BIOPHYSICS OF large membrane CHANNELS
|
Sergey
M. Bezrukov, PhD, Head, Section on
Molecular Transport Philip
A. Gurnev, PhD, Postdoctoral Fellow a Ekaterina
M. Nestorovich, PhD, Postdoctoral Fellow Namdar
Kazemi, BS, Postbaccalaureate Fellow b Tatiana K. Rostovtseva, PhD, Research Fellow |
|
|
We
investigate the physical principles of channel-facilitated transport of
metabolites and other large solutes across cell and organelle membranes. To
study channels under precisely controlled conditions, we reconstitute
channel-forming proteins into planar lipid bilayers. The proteins we work
with include VDAC (Voltage-Dependent Anionic Channel) from the outer membrane
of mitochondria, OmpF (general bacterial porin), LamB (sugar-specific
bacterial porin), alpha-hemolysin (toxin from Staphylococcus aureus),
alamethicin (amphiphilic peptide toxin from Trichoderma viride),
syringomycin E (lipopeptide toxin from Pseudomonas syringae), and
anthrax protective antigen. Channels formed by these proteins and peptides
have aqueous pores that are 1 nm or larger in diameter. We approach large
channels by complementing traditional electrophysiological methods with an
original concept of ion channels as molecular Coulter counters,
which distinguishes us from the main body of contemporary ion channel
researchers. Specifically, we focus on studying metabolite transport at the
level of single molecules. Using this strategy, we elucidate the molecular
mechanisms responsible for metabolite flux regulation under normal conditions
and in pathology. Apoptosis:
VDAC regulation by Bcl-2 family of proteins Rostovtseva, Bezrukov;
in collaboration with Antonsson, Colombini, Suzuki, Youle The
mitochondrion’s crucial role in the initiation of apoptosis is well
established. Triggered by a number of different stimuli, the mitochondrial
outer membrane (MOM) becomes permeable to apoptogenic factors such as
cytochrome c. The Bcl-2 family proteins regulate the permeabilization of MOM.
Pro-apoptotic proteins such as Bax and Bid induce the release of apoptogenic
factors, whereas anti-apoptotic proteins such as Bcl-xL prevent
their release. Two working models associate these proteins with the VDAC, the
existing major channel in the MOM. Given that this channel is known to be
responsible for most of the metabolite flux across the membrane, it is an
attractive candidate for a pathway for cytochrome c release. In the first
model, the pro-apoptotic protein Bax induces formation of the large
VDAC-based channels permeable to cytochrome c. In the second model, it is the
closure of VDAC channels that leads to MOM rupture. Therefore, although both
models explain the ability of Bcl-2 proteins to regulate the state and
integrity of VDAC and function as either anti- or pro-apoptotic agents, the
proposed mechanisms of VDAC channel regulation by Bcl-2 proteins are
diametrically opposite. To
resolve this matter, we studied the effect of the proteins on the properties
of VDAC channels reconstituted into the planar phospholipid membranes. We
first demonstrated that, contrary to general belief, there is no functionally
significant interaction between VDAC channels and the pro-apoptotic protein
Bax. A detailed analysis of the characteristic properties of VDAC channels
such as voltage gating, ion selectivity, single-channel conductance, and
water-soluble polymer exclusion did not show any change after Bax addition,
regardless of lipid composition, medium pH, or ionic content. We conclude
therefore that there is no functionally detectable interaction between VDAC
channels isolated from mammalian mitochondria and either monomeric or oligomeric
forms of Bax. However, we found that another pro-apoptotic protein, tBid,
affects the voltage-gating properties of VDAC by inducing channel closure and
that tBid induces closure of VDAC channels, both on single and multichannel
membranes, in a dose-dependent manner. By decreasing the probability of VDAC
opening, tBid would reduce the flux of adenine nucleotides and other
negatively charged metabolites across the MOM, which may affect mitochondrial
functions in different ways. One of the possible consequences of VDAC channel
closure is the disruption of metabolite exchange across the MOM and the
resultant accumulation of small metabolites in the intermembrane space,
followed by mitochondrial swelling and consequent loss of MOM integrity. The
mechanism by which tBid alters the gating properties of VDAC remains to be
understood. A direct interaction of VDAC with tBid (as well as with Bax) has
never been demonstrated. Based on our observations, we suggest that tBid most
likely affects VDAC following tBid insertion into the lipid membrane. Rostovtseva TK, Antonsson B, Suzuki M, Youle RJ, Colombini M,
Bezrukov SM. Bid but not Bax regulates VDAC channels. J Biol Chem
2004;279:13575-13583. Water-soluble
polymers as molecular probes Bezrukov; in collaboration
with Berezhkovskii, Krasilnikov Polymer
partitioning into nanoscale cavities is crucially important in many areas of
science and technology. Chromatography is probably the oldest example in
which the mechanisms of polymer chain distribution between a mobile solution
phase and the small voids of a stationary medium underlie the theoretical
basis of a number of methods of macromolecule separation. Among the newest
examples is size-dependent partitioning as a means of sizing aqueous pores of
ion channels. During the past year, we extended our studies in this direction
to address the problem of polymer solution non-ideality and to compare
partitioning of linear and cyclic polymers. We
studied the thermodynamics of polymer partitioning in the regime of advanced
solution non-ideality (de Cloizeaux regime) with the alpha-hemolysin channel.
Using the change in conductance of a nanometer-wide protein pore of the
channel to detect pore occupancy by polymers, we measured the equilibrium
partitioning of differently sized linear poly(ethylene glycol)s (PEGs) as a
function of polymer concentration in the bulk solution. In the semidilute
regime, increased polymer concentration resulted in a sharp increase in
polymer partitioning. Quantifying solution non-ideality by osmotic pressure
and taking the free energy of polymer confinement by the pore at infinite
dilution as an adjustable parameter allowed us to describe polymer
partitioning only at low polymer concentrations. At higher concentrations,
the increase in partitioning is much sharper than predicted by the model. The
nature of the sharp transition between strong exclusion and strong
partitioning might be rationalized within the concepts of scaling theory,
which predicts such behavior whenever the correlation length of the monomer
density in the semidilute bulk solution becomes smaller than the pore radius.
Specific attractive interactions between the protein pore and the polymer, in
addition to the entropic repulsion accounted for in the present study, may also
play a role. Closing
linear poly(ethylene glycol) into a circular “crown” dramatically
changes its dynamics in the alpha-hemolysin channel. In the electrically
neutral crown ether (C2H4O)6, six ethylene
oxide monomers are linked into a circle that gives the molecule
ion-complexing capacity and increases its rigidity. As with linear PEG, the
addition of the crown to the membrane-bathing solution decreases the ionic
conductance of the channel and generates additional conductance noise.
However, in contrast to linear PEG, both the conductance reduction
(reflecting crown partitioning into the channel pore) and the noise
(reflecting crown dynamics in the pore) now depend on voltage both strongly
and nonmonotonically. Within the frequency range accessible in channel
reconstitution experiments, the noise power spectrum is “white,”
showing that crown exchange between the channel and the bulk solution is
rapid. Analyzing the data in the framework of a Markovian two-state model, we
are able to characterize the process quantitatively. We showed that the
lifetime of the crown in the channel reaches its maximum (a few microseconds)
at about the same voltage (about 100 mV, negative from the side of protein
addition) at which the crown’s reduction of the channel conductance is
most pronounced. Our interpretation of these observations is that, given its
rigidity, the crown feels an effective steric barrier in the narrowest part
of the channel pore. The barrier together with crown-ion complexing and
resultant interaction with the applied field leads to behavior usually
associated with voltage-dependent binding in the channel pore. Thus, studies
of the crown ether effects on the single-channel conductance appear to be a
promising tool for investigating channel-facilitated membrane transport. They
also provide a better understanding of the pharmacological effects of the
crown ether superfamily at the molecular level. Bezrukov SM. Noise analysis in studies of protein dynamics and
molecular transport. Fluctuation and Noise Letters 2004;4:L23-L31. Bezrukov SM, Krasilnikov OV, Yuldasheva LN, Berezhkovskii AM,
Rodrigues CG. Field-dependent effect of crown ether (18-crown-6) on ionic
conductance of alpha-hemolysin channels. Biophys J 2004;87:3162-3171. Krasilnikov OV, Bezrukov SM. Polymer partitioning from non-ideal
solutions into protein voids. Macromolecules 2004;37:2650-2657. Electrostatics
in ion transport through large channels Nestorovich, Bezrukov;
in collaboration with Aguilella, Alcaraz Although
the crystallographic structure of the bacterial porin OmpF has been known for
a decade, the physical mechanisms underlying its ionic selectivity are still
under investigation. We addressed this issue in a series of experiments with
varied pH, salt concentrations, inverted salt gradient, and charged and
uncharged lipids. Measuring reversal potential, we showed that OmpF
selectivity (traditionally regarded as slightly cationic) depends strongly on
pH and salt concentration and is conditionally asymmetric, that is, it is
sensitive to the direction of salt concentration gradient. At neutral pH and
subdecimolar salt concentrations, the channel exhibits nearly ideal cation
selectivity (t+= 0.98 ± 0.01). Substituting neutral DPhPC
with DPhPS, we demonstrate that the fixed charge of the host lipid has a
small but measurable effect on the channel reversal potential. The available
structural information allows for a qualitative explanation of our
experimental findings, which led us to re-examine the ionization state of 102
titratable sites lining the OmpF channel. Using standard methods of continuum
electrostatics tailored to our purpose, we found that the charge distribution
in the channel is a function of solution acidity and could relate the
pH-dependent asymmetry in channel selectivity to the pH-dependent asymmetry
in charge distribution. In an attempt to find a simple phenomenological
description of our results, we also evaluated different macroscopic models of
electrodiffusion through large channels. Alcaraz A, Nestorovich EM, Aguilella-Arzo M, Aguilella VM,
Bezrukov SM. Salting out the ionic selectivity of a wide channel: the
asymmetry of OmpF. Biophys J 2004;87:943-957. Nestorovich EM, Bezrukov SM. Voltage-induced
“gating” of bacterial porin as reversible protein denaturation.
In: Abbott D, Bezrukov SM, Der A, Sanchez A, eds. Second Intl. Conf. on
Fluctuations and Noise in Biological, Biophysical, and Biomedical Systems.
Proc SPIE 2004;5467:42-53. aJoined Section in January,
2004. bJoined Section in July, 2004. COLLABORATORS Vicente M. Aguilella, PhD, Universidad
Jaume I, Antonio Alcaraz, PhD, Universidad Jaume I, Bruno Antonsson, PhD, Serono Pharmaceutical
Research Institute, Alexander M. Berezhkovskii, PhD, Center for
Information Technology, NIH, Marco Colombini, PhD, University of
Maryland, Oleg V. Krasilnikov, PhD, Universidad
Federal de Pernambuco, Motoshi Suzuki, PhD, Surgical Neurobiology
Branch, NINDS, Richard Youle, PhD, Surgical Neurobiology
Branch, NINDS,
|
|
