW (1996). Characterization ofindividual polynucleotide molecules usinga membrane channel. Proceedings of theNational Academy of Sciences, USA, 93:13770-13773.

89. Krasilnikov OV, Carneiro CM, YuldashevaLN, Campos de Carvalho AC & NogueiraRA (1996). Diameter of the mammalianporin channel in open and “closed”states: direct measurement at the singlechannel level in planar lipid bilayer. Brazil-ian Journal of Medical and Biological Re-search, 29: 1691-1697.

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