Recent Advances in Nuclear Electrophysiology
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References,
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Bustamante, J.O.
The Nuclear Physiology Lab-ITP-UNIT
The Millenium Institute of Nanosciences-CNPq-MCT
Rua B-508, Praia Aruana, Aracaju, SE 49037-610, Brazil
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Sunday, June 16, 2002
Key Words/Phrases:
nuclear pore, nuclear envelope, ion channel, electrophysiology,
patch-clamp, gene activity, gene expression, DNA, RNA; physiological genomics
Symbols:
NE, nuclear envelope; INM, inner nuclear membrane;
ONM, outer nuclear membrane;
NPC, nuclear pore complex;
RNE, NE electrical resistance;
GNE, NE electrical conductance;
RNPC, NPC electrical resistance;
gNPC, NPC electrical conductance; VNE,
voltage across the NE;
INE, electrical current across the NE;
Vcytoplasm, voltage at the cytoplasmic side of the NE;
Vnucleoplasm, voltage at the nucleoplasmic side of the NE;
Popen, probability of having a number of channels open in a channel population;
popen, probability of finding a channel in the open state;
pclosed, probability of finding a channel in the closed state;
EM, electron microscopy;
ER, endoplasmic reticulum;
NHT, nuclear hourglass technique
The recent synergism of the Coulter counter principle with patch-clamp for the quantification of macromolecular translocation, as well as the application of nanopore-based electrical and atomic force microscopy techniques for the analysis and manipulation of molecular and supramolecular structures, hold the promise of producing significant contributions in the nanobiotechnology of gene control and expression, DNA and RNA sequencing, cellular cloning, etc.
At the moment, two electrophysiological techniques are applied to the whole cell nucleus for the study of its function.
One technique, patch-clamp, uses a microscopic approach to directly measure single channel activity at the nuclear envelope.
From patch-clamp measurements one derives the macroscopic characteristics.
The other technique, the nuclear hourglass, uses a macroscopic approach to indirectly measure the single channel activity at the
envelope.
From its measurements, the microscopic characteristics are derived.
As efforts have just begun to develop microchips for patch-clamp at large scale, and as the post-genomic era has stimulated
physiological genomics research, it seems timely to review the basic concepts on which these two leading electrophysiological
techniques are based.
This review is conceived to complement recent in-depth reviews in the area of nuclear electrophysiology.
Finally, for sake of clarity and space, since published cell and molecular biology work in the field is copious
(click here to have a look at the PubMed database),
where possible, I have chosen the most relevant and/or the references with easiest internet access and set a cutoff date prior to the year 2000.
PREFACE
(Click here to go back to Table of Contents)
TABLE OF CONTENTS
(Click on items to go to specific sections).
Basic Principles of Electrophysiological Approaches
Patch_Clamp: The Microscopic Approach
The Nuclear Hourglass Technique, NHT: The Macroscopic Approach
Identifying Nuclear Pores As The Source For The Recorded Ion Channel Activity
Identifying Ion Selectivity for Nuclear Ion Channels
In the turbulent political decade of the
1960s, Loewenstein and colleagues published a series of papers that
demonstrated the use of electrophysiological methods for the assessment of
nuclear envelope (NE) permeability and, therefore, of nuclear pore complex
(NPC) ion conductance (gNPC). The application of electrophysiological
approaches to the analysis of nuclear function (i.e. nuclear electrophysiology)
was practically discontinued because the measurement of NE resistance (RNE)
was carried out with microelectrodes and the concern prevailed with
microelectrode plugging during their insertion into the cell and nucleus
(recently reviewed in Mazzanti et al., 2001).
The electrical approach for the assessment of NE permeability was then
resumed when the Nobel prize-winning patch-clamp technique (e.g. Neher and
Sakmann, 1991, 1992; Neher, 1992; Sakmann, 1992) was applied to the isolated
whole nucleus (Matzke et al., 1990; Mazzanti et al., 1990 – reviewed in
Mazzanti et al., 2001). Unfortunately, these
patch-clamp investigations were carried out under saline solutions, without the
necessary substrates for NPC-mediated macromolecular transport (see, for
example, Adam, 2001; Fahrenkrog et al., 2001; Komeili and O´Shea, 2001; Macara,
2001; Ribbeck and Görlich, 2001; Fahrenkrog and Aebi, 2002; Smith et al.,
2002). Fortunately, with the recent
introduction of naturally occurring living syncytial nuclei (Bustamante, 2002),
for patch-clamp and nucleocytoplasmic research, potential questions about lack
of substrates are eliminated. Since 1999, some of the patch-clamp data and
their interpretation have been either confirmed, complemented or challenged by
an indirect, macroscopic approach for the measurement of gNPC: the nuclear
hourglass technique (NHT - Danker et al., 1999; 2001; Shahin et al., 2001). The NHT derives gNPC from a simple
geometrical formula for electrical resistance, under the assumption of a
high-quality seal between the nuclear surface and the tube within which it is
placed (a concept reminiscent of the gigaseal that we will discuss later). Thus, due to the indirect nature of the NHT,
one must address the same questions raised for the few techniques
contemporaries to patch-clamp (e.g. Kostyuk, 1982, 1984; Kostyuk and Krishtal,
1984). Specifically, is the
electrolyte leakage or shunt sufficiently low to prevent proper extrapolation
to the microscopic world of the single NPC?
Or, in electrical circuit terms, is the shunt resistance (Rshunt)
much higher than the NE resistance (RNE)? The potential benefits of electrical
measurements of ion channel activity deriving from NPCs and non-NPCs are great
and far-reaching (e.g. Kasianowicz et al., 1996; Matzke and Matzke, 1996; Hanss
et al., 1998; Meller et al., 2000; Howorka et al., 2001; Marziali and Akeson,
2001; Matzke et al., 2001; Vercoutere et al., 2001; Wang and Branton, 2001;
Fertig et al., 2002; Klemic et al., 2002; Sigworth and Klemic, 2002). Therefore, we must discuss the foundations
and the current issues of nuclear electrophysiology. This is especially relevant to patch-clamp data obtained with
pipettes placed on the cytoplasmic side of the outer membrane of the NE and to
new techniques introduced to assess the macroscopic behavior of the NE. The ideas presented here are aimed at a wide
audience (from biologists and clinicians to physicists and engineers). They are also intended to complement reviews
presented elsewhere (e.g. Bustamante, 1996; DeFelice and Mazzanti, 1997;
Bustamante and Varanda, 1998; Mazzanti et al., 2001).
INTRODUCTION
(Click here to go back to Table of Contents)
Most of a cell’s electrical resistance is due
to the resistance of the cell surface membrane: the plasmalemma.
In the same manner, most of the electrical
resistance of the nucleus is due to the permeability barrier offered by the NE
to the movement of physiological ions.
The NE, however, is more complex than the plasmalemma in several ways
(see, for example, Fahrenkrog et al., 2001; Fahrenkrog and Aebi, 2002).
The envelope consists of two membranes: the
inner and the outer nuclear membranes (INM and ONM, respectively), that
delimit a cisterna known as the perinuclear space.
The cisterna seems to be reservoir for
important constituents and signaling molecules (e.g. Garner, 2001).
Both NE membranes have been shown to contain
receptors and pumps similar to those found in the plasmalemma and other
organelles:
(1) losartan-senstivity angiotensin II receptors (AT1 –
e.g. Booz et al., 1992; Tang et al., 1992; Eggena et al., 1993; Jímenez et al.,
1994; Merjan et al., 2001),
(2) IP3 receptors (IP3R -
e.g. Guihard et al., 1997; Adebanjo et al., 2000; Jaimovich et al., 2000; Tovey
et al., 2001),
(3) ryanodine receptors (RyR – e.g. Adebanjo et al., 1999, 2000;
Kapiloff et al., 2001),
(4) Na+-K+-pumps (e.g. Garner,
2001),
(5) Ca2+-pumps (e.g. Rogue et al., 1998; Abrenica and
Gilchrist, 2000), etc. (reviewed in Bustamante, 1994b).
The ONM contains ribosomes and is continuous
with the endoplasmic reticulum, ER.
This has led to the idea that, when a patch-clamp pipette is placed at
the cytoplasmic side of the NE, the ion channel activity derives only from ONM
or ER sources (see Mazzanti et al., 2001).
At discrete locations, the NPCs join these two membranes in a manner
resembling dual-membrane channels such as gap junction and plasmodesmata ion
channels.
Under the electron microscope,
EM, the large mass of NPCs (over 125 MDa in vertebrates) makes these
supramolecular structures the only conspicuous channels in the NE (e.g.
Mazzanti et al., 2001).
The NPCs are
decorated with filaments that protrude into the cytoplasm: the cytoplasmic
filaments (e.g. Fahrenkrog et al., 2001; Fahrenkrog and Aebi, 2002). On the nucleoplasmic face, filaments
emanating from the NPC form a basket: the nuclear baskets (e.g.
Fahrenkrog et al., 2001; Fahrenkrog and Aebi, 2002).
The NPC has a large central aqueous channel whose diameter for
passive diffusion is in the order of 10 nm.
This large nanochannel is present in all structural models of the NPC,
whether or not they have a central plug.
Some investigators have interpreted the plug as a transporter for
macromolecular translocation.
The most
discriminating structural data on the the central plug has been produced with
atomic force microscopy (AFM), a technique that uses a molecular-sized tip to
image the topology in chemically unfixed preparations (elasticity and other
parameters can be imaged but this has yet to be reported for the NE).
With AFM, Rakowska et al. (1998) imaged
ATP-dependent changes in the central plug whereas Perez-Terzic et al. (1996, 1999)
and Wang and Clapham (1999) imaged Ca2+-dependent changes in the
plug that they interpreted as conformational changes on a plug that forming
part of the NPC.
The central plug,
however, was not seen under AFM when macromolecular transport substrates were
provided to the preparation (Stoffler et al., 1999a; reviewed in Stoffler et
al., 1999b and Fahrenkrog et al., 2001; Fahrenkrog and Aebi, 2002).
Therefore, the controversy on whether the
central plug is an integral part of the NPC appears to have reached its final
conclusion: the central plug is a macromolecule in transit through the NPC
diffusional channel.
Accordingly,
the Coulter counter principle can be used as a working patch-clamp paradigm, to
characterize the dynamics of macromolecular translocation along the NPC
(Bezrukov, 2000).
Diffusion through the
large channel seems to operate via hydrophobic exclusion (Ribbeck and Görlich,
2002).
A decade ago, peripheral channels for ion
diffusion were proposed based on EM reconstruction studies
(Hinshaw et al., 1992 – see Fahrenkrog and Aebi, 2002).
Corroborating data from other EM laboratories have not been forthcoming.
Thus, most recent
structural models of NPCs do not incorporate this feature (e.g. Rout and Aitchison,
2001; see review by Fahrenkrog and Aebi, 2002).
Peripheral channels are tantamount to interpreting the background
or leakage current obtained with the indirect, macroscopic approach of the NHT
(Shahin et al., 2001) as the authors have recently interpreted this leakage as
resulting from ion flow along these peripheral channels.
In contrast, only a single diffusional
channel was derived from direct EM observations (Felherr and Akin, 1997) and
from high-resolution fluorescence microscopy (Keminer and Peters, 1999).
Therefore, when taken together, both EM and
fluorescence microscopy do not support the existence of these peripheral
channels.
Furthermore, since
patch-clamp also does not support the existence of the peripheral channels
(e.g., Bustamante et al., 1995a, 2000a; Bustamante, 2002), we are forced to
analyze the assumptions on which NHT is based.
As some of the disagreement between patch-clamp and NHT data
(existence of peripheral channels, leak or shunt resistance, etc.)
appears to originate from different interpretations of the
electrical circuit theory, we need to discuss the principles that underlie
these two leading techniques of nuclear electrophysiology.
I shall, however, refer the reader to other
publications that deal with a more in-depth analysis of the general methodology
(e.g. DeFelice, 1997; Ypey and DeFelice, 1999).
In the end, we shall learn what biologists know well:
that not all in nature is black and white
as formulas and equations would have us believe.
Seminal to the measurement of electrical
resistance is Ohm’s law which tells us that the movement of positive electrical
charges per unit time (i.e. electrical current, I) between two points, say 1
and 2 (i.e. I12), is given by the voltage drop across these two
points (V12) divided by the resistance between these two points (R12):
In terms of the NE, we have (see also DeFelice and Mazzanti, 1997;
Bustamante and Varanda, 1998):
(2)
INE = VNE / RNE
or
INE = GNE x VNE
where INE is the electrical current
(produced by the flow of ions, the major electrical charge carriers)
between the outside and inside faces of the NE,
VNE is the voltage drop across these two faces and
RNE is the electrical resistance posed by the NE to the ion flow
(the inverse of NE conductance:
GNE
).
If Vcytoplasm is the voltage on
the cytoplasmic side of the NE and Vnucleoplasm is the voltage at
the nucleoplasmic side of the NE, then:
(3)
VNE
= Vnucleoplasm – Vcytoplasm
Under no nucleocytoplasmic concentration gradient for the electrical charges
(e.g. if K+ were the charge carriers, [K+]cytoplasm=[K+]nucleoplasm),
when Vcytoplasm>Vnucleoplasm, positive charges will
move toward the nucleoplasm.
For negative charges (e.g. Cl-, with [Cl-]cytoplasm=[Cl-nucleoplasm)
their movement will be towards the nuclear exterior.
That is, in both cases the electrical current is inward
(inward movement of positive electrical charges)
although the flow of ions may not be
inward if the ions are negative.
Thus, one must not confound the direction of the flow (defined as the movement of
particles of any charge) with the direction of the ion current (defined as the
movement of positive electrical charges).
Fig. 1
illustrates the flow of positive charges for
Vcytoplasm>Vnucleoplasm.
When Vcytoplasm<Vnucleoplasm,
and there is no concentration gradient, the flow of ions is reversed and the
currents are outward (negative ions will move inwardly).
Finally, in the absence of gradients for
both the chemical potential (e.g. [K+]cytoplasm=[K+]nucleoplasm)
and the electrical potential
(i.e. Vcytoplasm=Vnucleoplasm)
there is no electrical driving force for the charge carriers is absent and
there is no ion flow if there is no concentration gradient.
Note that, as established in the classical ion channel theory
(e.g. Hodgkin and Huxley, 1952 – see Huxley, 2002),
the force determining the movement of ions is of electrochemical origin.
For an NE patch, the total current due to an ion species X is given by Ohm’s law:
(4)
IX =
GX (VNE-VX)
where GX is the patch
conductance for the ionic species X and VX is the equilibrium
potential for this ionic species (also known as Nernst potential, Nernst-Planck
potential, etc.).
In its simplest form, commonly found in textbooks:
(5)
VX = (RT/FzX) ln ([Xcytoplasm]/[Xnucleoplasm])
where R is the gas constant,
T is the absolute temperature, zX is the valence of the ion species
X, F is the Faraday constant, and [Xcytoplasm] and [Xnucleoplasm]
are, respectively, the concentration of ions on the cytoplasmic and
nucleoplasmic sides of the NE.
This equation will be relevant to our discussion on ion selectivity, later in this chapter.
To date, there appears to be a
general agreement that the equilibrium potential for the major electrical
charge carriers (e.g. K+) is negligible (e.g. Bustamante, 1992,
1993; Boehning et al., 2001).
Since ions in solution are the major
electrical charge carriers in nuclear electrophysiology, for large preparations
generating large currents, two pairs of half-cell electrodes (e.g. Ag-AgCl) are
used to avoid effects of electrode polarization due to electrolysis and
electrodeposition (e.g. Sherman-Gold, 1993).
For patch-clamp measurements of single ion
channel gating, electrode polarization is not a concern due to the low values
of the currents generated and, therefore, only two Ag-AgCl electrodes are used
(e.g. Sherman-Gold,
1993).
The two electrodes are connected to the
probe of an instrumentation amplifier that operates like a virtual-ground (an
operational amplifier connected in such a manner that forces the input to the
ground potential).
The electrode used
to record current, the one inside the patch-clamp pipette, is forced to a known
voltage (for this reason, it is not a virtual ground configuration).
The bath electrode is used as reference
(Vreference =Vbath = 0).
Therefore, the
potential difference applied to the NE is the difference between the potential
inside the pipette, Vpipette, and Vreference.
Note, however, the potential experienced by
the NE will be influenced by the NE potential when there is no potential
applied: the NE resting potential.
As mentioned above, so far, the experiments have shown that this potential is negligible
under steady-state conditions (when the nucleocytoplasmic concentration
difference has had time to dissipate).
Consequently, one is to expect that, in patch-clamp, the ion flow will
be controlled by Vpipette only because Vreference = 0.
A major problem with any electrophysiological
technique is the presence of a leak or shunt resistance (Rshunt), in
parallel to the target of measurement (the nucleus in our case), through which
electrical charge carriers escape.
Another problem, dealt with since the times of Hodgkin and Huxley
(1952), is the presence of a series resistance (Rseries) through
which there will be a voltage drop proportional to the current.
As a result of Rseries, the
resistance offered to the movement of electrical charges is increased by this
amount and the resistance that is the target of our measurement (i.e. RNE)
is underestimated.
In mathematical
terms, the resistance measured (Rmeasured) is:
(6)
Rmeasured = Rseries +
[ (Rshunt)-1 + (RNE)-1 ]-1
As a consequence of the above relationship,
there are two golden rules of electrical circuits.
First, the measured resistance, Rmeasured, of a
circuit of resistors in series will always be greater than the value of the
largest resistor.
Second, and most relevant to our case, the measured resistance, Rmeasured, of a
circuit of parallel resistors can never be greater than the smallest component
resistance: Rshunt or RNE.
Finally, other components of the electrical
circuit (e.g. access resistance), are of importance but not relevant to our
central theme.
For this reason, the
interested reader should consult the excellent textbooks on the topic (e.g.
Sakmann and Neher, 1995).
The study of native, in situ single
ion channel behavior was greatly facilitated with the introduction of the
patch-clamp technique for the imposition of a voltage gradient across a
membrane (i.e. voltage-clamp –see Sakmann & Neher, 1995).
Patch-clamp succeeded when the possibility
was discovered to attain Rshunt in the order of gigaohms (GW, 109
ohm).
The gigaohm value indicated that
the seal resistance, Rseal, between the tip of the glass pipette and
the biological membrane was in the order of GW.
This seal was henceforth coined the gigaseal.
This breakthrough took
patch-clamp to the spotlight, for the gigaseal was the last remaining
requirement for the precise analysis of ion channel function (the other
requirements, mainly concerned with solid-state electronic devices, had been
conquered).
The gigaseal was not just a
mere achievement of a high-valued Rshunt for it also dramatically
reduced the baseline noise (see Neher’s Nobel lecture in Neher and Sakmann,
1991 and Neher, 1992).
(7)
stheoretical = (4 kT Df)1/2 x Rsystem-1/2
from where
Rsystem =
constant x
stheoretical-2
where stheoretical is the
theoretical current noise of the signal source, k is the Boltzmann constant, T
is the absolute temperature (here assumed constant during an experiment), Df is the
frequency bandwidth (also assumed constant), and Rsystem is the
resistance of the system (i.e. Rmeasured, determined by RNE
and Rshunt as discussed above).
From this equation one reasons that for a bandwidth of 1 kHz, and a 1 pA
of ion channel current one should have an Rsystem>2 GW to guarantee a
10% resolution of the signal (see Neher, 1991).
This particular characteristic of the noise in patch-clamp
recordings is useful to monitor whether a gigaseal has been attained, even when
the NPCs of an NE patch are all simultaneously open and thus the Rshunt
cannot be measured.
Patch-clamp, reliably measures Rshunt
and this quantity gives the value of Rseal whenever there is no ion
channel opening and the membrane is not leaky to ions.
However, if we consider the contribution
from the ion flow through the channels at the NE (NPCs and channels at the ONM
and INM), Rshunt is derived from the parallel circuit formed by the
seal resistance, Rseal, and the NE resistance, RNE.
(8)
Rmeasured = Rshunt =
[ (Rseal)-1 + (RNE)-1 ]-1
Thus, when all channels are closed (i.e. RNE
>> 1), if Rmeasured > 1 GW, then we can be
sure that Rseal > 1 GW.
That Rseal > 1 GW in patch-clamp
investigations of the NE can be appreciated in the recordings of several
patch-clamp publications and, therefore, should not be taken as a mystery (e.g.
Bustamante, 1992, 2002).
Thus, the
patch-clamp researcher is neither required, nor should he/she expect, to have a
gigaseal prior to measuring the patch ion conductance if the number of NPCs
conducting ions is sufficiently high.
One may have to wait for a long time before a simultaneous closing of
all the channels is observed (e.g. Bustamante, 2002).
For example, if the patch has 2 NPCs of gNPC = 500 pS
simultaneously open, then the patch ion conductance (Gpatch) would be 1 nS
and Rmeasured ≤ 1 GW, even if Rseal
≥ 10 GW.
Likewise,
for 10 NPCs of 500 pS simultaneously open, Gpatch = 5 nS and Rmeasured
≤ 0.2 GW, even if Rseal ≥ 10 GW.
Thus, a general rule for patch-clamp of the
NE is that, when all NPCs are open, say N, Rmeasured ≤ (N x gNPC)-1,
even if Rseal ≥ 10 GW.
Patch-clamp must be applied and interpreted
with caution because the structure of the NE is more complicated than that of a
single membrane.
For example, for the
nucleus-attached patch configuration (i.e. pipette tip touching the ONM of a
whole nucleus), one has to check that the bath electrode makes a virtual
connection with the INM through the nucleoplasm.
As shown in
Fig. 2
this can be
demonstrated with fluorescence microscopy by placing small fluorescent probes
(e.g. 4-kD dextran, 5.4-nm dendrimer) on the cytoplasmic side of the nucleus
(Fig. 2a,c) and showing their
diffusion into the nucleus (Fig. 2b,d - see
Bustamante et al., 2000b; Bustamante, 2002).
Under these conditions, VNE is given by the voltage
difference between the bath and pipette electrodes (see discussion above).
It then follows that, in the
nucleus-attached configuration, if the NPCs are closed then any recorded ion
channel activity should derive from sources other than NPCs because these
sources should be the pathway to close the circuit between the pipette and bath
electrodes.
Since I have never seen ion
channel activity when the NPCs are closed or plugged (demonstrated by the lack
of entry of small dextran and dendrimer particles upon macromolecular transport
stimulation – e.g. Bustamante et al., 2000b, Bustamante, 2002), I cannot
exclude NPCs as sources of ion channel activity, even when the ion conductance
is below 100 pS.
For this reason, the
ion channel activity may be interpreted as a sub-state for ion conduction of
the NPC (Bustamante, 1994a).
Under this
context, it can then be reasoned that a change in ion activity induced by
hormones and other agonists (e.g. angiotensin II, IP3, ryanodine,
etc.) may be the result of either direct or indirect actions, mediated by their
corresponding receptors and whatever coupling mechanisms there may be such as
G-proteins and Ca2+ release feedback.
For the excised patch configuration (i.e. pipette tip touching
the ONM of a piece of excised NE), the requirement for demonstrating the status
of NPC ion conduction remains.
Only when we have proven that the NPCs are closed or plugged (e.g. with fluorescence
microscopy) we can say that the ion channel activity derives from sources other
than NPCs.
The understanding of this
requirement holds the key to understanding the relationship between
NPC-mediated nucleocytoplasmic transport of macromolecules and ion channel
activity recorded from the NE (i.e. the Rosetta stone of nuclear
electrophysiology).
Indeed, ever
since the first usage of a medium that supported macromolecular transport along
NPCs, as well as transcription and translation (e.g. TnT, Promega), it was observed that macromolecular transport
along NPCs restricts ion diffusion (Fig. 1 in Bustamante, 1994b; see also
Bustamante et al., 1995a-c 2000a,b; Bustamante, 2002).
Fig. 2
also illustrates an experimental fluorescence microscopy protocol for the
determination of the capacity of NPCs for macromolecular translocation and the
role of this phenomenon on NPC ion channel activity (e.g. Bustamante et al.,
1995a, 2000a,b; Bustamante, 2002).
Briefly, a nuclear-targeted macromolecule (one that contains a nuclear
localization signal or NLS) is placed on the cytoplasmic side (Fig. 2e).
A convenient probe that I have used (e.g. Bustamante et al., 1995a, 2000a,b; Bustamante, 2002) is the high quantum efficiency B-phycoerythrin (240 kDa, Molecular Probes), conjugated to the NLS of the SV40 large T antigen (Sigma Chemical).
If the NPCs are capable of macromolecular transport, then the macromolecular probe will go inside the nucleus (Fig. 2f).
If not, it will remain outside (Fig. 2e).
When used in the radio frequency range, the
same approach used by NHT can produce information on general cellular
characteristics such as the nuclear-to-cytoplasmic volume ratio – a parameter
relevant to various pathological states of cells (e.g. Coulter International,
1996; Beckmann-Coulter, 2002).
The radio frequency currents are electrically transparent to the cell membranes and
have the added advantage that they also appear to leave the cells undisturbed
(e.g. Coulter International, 1996; Beckmann-Coulter, 2002).
In the NHT (Danker et al., 1999, 2001 – no frequency details given),
a known value of current, Iinjected,
is injected to the lumen of the capillary tubing and the voltage drop between the
two ends of the tubing measured, Vmeasured.
From these values, the electrical resistance
of the system is calculated, Rmeasured:
(9)
Rmeasured = Vmeasured / Iinjected
If Iinjected were constant, then,
by simple differentiation, it can be shown that the variation in Vmeasured,
DVmeasured, caused by the introduction of
the nuclei inside the capillary, can be used to calculate the variation in Rmeasured,
DRmeasured (DR in Danker et
al., 1999, 2001):
(10)
DRmeasured = DVmeasured
/ Iinjected
Note, however, that since Iinjected
is alternating when not in the electrophoretic mode (see Danker et al., 2001),
the above relationship does not necessarily hold.
In setting the foundations for NHT, Danker et al. (1999) depart
from the undisputable premise that the measured resistance, Rmeasured,
is the result of the resistance of the cell nucleus (Rnucleus) in
parallel with Rshunt.
Although no explicit consideration of Rseries was made, this
seems of no major consequence as the analysis is made with changes in Rmeasured
(i.e. DRmeasured or DR) and Rseries
should vary very little upon the introduction of the nucleus inside the tubing.
(11)
Rmeasured = Rseries +
[ (Rshunt)-1 + (Rnucleus)-1 ]-1
(12)
Rnucleus = { [ DRmeasured-1 - Rshunt-1
}-1
In the NHT Rshunt is estimated by
using, instead of a nucleus, a stage I whole oocyte of Xenopus L. and by
applying a direct (rather than alternating) current to increase the value of Rmeasured.
With their most recent approach (Danker et al., 2001)
they arrived at a value of about 650 pS rather than the 1,700 pS
previously calculated (Danker et al., 1999).
Since their original approach (Danker et al., 1999) has been replaced
(Danker et al, 2001), I shall concentrate on their most recent data and their
interpretations.
There are several problems with the assumptions made in the NHT.
1st
The use of a whole oocyte is an approach that the investigators agreed is
controversial (see bottom of page 13532 in Danker et al., 1999).
2nd
The surface properties are probably not the same for the oocyte surface membrane and the NE.
Therefore, it is likely that stickiness
between the NHT tube and membrane surface of the oocyte and its NE are quite
different.
3rd
The responses
to physical and chemical maneuvers (e.g. voltage and antibodies) cannot be
assumed to be the same for both the oocyte plasmalemma and the NE.
4th
The large body of accumulated
experience with similar techniques (e.g. Fishman, 1975; Lopez-Barneo et al.,
1981; Kostyuk, 1982, 1984; Kostyuk and Krishtal, 1984) lead us to conclude that
the leak in the NHT must be of concern.
My own experience
(e.g. Bustamante, 1981, 1983; Bustamante and McDonald, 1983 – see
Fig. 3
in this paper)
is that current injection alone can be used to increase Rshunt (a
current of opposite polarity usually decreases Rshunt).
This issue will be revisited later in this chapter.
My patch-clamp observations (e.g. Bustamante et al., 1991; Ruknudin et al., 1993),
shared by the patch-clamp community
(e.g. Sherman-Gold, 1993) include
that current injection alone can be used to Rshunt,
(opposite polarity in Iinjection usually decreases Rshunt).
This phenomenon does not require NPC plugging.
Therefore, the Aquilles’ heel/tendon of the NHT is RShunt.
For this reason and for the significance of the interpretations of NHT
data, we must center our attention on NHT justification for their assumptions
on Rshunt.
In the NHT, a good experiment is defined as
one in which the lumen of the capillary neck (thus their terminology of hourglass)
has a diameter smaller than the diameter of the nucleus
(Danker et al., 2001).
This produces two pieces of NE in unrestricted contact with the physiological saline.
NHT assumed that the tubing and the nucleus were cylindrical,
that the width of gap between the nucleus and the capillary was w,
and that the surface of interest between the NE and the tubing wall
(the one where they had macroscopic contact) had a length L (Lgap),
and radii of r (rgap) and r-w (rnucleus), respectively.
NHT investigators determined that the electrical conductivities of their saline
(k)
and the nucleoplasm (k´)
had the same value of 13.2 mS/cm2 (I think that they meant to say mS/cm).
Based on the geometry of their system, they arrived at the following formulas.
(13)
Rshunt
= L k-1 p-1 (r2 - (r - w)2)-1
(14)
Rnucleus = RNE + Rintranuclear
(15)
Rintranuclear
= L k-1 p-1 (r - w)-2
They defined Rreplaced as the
resistance of the space replaced by the nucleus and the shunt. Thus:
(16)
Rreplaced
= L k-1 p-1 r-2
From these formulas, they derived the formula
for RNE:
(17)
RNE = ((DR+Rreplaced)-1
– Rshunt-1)-1 – Rintranuclear
NHT also assumed that all measurement errors
are negligible, except for w, because “they can be easily measured with fair
resolution” (Danker et al., 2001 – but see Appendix in this paper).
Note, however, that NHT proponents did not
consider that in the intact NE, the NPCs have protrusions into the cytoplasm
(the cytoplasmic filaments discussed above).
These protrusions may have interfered with the surface scan of their AFM
probe or may have collapsed during isolation of the oocyte nucleus.
Furthermore, as these investigators
previously showed with AFM (Danker et al., 1997), their Xenopus L.
oocyte nucleus was contaminated with ER and by other structural features.
It must be added that transmission EM
demonstrates that the NE surface is not a smooth surface (e.g. Feldherr and
Akin, 1997; Panté and Kann, 2001).
Therefore, it would appear that the gap width, w, used in the
mathematical model of Danker et al. (2001) could have been better, and much
more easily evaluated, with readily available fluorescent probes (e.g.
calibration probes from Molecular Probes).
NHT investigators concluded that Rshunt>50
Rnucleus (i.e. Rshunt>>RNE) and that
their measurement error was as small as 3.1% (Danker et al., 2001 – but, again,
see Appendix in this paper).
Since the calculations carried out by them are the basis for their justification, they
deserve a close scrutiny.
From their calculations, with L=270
mm, r=135 mm, w=1 mm and k=13.2 mS/cm2 (not mS/cm as it should be)
one has Rshunt = 24,000 W.m.
When the correct units for conductivity
(i.e. mS/cm) one obtains 2.4 MW.
Fig. 4
a shows a curve
drawn with the expected values from the contributions to NE resistance of the
NE areas involved (the caps created by squeezing the nucleus inside the
hourglass region of the tube). Please,
note that this curve is not drawn according to Danker et al. (1999, 2001). Say that the surface density of ion
conducting NPCs, sNPC, is 10 NPCs/mm2 and
that the single NPC channel conductance, gNPC, is 500 pS.
These values are in the order of the most
recent data from Oberleithner´s group: gNPC (Danker et al.,
2001) and sNPC (Schäfer et al., 2002).
The conductance of an NE segment, GNE, is determined
by sNPC, gNPC and by the area
of the NE surface, ANE:
(18)
GNE = sNPC x ANE
x gNPC
EXPERIMENT AND THEORY
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Structural Considerations
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Basic Principles of Electrophysiological Approaches
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Patch_Clamp: The Microscopic Approach
(Click here to go back to Table of Contents)
The Nuclear Hourglass Technique, NHT: The Macroscopic Approach
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When ANE is 10,000 mm2,
GNE will be 10-5
S and RNE will be 20 kW .
If we now consider that there are two electrically resistive surfaces connected in
series, then the contribution of these two segments of the NE will be 40 kW.
Values of RNE computed with this approach are given in
Fig. 4a
for radii of the NE caps (areas in unrestricted contact with the saline) between 5
and 100 mm.
As shown in Fig. 4a,
RNE values ranged between 12.7 kW and 5.1 MW. That is, they never fell below 10 kW
.
Fig. 4b
shows curves predicted by NHT formula for Rshunt (Danker et al.,
2001), using the conductivity of 10 mS.cm-1 and the geometry for
patch-clamp and NHT.
As clearly seen from Fig. 4b,
according to the NHT model, Rshunt should very high,
never be below 1 MW.
Contrasting their theoretical calculations are my unpublished
observations on Rshunt made during the experiments I carried out
between 1980 and 1990 with adult cardiac myocytes and with cultured
neuroblastoma cells (e.g. Bustamante 1981, 1983, 1985, 1989; Bustamante and
McDonald, 1983).
It is very likely that contemporary investigators using pipettes and tubes for voltage-clamp had similar experience to mine but that,
like me, considered them of no scientific
relevance.
The value of Rshunt
that I obtained when I perfused the tubing with physiological saline was in the
order of 1 MW while a cell was placed inside the small hole of
the intracellular perfusion tubing
(Fig. 3).
When I used the NHT formula to calculate the
Rshunt expected for my intracellular perfusion experiments with human
heart atrial myocytes (e.g. Bustamante and McDonald, 1983), I obtained a value
of 40.8 MW for L= 5 mm, r = 2 mm, and w=0.1 mm.
As I used enzymatic digestion for the
isolation of the myocytes (e.g. Bustamante et al., 1981, 1982a,b), the membrane
coat or glycocalyx was greatly removed and, therefore, the gap width between
the cell surface and the tubing should certainly be in the nanometer scale (see
EM images in Bustamante et al., 1981, 1982a).
Perhaps, more telling about the correctness of the model is that when I
applied the NHT formula for the calculation of Rshunt for
cell-attached patch-clamp experiments, the mathematical prediction was that the
indirect macroscopic approach of NHT had a significantly better seal than
patch-clamp.
Fig. 4b shows
the expected values of Rshunt for patch-clamp and NHT, as a function
of the gap width, w, gap lengths (Lgap) fixed to 100 and 0.1 mm, respectively,
and gap radius (rgap) fixed to 100 and 1 mm,
respectively.
The conductivity of the
fluid was set to 10 mS.cm-1 and, therefore, its resistivity (rfluid) was fixed at
100 W.cm.
Fig. 5
shows how
chemical and physical maneuvers can be used simultaneously to eliminate the
current leak.
In the specific experiment with adult human heart myocyte (Bustamante, 1980 - unpublished), Rshunt
was dramatically increased upon replacement of Cl- in the
intracellular perfusion with F- and of K+ with Cs+.
This maneuver also facilitated the study of
Na+-channel currents by blocking other ion channel currents (e.g.
Bustamante and McDonald, 1983).
The computations using the NHT formula
demonstrate that there must be other physical and/or chemical factors that were
not considered in the mathematical modeling of Rshunt.
One simple explanation is that their
simplifying formulas were originally conceived for systems of larger dimensions
and/or for diluted solutions and, consequently, these formulas are valid when
there is no molecular crowding and interactions, and other chemical and
physical principles that take effect at the molecular level.
Indeed, the conclusions presented here are
supported by their very own measurements of Rshunt when they used
the whole oocytes in stage I (Danker et al., 1999).
Their reported values for Rshunt were greater than 30
kW, a value that contradicts my computations with
their formula when the dimensions for conductivity are corrected (i.e.
geometrical formula predicts values greater than 1 MW – see Danker et
al., 2001).
Other explanations may also be given.
1st
Their injected currents produced voltage drops of opposite polarities at each of the two NE caps in unrestricted contact with the
saline solution.
According to their observations, the currents produced readily observable changes in the chromatin structure, suggesting NE
alterations.
2nd
The voltage drop caused by their currents could be substantial and could have
caused irreversible damage of the NE, leading to an artificially leaky NE.
Indeed, if their rationale that Rshunt>>RNE,
were correct, then most of injected current would flow through the NE and, with
their minimal value of Iinjected of 100 mA (maximal of
1,000 mA in Danker et al., 1999; 200 mA in Danker et
al., 2001), they would have caused a voltage drop with a lower limit of about
1,000 mV when RNE was 10 kW
(see calculations for RNE in the following paragraph).
In my experience, the NE from adult cardiac myocytes and cancer cells cannot
withstand voltages greater than ±50 mV for tens of seconds
(Danker et al., 2001 injected currents for several tens of seconds).
This observation has prompted me to favor
the use of 2-s voltage pulses in my experiments (e.g. Bustamante et al., 1995, 2000a,b).
3rd
Their placement of the nucleus inside the tubing may have caused unnatural
stretch-induced channel openings or closings (e.g. Bustamante et al., 1991;
Ruknudin et al., 1993).
4th
Their experimental system lacked control of the voltage across the NE, both
spatially and temporally (e.g. Johnson and Lieberman, 1971) because some of the
currents have an inactivation-like mechanism (e.g. Bustamane, 1992).
A conclusion made with NHT that deserves our
attention is the data interpreted as supporting the existence of the
diffusional channels peripheral to the large central channel of the NPC (Danker
et al., 1999, 2001; Shahin et al., 2001; Schäfer et al., 2001 - see Mazzanti et
al., 2001).
If valid, the conclusion
leads to the interpretation of the function of these putative channels as alternate
pathways for passive ion transport during macromolecular plugging of the NPC
(see Mazzanti et al., 2001).
However,
the accumulated experimental data from patch-clamp as well as from pipette- and
tubing-based voltage-clamp techniques suggest that the electrical leak in NHT
is not negligible (see above discussion).
Since the current NHT model does not fit the experimental observations
(see Fig. 4),
the interpretation of the leak current as originating from peripheral channels
will require further experimental, and not theoretical, work.
This conflict between experiment and theory
does not arise when the patch-clamp technique is applied to the cell nucleus
because no published patch-clamp data supports the existence of these channels.
Indeed, since EM shows that
NPCs has a preferred 8-fold geometry (see Fahrenkrog and Aebi, 2002), one is to
expect that if there were 8 identical channels peripheral to the large central
NPC channel, then one should observe a statistical probability function with 8
peaks corresponding to 8 identical channels per NPC (or to a multiple of 8) if
these peripheral ion channels function independently of one another.
Instead, such a probability function has
never been observed (e.g. Bustamante, 1992).
Let us see the statistical basis for this prediction.
For a steady-state system consisting of 8
identical units (the putative peripheral channels are identical) fluctuating or
gating (in the biophysical sense) between 2 states (say fully open and
fully closed) with two fixed or quantized values (say 0 and 500 pS,
corresponding to the open and closed states, respectively), we have a
distribution of peaks (each peak described by a normal distribution) that is
described by the binomial distribution:
(19)
Popen(n) = [ N! /
n! (N-n)! ] popenn (1-popen)N-n
)
where Popen is
the open probability of the channel population, popen is the
probability that a channel opens, N is the total number of functional ion
channels (whether open or closed) and n is the number of ion conducting, open
channels (see Bustamante, 1992).
Excellent simulation tools for the discrete binomial distribution
function, and for its analog continuous function (normal distribution), are
currently available in the internet (e.g. Narasimham, 2002; Stark, 2002). For a two state channel system, the
probability that a channel is open, popen, plus its probability of
being closed (pclosed) should always be one. Note that, as originally conceived (see
Hinshaw et al, 1992), these 8 peripheral channels are permanently open. Therefore, a constant current (i.e. very
similar to a technical artifact caused by electrical leak or shunt) should be
recorded. This is also not observed in
patch-clamp experiments.
Fig. 6
a shows
simulations of the probability function histograms for a population of 4, 8,
16 and 24 identical independent
channels with 2 states: open and closed, and for conditions in which popen
is 50% and 75%.
The 4, 8, 16 and 24 values of N would correspond to 0.5, 1, 2 and 3 times the number of peripheral channels assumed to exist in an
NPC.
Note that each bar in the histogram corresponds to a current level
(here set to an arbitrary unity, say 400 pA)
plus its variation (here half the unit value, say 200 pA).
That is, the bar graphs correspond to superimposed normal distributions.
Thus, for example, for the 4-channel histogram
(half the population of peripheral channels of an NPC)
we would have 4 normal, bell-shaped curves (see Bustamante, 1992).
As shown by panel b of Fig. 6,
the experimental data does not support the existence of these peripheral
channels because one never records such a Popen.
As mentioned at the beginning of this
chapter, an additional source of confusion is the fact that many patch-clamp recordings
are carried out in the nucleus-attached mode, a procedure in which the tip of
the patch-clamp pipette is placed against the outermost NE membrane, the
ONM.
Clearly, if the NPCs were
permanently closed or plugged, the ion channel activity recorded in this mode
would have to come necessarily from ion channels at the ONM.
Instead, it has been shown with fluorescence
microscopy that when nuclei are bathed in saline, small particles such as
dextrans and dendrimers do enter the nucleus (Bustamante et al., 2000a,b;
Bustamante, 2002).
Therefore, due to the large contribution of NPCs to nucleocytoplasmic ion flow,
electrophysiological measurements must be validated with an independent
technique, such as fluorescence microscopy, to test the state of conduction of
the NPC population.
While one should
not be skeptic about the existence of important ion channels at the ONM and INM
(e.g. Rousseau et al., 1996; Franco-Obregon et al., 2000; Guihard et al., 2000;
Valenzuela et al., 2000; Boehning et al., 2001; see Mazzanti et al., 2001), to
avoid potential conflict with prevalent cell biology concepts, the ion channel
investigator using whole nucleus must prove that NPCs are excluded as the
source of ion channel activity.
Note that this is not required for those using artificial systems such as lipid
bilayers.
Fig. 7
illustrates both large
and small conductance ion channel activity detected with patch-clamp from
NE patches of Dunning G prostate cancer cells (Bustamante, unpublished – see
also Bustamante et al., 2000a).
In my experience, the small conductance ion channel activity is a rare event
(in the order of 1 in 100 experiments).
It is not clear whether the small conductance ion channel activity results from a
partially occluded NPC or whether it is a genuine sub-state for ion conduction
along the NPC.
That it cannot correspond to the hypothetical peripheral channels is seen by the fact that the
value of the small conductance activity is not an 8th of the large conductance value.
The small conductance activity may also be interpreted as a sub-state of the channel
(Bustamante, 1994a).
Finally, the low
probability with which I observe these small ion channel openings may be due to
the fact that my techniques produce isolated nuclei with NPCs capable of
macromolecular transport.
In fact, I recently showed in intact living syncytial nuclei that when the extranuclear
environment is replaced with saline, the final state of the NPCs is one of 100%
open probability (Bustamante, 2002).
Identifying Nuclear Pores As The Source For The Recorded Ion Channel Activity
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Although all electrophysiological
observations are of great value, perhaps the most relevant to current cell and
molecular biology is the possibility of quantifying NPC gating and transport
capacity with electrophysiological techniques.
Since the techniques for the analysis of ion channels other than NPCs
are standard to channels found in other cellular structures (recently reviewed
in Mazzanti et al., 2001), it seems more productive for us to focus on the
description of how can we tell that NPCs, and not other channels, are
responsible for the observed ion channel behavior.
As discussed above, the fact that the recording
patch-clamp pipette is placed on the cytoplasmic face of the NE may be taken to
mean that the recorded ion channel activity derives from ion channels from the
ONM.
To determine if this is indeed the
case, one must carry out the fluorescence microscopy tests described above
(see
Fig. 2)
for determining the capability of NPCs for the passive diffusion of ions and
for the active transport of macromolecules.
It is important to note that NPCs appear to require the integrity of the
NE for leak of its cisterna (i.e. the perinuclear space) will result in the
impairment of NPC-mediated macromolecular transport
(e.g. by Ca2+ loss – see Bustamante, 1994b, Bustamante et al., 1994).
To further support that the source of ion
channel activity are the NPCs, one may apply an NPC monoclonal antibody that
has been shown effective on blocking ion channel activity (Bustamante et al.,
1995a; Prat and Cantiello, 1996).
Finally, one may use the concept developed for other preparations that
the organelle-targeted proteins block ion channel activity
(e.g. Thieffry et al., 1992; Lohret and Kinnally, 1995;
Pelleschi et al., 1997; Teter and Theg, 1998; Heins et al., 2002 –
see reviews by
Neuhaus and Wagner, 2000; Bölter and Soll, 2001)
which, in our case, would be macromolecules
known to contain nuclear localization signals (NLS) or artificially conjugated
to contain an NLS (Bustamante et al., 1995a, 2000a,b; Bustamante, 2002 - see
Fig. 2).
To determine whether ER channels are present
in the patch from which one is recording activity from fewer simultaneous
channel openings than the NPC surface density (e.g. less than 5 channel
openings when the NPC density is 10 per
mm2),
one may use puromycin to clear out protein conducting channels of the ER from
nascent proteins (Simon and Blobel, 1991).
In my hands, however, the application of puromycin to NE patches has
neither resulted in the appearance of new, added ion channels nor in the
alteration of the recorded ion channel activity (e.g. Bustamante et al., 1995a – Bustamante, unpublished data).
It is for these reasons that, under these conditions, it is possible to state without
hesitation that the observed derives from NPCs and not from other
structures.
Therefore, patch-clamp is a
useful tool to investigate and quantify NPC function.
One should expect that NHT would also be very useful
(like the loose patch-clamp technique has been)
once independently supported with the
fluorescence microscopy tests that I proposed above and illustrated in
Fig. 2.
Identifying Ion Selectivity for Nuclear Ion Channels
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So far, it appears that only K+,
under normal conditions, is the major charge carrier for currents purportedly
resulting from the NPC (e.g. Bustamante, 1992, 1993). IP3R-operated channels have been assigned Ca2+
selectivity (Boehning et al., 2001).
Selectivity for Cl- has been assigned to channels from other
preparations (e.g. Franco-Obregón et al., 2000; Valenzuela et al., 2000 – see
Mazzanti et al., 2001 for a complete list).
To estimate the selectivity of an ion channel one departs from the
concept that, in a complex system, the equilibrium or resting potential is
given by the relative contributions of each ion species diffusing through the
channel:
(20)
Vsystem = S CjVj
where Cj and Vj
are, respectively, the relative conductance coefficient and the equilibrium
potential for the jth ion species.
Note that the equilibrium potential of a system can be measured as the
value of potential at which the current reverses direction. For this reason, this potential is also
known as the reversal potential (Vreversal). The relative conductance coefficient, Cj,
is given by:
(21)
Cj = Gj/Gtotal and
Gtotal = S Gj
where Gtotal is
the sum of all the relative coefficients.
One next has to take into account the fact
that the permeability is a quantity that refers to particles and not to charges
(this is the reason why we have the valence of the ion in the Nernst
equation). Therefore, the results have
to be calibrated accordingly. For a
system consisting of K+ and Ca2+ ions, one has, according
to the Goldman-Hodgkin-Katz equation (see Hille, 2001):
(22)
Vreversal = (RT/F)
ln{(PK[K+]cytoplasm+2PCa
[Ca2+]cytoplasm)/(PK[K+]nucleoplasm+2PCa
[Ca2+]nucleoplasm)}
where the sub-index for each
parameter indicates the compartment to which one refers to. Note that if there were an electrogenic pump
(an ATP-dependent mechanism that generates an electrical current during
exchange of ions between the two sides of the membrane), an additional term
would have to be added or subtracted to the factor containing the
permeabilities. So far, no report has
appeared on electrogenic pump of the NE.
To simplify the biophysical analysis, one
uses bi-ionic conditions (e.g. Hille, 2001).
Bi-ionic conditions mean that, at one side of the membrane one replaces
the reference ion (e.g. K+) with the tested ion (e.g. Ca2+). Thus, bi-ionic conditions do not appear to
be a friendly environment for NPCs. The
reversal potential under bi-ionic conditions is then used with the equation to
determine the permeability ratio for the two ion species tested. Thus, for K+ and Ca2+:
(23)
Vreversal = (RT/F) ln{(PK[K+]cytoplasm)/(2PCa
[Ca2+]nucleoplasm)}
Using this approach, Boehning et al. (2001)
determined that K+ was 15 times more permeant than Cl-
and that Ca2+ was 4 times more permeant than K+.
Note, however, that the bi-ionic conditions
used for the calculation of Ca2+ permeability relative to K+,
Boehning et al. (2001) used 50 mM Ca2+.
The use of this high level of Ca2+ may be too extreme for the NE to handle.
Indeed,
Stehno-Bittel et al. (1995) reported that Ca2+ levels higher than 1
mM destroyed the NE of Xenopus L. oocytes.
Therefore, it would appear that a less stressful approach would
be to still use the two ions but under less demanding conditions for the NE
structure (i.e. using the more complex equation 22).
One must also be careful when making ion
replacements because this maneuver may seriously affect the junction potentials
caused by ion species difference (a phenomenon similar to the potentials
developed at metal junctions such as those created when copper and aluminum are
joined).
Therefore, one has to correct
for this with a proper equation (e.g., Neher, 1995; Barry, 2002).
But variations of divalent ions like Ca2+
are known to affect the surface charge of the cell membranes and this effect
alone can affect the voltage sensed by the ions, having greater effect with
larger variations (e.g. Ehrenstein, 2001).
There is also a pharmacological approach to identifying the selectivity of an ion channel.
This may be useful when considering the questions just mentioned.
For example, drugs known to block Cl--channels
were used to identify the activity recorded from hepatocyte nuclei (e.g.
Tabares et al., 1991) and heparin, a favorite probe for blocking IP3R-operated
channels, was also used to identify the ion channel activity (e.g. Boehning et
al., 2001). The pharmacological
approach, like others, has also its drawbacks.
Thus, the once though specific Ca2+-antagonists drugs were
shown to be less than specific and the hunt has always been for specific drugs
and/or definitions of channels according to the pharmacological blocker (e.g.
Bustamante, 1983, 1985 – Bustamante, unpublished observations; Ren et al.,
2001). Heparin is another example of a
pharmacological tool that must be used with caution for it is used to extract
fractions from the NE (e.g. Strambio-de-Castillia et al., 1995; Rout and Field,
2001) and has been shown to alter ion channel behavior (e.g. Knaus et al.,
1990; Krasilnikov et al., 1999). Taken
together, it appears that one should exercise extreme care when using
pharmacological tools with the NE, and specially, when recording from whole
nucleus.
Four decades have passed since the first
publication on NE electrical resistance (e.g. Loewenstein et al., 1962). Since 1990, efforts in applying patch-clamp
to the cell nucleus (Matzke et al., 1990; Mazzanti et al., 1990) have led us
through an arduous path that promises to fuse electrophysiology with the
cell/molecular biology of the nucleus.
Despite the efforts, however, some questions remain to be solved before
the electrophysiological observations can be applied to other fields (see
Bustamante, 2001). One vexing problem discussed here is that of
the shunt resistance, Rshunt, another is the identification of the
source for the recorded ion channel activity.
Ever since the time that Nobel laureates Hodgkin and Huxley introduced
the voltage-clamp technique, Rshunt has been a parameter to reckon
with (see Hodgkin and Huxley, 1963).
The application of glass capillaries and plastic tubes for the recording
of ionic currents has met the challenge with certain preparations because the
input impedance of the system (preparation together with the pipette/tubing)
has been shown to be sufficiently high (e.g. Kostyuk, 1982, 1984; Kostyuk and
Krishtal, 1984). The achievement of
this high impedance has been possible, in many preparations, with the usage of
un-physiological ions such as fluoride (e.g. Kostyuk, 1982, 1984; Kostyuk and
Krishtal, 1984). It is for this reason
that patch-clamp has succeeded in popularity over these alternate
techniques. I have no doubt of the
merit of the NHT approach (see introductions in Danker et al., 1999,
2001). However, the major vexing
problem challenging the NHT is that there is no frame of reference but a leak
current that can not be assumed to derive from channels peripheral to the NPC. A justification similar to that of the NHT
was used for the loose patch-clamp (e.g. Almers et al., 1983; 1984; Anson and
Roberts, 1998 – see review by Roberts and Almers, 1992). As originally conceived, this technique was
applied to the study of voltage-dependent plasmalemmal Na+-channels. The loose patch-clamp technique could be
validated because the behavior of these Na+-channels were well
established. At difference from
pipettes and tubing techniques used in the past for the recording of classical
voltage-dependent ion currents (e.g. Bustamante and McDonald, 1983) the
macroscopic ion currents through NPC populations look very much like leak
currents (e.g. Bustamante, 1992, 1993, 1994a; Bustamante et al., 1995a;
2000a,b). As discussed here, a solution
to this current limitation of both the NHT and of some of the patch-clamp data
being produced is the incorporation of fluorescence microscopy, an approach not
uncommon to many electrophysiological laboratories working with isolated cells
(e.g. Bustamante et al., 1995a, 2000a,b; Bustamante, 2002). The major criticism on the artificiality of
patch-clamp experiments due to the physiological solutions used without
NPC-mediated macromolecular substrates appears to have found a remedy with the
recent introduction of living syncytial nuclei in their native environment
(Bustamante, 2002). In addition, the
new preparation demonstrates a high rate of macromolecular transport as well as
of transcription and translation. This
was shown by their transport of nuclear-targeted proteins and by their
expression of foreign cDNA for the green fluorescent protein (GFP – Bustamante,
2002). It must be noted that, when the
NPCs of this intact preparation are engaged in macromolecular transport, both
patch-clamp and fluorescence microscopy (low-sensitivity CCD – Bustamante, 2002
and unpublished observations) show that the NE restricts the diffusion of
monoatomic ions and of small fluorescent particles such as 4 kDa dextrans and
5.4 nm diameter dendrimers (Bustamante, 2002).
This observation requires further testing with high-sensitive light
detectors since it challenges the dominant thinking of NPCs freely permeable to
monoatomic ions. This dominant thinking
has been consolidated, in recent years, with data from permeabilized cells, from
isolated nuclei in cell extract, and from cells and nuclei injected with
micropipettes. Therefore, as done for
patch-clamp experiments identifying NPC ion channel behavior (e.g. Bustamante,
2002; Bustamante et al., 1995a-c, 2000a,b), NHT experiments could greatly
benefit from the integration of fluorescence microscopy approaches. The advanced level of progress of the
fluorescence microscopy industry (e.g. Molecular Probes) should facilitate this
integration. The identification (i.e. inclusion or
exclusion) of the NPC as the source of ion channel activity appears to be of
great importance. The
electrophysiological thinking, which has been mostly biophysical and
pharmacological, must be complemented with the accumulated knowledge of
nucleocytoplasmic experts in cell and molecular biology. If NPCs engaged in heavy macromolecular
traffic do indeed block physiological ions (e.g. Bustamante et al., 1995a;
Bustamante, 2002), then many of the phenomena observed on nucleocytoplasmic
gradients of ions will no longer need to be considered a technical artifact and
may receive undivided attention. The
application of single-channel statistics and the molecular Coulter counter
principle (e.g. Bezrukov, 2000) has conferred electrophysiological methods with
high hopes for the study of not only functional but also structural genomics
(e.g. Kasianowicz et al., 1996; Matzke and Matzke, 1996; Hanss et al., 1998;
Meller et al., 2000; Howorka et al., 2001; Marziali and Akeson, 2001;
Vercoutere et al., 2001; Wang and Branton, 2001; Fertig et al., 2002; Klemic et
al., 2002; Sigworth and Klemic, 2002).
Since several diseases have been reported to be connected to nuclear
structures relevant to NPCs (e.g. Nagano and Arahata, 2000; Wilson, 2000; Berry
et al., 2001; Burke et al., 2001; Hutchison et al., 2001; Invernizzi et al.,
2001; Morris, 2001; Mounkes et al., 2001; Raharjo et al., 2001; Arbustini et
al., 2002;), electrophysiological measurements may be also relevant to future
tools for the diagnosis of cellular pathologies (see Bustamante, 2001). I thank Dr. Timm Danker
(University of Münster) for discussing with me the Rshunt and other
technical issues of his NHT. This work
was supported by a grant from the Millenium Science Initiative of the Brazilian
National Research Council (CNPq) of the Ministry of Science and Technology
(MCT) and a Senior Scientist Scholarship from the same.
CONCLUSIONS
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Common to all measurements is the fact that errors add up, they never cancel each other
(e.g. Barford, 1985; Baird, 1988).
To illustrate this principle of physical measurements, let us take the simple example of
the error made in deriving the area of a rectangle, Arectangle,
(24)
Arectangle= W x L
In order to derive the relative error, we can
apply the L’Hospital rule:
(25)
log (Arectangle) = log (W x L) =
log(W) + log(L)
Note that the logarithm is taken due to the differential calculus properties of this function
(see next equation).
(26)
(DArectangle/Arectangle) = | (DW/W) | + | (DL/L) |
Note also that the absolute values (indicated
by the vertical bars) are taken because errors always add up, they do not
cancel each other. Likewise, the error made in calculating the
area of a circle, Acircle, of radius r is
(27)
Acircle = p r2
applying the L’Hospital rule:
(28)
log (Acircle) = log (p
r2) = log(p) + 2 log(r)
APPENDIX: Measurement Theory
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(29)
(DAcircle/Acircle) = 2 | (Dr/r) |
Thus, for the above formulas, the relative
error in the calculated quantity equals the sum of the absolute values of the
individual relative errors.
From the above it is clear that with smaller quantities, the greater the relative
resulting error.
This concept must be
used for proper assessment of the relative error in Rshunt, whether
patch-clamp or NHT.
In the end, we have to apply common sense to decide what the error is.
For example, even if our statistical error in Acircle
is 0.000001%, (e.g. as a result of a large number of measurements) if our
relative error is 10%, our final error (the one we must report to other
scientists) cannot be better than 10%.
In the particular case of patch-clamp or NHT, since we have to consider
the complex geometry and forces for the molecular interactions between the NE
and the glass of the pipette (patch-clamp) or tube (NHT), it seems easier to
use fluorescent probes to directly assess the dimension of the leak or gap for
the two techniques.
Fig. 1.
Ion channels at the nuclear envelope.
Fig. 2.
Use of fluorescence microscopy to determine the state of ion and macromolecular conduction of nuclear pores.
Fig. 3.
Tubing system for simultaneous single cell voltage-clamp and intracellular perfusion.
Fig. 4.
Computed values for the resistance contributed by the NE and for the shunt resistance using the NHT model.
Fig. 5.
Reduction in shunt resistance induced by current injection simultaneous to intracellular perfusion with fluoride ions.
Fig. 6.
Theoretical and experimental amplitude histograms for patch-clamp recordings from the nuclear envelope.
Fig. 7.
Patch-clamp detection of NE large and small conductance ion channels.
FIGURES
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