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R Z Sabirov

Publications and source records attributed to R Z Sabirov.

At least 19 recordsLinked to original sources
PubMed2005

Cells die with increased cytosolic ATP during apoptosis: a bioluminescence study with intracellular luciferase.

Apoptosis is a distinct form of cell death, which requires energy. Here, we made real-time continuous measurements of the cytosolic ATP level throughout the apoptotic process in intact HeLa, PC12 and U937 cells transfected with the firefly luciferase gene. Apoptotic stimuli (staurosporine (STS), tumor necrosis factor alpha (TNFalpha), etoposide) induced significant elevation of the cytosolic ATP level. The cytosolic ATP level remained at a higher level than in the control for up to 6 h during which activation of caspase-3 and internucleosomal DNA fragmentation took place. When the STS-induced ATP response was abolished by glucose deprivation-induced inhibition of glycolysis, both caspase activation and DNA laddering were completely inhibited. Annexin V-binding induced by STS or TNFalpha was largely suppressed by glycolysis inhibition. Thus, it is suggested that the cells die with increased cytosolic ATP, and elevation of cytosolic ATP level is a requisite to the apoptotic cell death process.

Adenosine Triphosphate↗
PubMed2003

Osmotic water permeability and regulatory volume decrease of rat thymocytes.

Rat thymocytes displayed robust regulatory volume decrease (RVD) when suspended in NaCl-based hypotonic Ringer solutions. The RVD of thymocytes was completely abolished upon replacement of external Na+ ions with K+, indicating a role of coupled efflux of K+ and Cl- ions as a driving force of regulatory volume decrease. Osmotic water permeability (Pf) measured in KCl-based hypotonic solutions was (1.3 +/- 1.0 x 10(-4) cm/s at 25 degrees C and was temperature-dependent with low activation energy (Ea = 4.65 +/- 0.77 kcal/mol) characteristic to water transport through pores. HgCl2 and a sulfhydryl-blocking reagent, methyl methanethiosulphonate (MMTS), modulated the water permeability of thymocytes in a biphasic manner: inhibited at low dose (0.1-1 micromol/l) and restored or even enhanced at higher (10-100 micromol/l) concentrations. RVD paralleled the Pf: it was greatly suppressed at low dose of MMTS (sufficient to attenuate the water transport), but recovered at higher dose, when the water movement was restored. Therefore we suggest that thymocytes require the effective water transport for functional regulatory volume decrease.

Animals↗
PubMed2001

Volume-dependent ATP-conductive large-conductance anion channel as a pathway for swelling-induced ATP release.

In mouse mammary C127i cells, during whole-cell clamp, osmotic cell swelling activated an anion channel current, when the phloretin-sensitive, volume-activated outwardly rectifying Cl(-) channel was eliminated. This current exhibited time-dependent inactivation at positive and negative voltages greater than around +/-25 mV. The whole-cell current was selective for anions and sensitive to Gd(3)+. In on-cell patches, single-channel events appeared with a lag period of approximately 15 min after a hypotonic challenge. Under isotonic conditions, cell-attached patches were silent, but patch excision led to activation of currents that consisted of multiple large-conductance unitary steps. The current displayed voltage- and time-dependent inactivation similar to that of whole-cell current. Voltage-dependent activation profile was bell-shaped with the maximum open probability at -20 to 0 mV. The channel in inside-out patches had the unitary conductance of approximately 400 pS, a linear current-voltage relationship, and anion selectivity. The outward (but not inward) single-channel conductance was suppressed by extracellular ATP with an IC(50) of 12.3 mM and an electric distance (delta) of 0.47, whereas the inward (but not outward) conductance was inhibited by intracellular ATP with an IC(50) of 12.9 mM and delta of 0.40. Despite the open channel block by ATP, the channel was ATP-conductive with P(ATP)/P(Cl) of 0.09. The single-channel activity was sensitive to Gd(3)+, SITS, and NPPB, but insensitive to phloretin, niflumic acid, and glibenclamide. The same pharmacological pattern was found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel serves as a conductive pathway for the swelling-induced ATP release in C127i cells.

ATP-Binding Cassette Transporters↗
PubMed2000

Extra- and intracellular proton-binding sites of volume-regulated anion channels.

We have investigated the effects of extracellular and intracellular pH on single channel and macroscopic (macropatches) currents through volume-regulated anion channels (VRAC) in endothelial cells. Protonation of extracellular binding sites with an apparent pK of 4.6 increased voltage independent of the single-channel amplitude. Cytosolic acidification had a dual effect on VRAC currents: on the one hand, it increased single channel conductance by approximately 20% due to protonation of a group with an apparent pK of 6.5 and a Hill coefficient of 2. On the other hand, it reduced channel activity due to protonation of a group with an apparent pK of 6.3 and a Hill coefficient of 2.1. This dual effect enhances the macroscopic current at a slightly acidic pH but inhibits it at more acidic pH. Cytosolic alkalization also reduced channel activity with a pK of 8.4 and a Hill coefficient of 1.9, but apparently did not affect single-channel conductance. These data show that VRAC channels are maintained in an active state in a narrow pH range around the normal physiological pH and shut down outside this range. They also show that HEPES-buffered pipette solutions do not effectively buffer pH in the vicinity of the VRAC channels.

Animals↗
PubMed2000

Reduction of ionic strength activates single volume-regulated anion channels (VRAC) in endothelial cells.

We have previously shown that a reduction of intracellular ionic strength is involved in the activation of volume-regulated anion channels (VRAC). Here we show in a single-channel study that VRAC can be activated in a cell-attached patch when the cell interior is dialyzed with a solution of decreased ionic strength. For this purpose, bovine pulmonary endothelial (CPAE) cells) were permeabilized with alpha-staphylotoxin (alphaST) which has a molecular weight cut-off size of 2 kDa. If the ionic strength in the bath solution is reduced from 160 mM to 95 mM, single-channel activity is initiated in cell-attached patches sealed before permeabilization. Conductance is outwardly rectifying with approximately 17 pS at negative and 57 pS at positive potentials. Single-channel currents reverse near the calculated equilibrium potential for Cl-. The averaged current shows inactivation at positive potentials. The current is blocked by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB). An increase in ionic strength reversibly inhibits current activation. It is concluded that a decrease in ionic strength activates single-channel currents through VRAC rather than shifting the set point of a hypothetical volume sensor.

Animals↗
PubMed1999

Na(+) sensitivity of ROMK1 K(+) channel: role of the Na(+)/H(+) antiporter.

To examine the extracellular Na(+) sensitivity of a renal inwardly rectifying K(+) channel, we performed electrophysiological experiments on Xenopus oocytes or a human kidney cell line, HEK293, in which we had expressed the cloned renal K(+) channel, ROMK1 (Kir1. 1). When extracellular Na(+) was removed, the whole-cell ROMK1 currents were markedly suppressed in both the oocytes and HEK293 cells. Single-channel ROMK1 activities recorded in the cell-attached patch on the oocyte were not affected by removal of Na(+) from the pipette solution. However, macro-patch ROMK1 currents recorded on the oocyte were significantly suppressed by Na(+) removal from the bath solution. A blocker of Na(+)/H(+) antiporters, amiloride, largely inhibited the Na(+) removal-induced suppression of whole-cell ROMK1 currents in the oocytes. The pH-insensitive K80M mutant of ROMK1 was much less sensitive to Na(+) removal. Na(+) removal was found to induce a significant decrease in intracellular pH in the oocytes using H(+)-selective microelectrodes. Coexpression of ROMK1 with NHE3, which is a Na(+)/H(+) antiporter isoform of the kidney apical membrane, conferred increased sensitivity of ROMK1 channels to extracellular Na(+) in both the oocytes and HEK293 cells. Thus, it is concluded that the ROMK1 channel is regulated indirectly by extracellular Na(+), and that the interaction between NHE transporter and ROMK1 channel appears to be involved in the mechanism of Na(+) sensitivity of ROMK1 channel via regulating intracellular pH.

Animals↗
PubMed1998

Probing the water permeability of ROMK1 and amphotericin B channels using Xenopus oocytes.

Water permeability of ion channels in the plasma membrane of Xenopus oocytes was studied by simultaneously measuring the membrane conductance under two-electrode voltage-clamp and the cell size by video-imaging technique. The basal level of osmotic water permeability of oocyte plasma membrane was 15.9+/-0.98 microm/s (SE, n = 5). Extracellular application of pore-forming antibiotic amphotericin B at 5 microM developed macroscopic conductance of 995+/-70 microS (n = 5) and increased the osmotic water permeability of cell membrane by 44.9+/-4.1 microm/s. Meanwhile, after expressing ROMK1 channels, originally cloned from kidney, virtually no increase in the water permeability was observed even at the conductance level as high as 1113+/-47 microS (n = 5). This result suggests that even though potassium channels, like any others, are considered to be water-filled pores, K+-selective ion-transporting pathway remains virtually water-impermeable in physiological conditions, such as in kidney epithelia where huge water transport takes place at both apical and basolateral sides.

Amphotericin B↗
PubMed1997

Two-sided action of protons on an inward rectifier K+ channel (IRK1).

A cloned inwardly rectifying potassium channel, IRK1, expressed in Xenopus oocytes was found to be sensitive to an extracellular acidic pH level of below 6, achieved by buffering with a membrane-impermeable buffer, phthalate. The voltage dependency of the suppressive effect of pH on the macroscopic current suggested that the location of the proton-sensitive site was at approximately 5% of the distance from the outer entrance to the pore. The single-channel conductance was reduced by protonation of the channel on the extracellular side. The external proton-binding site appears to consist of a single class of negatively charged groups with a pK of around 4.6. An intracellular acidic pH, buffered with membrane-permeable acetate, was found to inhibit, in a voltage-independent manner, the macroscopic IRK1 current with an approximate apparent pK of 5.6 and an approximate apparent Hill coefficient of 2.3. The single-channel activity was abolished by intracellular acidification down to pH 5.0.

Animals↗
PubMed1997

A conserved arginine residue in the pore region of an inward rectifier K channel (IRK1) as an external barrier for cationic blockers.

The number, sign, and distribution of charged residues in the pore-forming H5 domain for inward-rectifying K channels (IRK1) are different from the otherwise homologous H5 domains of other voltage-gated K channels. We have mutated Arg148, which is perfectly conserved in all inward rectifiers, to His in the H5 of IRK1 (Kir2. 1). Channel activity was lost by the mutation, but coexpression of the mutant (R148H) along with the wild-type (WT) mRNA revealed populations of channels with reduced single-channel conductances. Long-lasting and flickery sublevels were detected exclusively for the coexpressed channels. These findings indicated that the mutant subunit formed hetero-oligomers with the WT subunit. The permeability ratio was altered by the mutation, while the selectivity sequence (K+ > Rb+ > NH4+ >> Na+) was preserved. The coexpression made the IRK1 channel more sensitive to extracellular block by Mg2+ and Ca2+, and turned this blockade from a voltage-independent to a -dependent process. The sensitivity of the mutant channels to Mg2+ was enhanced at higher pH and by an increased ratio of mutant:WT mRNA, suggesting that the charge on the Arg site controlled the sensitivity. The blocking rate of open channel blockers, such as Cs+ and Ba2+, was facilitated by coexpression without significant change in the steady state block. Evaluation of the electrical distance to the binding site for Mg2+ or Ca2+ and that to the barrier peak for block by Cs+ or Ba2+ suggest that Arg148 is located between the external blocking site for Mg2+ or Ca2+ and the deeper blocking site for Cs+ or Ba2+ in the IRK1 channel. It is concluded that Arg148 serves as a barrier to cationic blockers, keeping Mg2+ and Ca2+ out from the electric field of the membrane.

Animals↗
PubMed1993

Relation between ionic channel conductance and conductivity of media containing different nonelectrolytes. A novel method of pore size determination.

The effects of nonelectrolytes on conductivity and viscosity of KCl solutions as well as on ion channel conductance were studied. Mobility of ions in solutions were found to solely depend on percent concentration (w/w) of the nonelectrolytes added and to be effectively independent on their chemical nature (sugars or polyglycols) and molecular size. Proportional changes in both the ion channel conductance and the conductivity of bulk solution induced by low m. w. nonelectrolytes may be used as a criterion of diffusion mechanism of ion transport through channels. The slope of the dependence of ion channel conductance on conductivity of bulk solution containing different concentrations of nonelectrolytes is a good measure of channel permeability for nonelectrolyte. A new method of pore size determination is introduced. Results of practical application of this simple method to three types of ion channels (formed by alpha-latrotoxin, staphylococcal alpha-toxin and its N-terminal fragment) are shown. The advantages and disadvantages of the method are discussed.

Bacterial Toxins↗
PubMed1993

The interaction of amphotericin B with cell membrane of rat thymocytes.

Amphotericin B (AB) at micromolar concentrations increases cell membrane permeability and induced swelling of rat thymus lymphocytes. Potassium efflux is a precondition for AB to induce swelling of the cells. The rate constants for potassium loss and volume changes were proportional to the 1.24th and the 2nd power of the antibiotic concentration respectively. The reflection coefficients for nonelectrolytes with different hydrodynamic radii were determined, and the equivalent radius of the amphotericin pore in the thymocyte cell membrane was estimated to be 4.1 +/- 0.3 A at polyene concentrations varying between 2.5 mumol/l and 80 mumol/l. It is suggested that channel formation by AB in cell membranes is actually able to modulate immune responses.

Amphotericin B↗
PubMed1992

Comparative analysis of latrotoxin channels of different conductance in planar lipid bilayers. Evidence for cluster organization.

It has been established that channels induced by Latrodectus tredicimguttatus alpha-toxin (LT) in lipid bilayers have a cluster organisation. So far as: (i) the LT-channels had practically identical sizes of its water pores (r = 9.4 +/- 0.6 A) independently on the lipid composition of planar bilayer lipid membrane (BLM) although their conductances might differ from each other more than 10 times (100 mM KCl (pH 7.5)). (ii) affinity of permeable ions to channels had a small variation with distinct group of BLM, although LT-channels conductances varied from 112 +/- 8 pS till 1110 +/- 40 pS for phosphatidylcholine-BLM and from 75 +/- 6 pS till 170 +/- 14 pS for phosphatidylserine-BLM. (iii) Ca/K selectivity was greater in negatively charged membranes but did not also depend on the channel amplitude for the same BLM. Cation-anionic selectivity was identical for all studied channels.

Lipid Bilayers↗
PubMed1992

A simple method for the determination of the pore radius of ion channels in planar lipid bilayer membranes.

A new method of pore size determination is presented. The results of applying this simple method to ion channels formed by staphylococcal alpha-toxin and its N-terminal fragment as well as to cholera toxin channels are shown. The advantages and the difficulties of this method are discussed. It was found that (i) the mobility of ions in solutions depends only on the percentage of concentration of added non-electrolytes and practically not on their chemical nature (sugars or polyglycols) and molecular size; (ii) the proportional change of both ion channel conductance and bulk solution conductivity by low M. nonelectrolytes may be used as an indication of a diffusion mechanism of ion transport through channels; (iii) the slope of the dependence of the ion channel conductance on the bulk conductivity of solutions containing different concentrations of non-electrolyte is a good measure of channel permeability for non-electrolytes.

Bacterial Toxins↗
PubMed1990

Memory is a property of an ion channels pool: ion channels formed by Staphylococcus aureus alpha-toxin.

The short-time depolarization effects on the integral conductance induced by S. aureus alpha-toxin (ST) in planar lipid bilayer membranes has been studied. Ion channels formed by ST were found to have several potential-induced nonconductance (closed) states. The transitions of ion channels between the states are only through one conductance state. The transition of ST-channels from closed to open state is induced by membrane depolarization. The amplitude current after a series of voltage pulses is a function of pulse number, and is effectively independent of the time interval between the neighbouring pulses. Therefore, a membrane which contains a pool of ion channels "remembers" its previous existence. A simple model can be used to explain this phenomenon.

Bacterial Toxins↗
PubMed1989

Ion transport through channels formed in lipid bilayers by Staphylococcus aureus alpha-toxin.

Staphylotoxin channel appears to be predominantly anion-selective with non-linear and asymmetric current-voltage characteristics (CVC) at neutral pH. Increased salt concentrations induce linearity and asymmetry of CVC and loss of selectivity. At lower pH both the channel conductivity and anion selectivity increase. Higher temperatures raise the channel conductivity in parallel with the changes in electrical conductivity of the salt solution, but do not change selectivity. Experimental dependences are described obtained by approximation of electrical diffusion and considering the interactions of penetrating ions with fixed charges at the entrances and the channel energy profile.

Bacterial Toxins↗