Biomedical subjects

T K Rostovtseva

Publications and source records attributed to T K Rostovtseva.

8 recordsLinked to original sources

PubMed2002

VDAC channels differentiate between natural metabolites and synthetic molecules.

VDAC provides the major permeability pathway through the mitochondrial outer membrane by forming voltage-gated channels with pore radius of 1.2-1.5 nm. We find that VDAC can select among comparably-charged molecules with a much smaller effective radius, 0.4-0.5 nm. The molecules studied were the nucleotides, ATP, UTP, NADH and synthetic anions, tetraglutamate (T-Glu) and 1-hydroxypyrene-3,6,8-trisulfonate (HPTS). VDAC channels were reconstituted into planar phospholipid membranes bathed in 1.0 M NaCl (buffered to pH 8.0). The nucleotides decreased the conductance of VDAC for NaCl demonstrating that they could permeate into the channel. In contrast, T-Glu and HPTS did not change the single-channel conductance, indicating exclusion from the channel. Reversal potential measurements report near ideal selectivity of Na + over T-Glu. The nucleotides increased single-channel noise as they penetrated into the channel, while T-Glu had no effect. HPTS increased noise, but unlike NADH, this was not voltage-dependent when HPTS was added asymmetrically, indicating no penetration into the channel. The differences in effective size and charge cannot explain the difference in permeation characteristics. Thus VDAC must select among these based on shape and charge distribution. We propose that the electrostatic environment within the channel has been evolutionarily selected to favor the passage of adenine nucleotides.

Adenosine Triphosphate↗

PubMed2000

Positive cooperativity without domains or subunits in a monomeric membrane channel.

The monomeric VDAC channel shows an accelerated pH titration of its transport properties with a Hill coefficient of about 2. This manifests itself as a sharp peak in conductance noise as well as a fast change in channel selectivity with pH. On the basis of the known structure of this channel, we propose that this cooperativity arises from a mechanically linked mobile pair of ionizable groups. Concerted movement of these groups between two states changes the distance from nearby electrostatic charge to influence the pK of the groups. This model of pH-dependent motion produces positive cooperative behavior that fits the observations without need for subunits or identifiable domains within the protein. The mathematical formalism has never required such domains, but these are generally considered an essential part of cooperative behavior in proteins. The present proposal reduces the size of a cooperative unit to a minimum, extending the limits of what is perceived to be possible. Together with large-scale conformational transitions, these subtle cooperative structural changes may allow proteins to adapt, with high sensitivity, to changes in their environment. They might also be relatively easy to engineer into a protein.

Allosteric Regulation↗

PubMed1998

Membrane surface-charge titration probed by gramicidin A channel conductance.

We manipulate lipid bilayer surface charge and gauge its influence on gramicidin A channel conductance by two strategies: titration of the lipid charge through bulk solution pH and dilution of a charged lipid by neutral. Using diphytanoyl phosphatidylserine (PS) bilayers with CsCl aqueous solutions, we show that the effects of lipid charge titration on channel conductance are masked 1) by conductance saturation with Cs+ ions in the neutral pH range and 2) by increased proton concentration when the bathing solution pH is less than 3. A smeared charge model permits us to separate different contributions to the channel conductance and to introduce a new method for "bilayer pKa" determination. We use the Gouy-Chapman expression for the charged surface potential to obtain equilibria of protons and cations with lipid charges. To calculate cation concentration at the channel mouth, we compare different models for the ion distribution, exact and linearized forms of the planar Poisson-Boltzmann equation, as well as the construction of a "Gibbs dividing surface" between salt bath and charged membrane. All approximations yield the intrinsic pKain of PS lipid in 0.1 M CsCl to be in the range 2.5-3.0. By diluting PS surface charge at a fixed pH with admixed neutral diphytanoyl phosphatidylcholine (PC), we obtain a conductance decrease in magnitude greater than expected from the electrostatic model. This observation is in accord with the different conductance saturation values for PS and PC lipids reported earlier (, Biochim. Biophys. Acta. 552:369-378) and verified in the present work for solvent-free membranes. In addition to electrostatic effects of surface charge, gramicidin A channel conductance is also influenced by lipid-dependent structural factors.

Gramicidin↗

PubMed1998

ATP transport through a single mitochondrial channel, VDAC, studied by current fluctuation analysis.

The "molecular Coulter counter" concept has been used to study transport of ATP molecules through the nanometer-scale aqueous pore of the voltage-dependent mitochondrial ion channel, VDAC. We examine the ATP-induced current fluctuations and the change in average current through a single fully open channel reconstituted into a planar lipid bilayer. At high salt concentration (1 M NaCl), the addition of ATP reduces both solution conductivity and channel conductance, but the effect on the channel is several times stronger and shows saturation behavior even at 50 mM ATP concentration. These results and simple steric considerations indicate pronounced attraction of ATP molecules to VDAC's aqueous pore and permit us to evaluate the effect of a single ATP molecule on channel conductance. ATP addition also generates an excess noise in the ionic current through the channel. Analysis of this excess noise shows that its spectrum is flat in the accessible frequency interval up to several kilohertz. ATP exchange between the pore and the bulk is fast enough not to display any dispersion at these frequencies. By relating the low-frequency spectral density of the noise to the equilibrium diffusion of ATP molecules in the aqueous pore, we calculate a diffusion coefficient D = (1.6-3.3)10(-11) m2/s. This is one order of magnitude smaller than the ATP diffusion coefficient in the bulk, but it agrees with recent results on ATP flux measurements in multichannel membranes using the luciferin/luciferase method.

Adenosine Triphosphate↗

PubMed1996

Diffusion through narrow pores: movement of ions, water and nonelectrolytes through track-etched PETP membranes.

The rates at which ions (86Rb+, [3H]-choline, 36Cl), 3H2O and nonelectrolytes ([14C]-urea, [14C]-glycerol, and [14C]-sugars) equilibrate across track-etched polyethyleneterephthalate (PETP) membranes (isotopic diffusion) have been measured by a 'static' and a 'dynamic' technique under conditions where no net flow takes place; the two techniques give essentially the same results. All tracers diffuse faster the longer the membranes are etched, consistent with an increase in pore size. Water and neutral solutes diffuse at rates that are relatively independent of ionic strength, pH or the presence of divalent cations. Diffusion of cations is decreased by high ionic strength, by reducing pH or by addition of divalent cations; diffusion of chloride is increased by these procedures. Treatment of the membrane with diazomethane to reduce the negative fixed charge decreases diffusion of cations and increases that of anions; diffusion of water and neutral solutes is unaffected by methylation except in the membranes with the narrowest pores (i.e., those etched for the shortest time), in which case diffusion is reduced. We conclude (1) that the special features of flow near a charged surface apply to ions but not to water or nonelectrolytes and (2) that calculation of absolute rates of diffusion leads to values for the radii of pores through track-etched PETP membranes that are in remarkably good agreement with measured values.

Anions↗

PubMed1994

Triton channels are sensitive to divalent cations and protons.

Addition of Triton X-100 to planar bilayers composed of dioleoyl phosphatidyl choline, diphytanoyl phosphatidyl choline or mono-oleoyl glycerol induces single channel-like events when electrical conductivity across the bilayer is measured. Addition of divalent cations or protons causes channels to disappear; single channel conductance of remaining channels is not significantly altered; addition of EDTA or alkali (respectively) reverses the effect. It is concluded that sensitivity to divalent cations and protons need not be dependent on specific channel proteins or pore-forming toxins, but may be a feature of any aqueous pore across a lipid milieu.

Calcium↗

PubMed1993

Rapid switching of ion current in narrow pores: implications for biological ion channels.

Ions flowing through purely synthetic filters made of polyethylene terephthalate which have been etched to produce narrow pores show: (i) rapid transitions between a high-conducting and a low-conducting state; (ii) selectivity of ion flow; and (iii) inhibition by divalent cations and protons. These features resemble those displayed by many biological ion channels. We interpret our results in terms of the special properties of ion conductance at an interface that may be observed whenever the contribution of bulk conductance is minimal.

Cations, Divalent↗

PubMed1992

Membrane damage: common mechanisms of induction and prevention.

Common features in the induction of pores by various agents are as follows: induction is stochastic and progressive; damage by different agents is often synergistic and limited. The prevention of membrane damage is affected by trivalent and divalent cations, by low pH, by low ionic strength and by high osmotic pressure. The inhibitory role of protons and divalent cations is considered in greater detail: pore-forming agents can be classified into two groups: channels across planar lipid bilayers induced by the first group display voltage-sensitive, reversible inhibition by divalent cations; channels of the second group show voltage-insensitive, irreversible inhibition by divalent cations. A search for the ligands to which divalent cations and protons bind has proved elusive. Comparison with the phenomenon of 'surface conductance' through narrow apertures, that is manifest in the absence of any pore-forming agent, may prove fruitful.

Animals↗