Fluorescent styryl dyes of the RH series affect a potential drop on the membrane/solution boundary

AbstractThe effects of the adsorption of the fluorescent potential-sensitive dyes RH-421, RH-237 and RH-160 on the bilayer lipid membrane were studied. It was shown that a dipole potential drop, positive in the hydrophobic part of the membrane, arose due to the dye adsorption. The dye adsorption led to a considerable increase of the rate constant of hydrophobic anion translocation through the membrane, but did not affect their partition coefficient between membrane and water. It implies that the region of the membrane where the potential drops is located deeper than the adsorption plane of hydrophobic ions. The values of the boundary potential differences were estimated by two independent methods with unilateral and bilateral application of the dyes to lipid bilayer membranes. The results suggest that RH dye molecules penetrate through the lipid bilayers. The values of ζ-potential in liposomes did not change on dye adsorption. Hence, dye molecules are adsorbed in a form that does not change the surface charge. We estimated the effects of the electric field of dye dipole layer on an individual dipole located in the same layer and on ion transport through a membrane protein Na+/K+-ATPase. It turned out that the local electric field of each dye dipole decayed so rapidly that a neighbouring dye molecule did not feel it. It also appeared that RH dyes could have but a minor effect on the electrogenic transport performed by the sodium pump in the examined range of dye concentrations

Assignment of charge movements to electrogenic reaction steps of Na,K-ATPase by analysis of salt effects on the kinetics of charge movements

Na,K-ATPase-enriched membrane fragments adsorbed to lipid bilayers were used to study electrogenic Na+ movements induced by anzyme phosphorylation when ATP was photo-relased from inactive caged ATP, and simultaneously by externally applied alterating voltages which allowed the measurement of small ATP-induced membrane admittance changes. A detailed analysis of frequency dependence of the capacitance and conductance increments showed that the observed peocess consists of more than one electrogenic step. The frequency dependence could be described by the sum of two Lorentzian functions and y constant term. Preferentially, the slower process showed a dependence on the ion concentration of choline salts with different anions. This observation matches their effect on the partition between two phosphoenzyme states of the Na,K-ATPase, as established previously

ИсслЕдованиЕ напряженнО–дефОрмирОваннОгО состоЯниЯ элементоВ палубЫ контейнеровозА С использованиеМ системЫ дифференциальныХ уравнениЙ КАрмАнА

Рассматриваются современные методы и средства решения задачи расчета напряженно–деформированного состояния палубных элементов судов. Проведен анализ современных методов оценки напряженно–деформированного состояния судовых пластин на стадии проектирования и эксплуатации судна. Для этого в качестве обзора рассмотрены основные программные комплексы, позволяющие проводить подобные расчеты, их положительные и отрицательные стороны. Приведены основные этапы создания программного обеспечения для проведения расчетов, основанных на использовании системы Кармана

Negative Changes of the Membrane Capacitance due to Electrogenic Na Transport by the Na, K-ATPase

Electrogenic Na+ transport was investigated in membrane fragments containing Na,K-ATPase adsorbed to bilayer lipid membranes (BLM) triggered by fast ATP release from caged ATP. The influence of voltage on the transport after ATP release was determined as small increaments of capacitance and conductance by applying an alternating voltage to the membrane. An electrogenic Na+ transport through a cytoplasmic access channel of the Na,K-ATPase was detected that disappeared after enzyme phosphorylation, and thus produced a negative capacitance and conductance increaments at Na+ concentrations below 5 mM3. This effect was studied now in more detail by measuring the frequency dependence of the capacitance and conductance increaments at different Na+ concentrations

Influence of the salts of choline on the rate limiting step of active transport of sodium ions by Na,K-ATPase

Изучено влияние температуры и концентрированных растворов солей иодида, бромида и хлорида холина с различными анионами на кинетику нестационарного электрогенного транспорта ионов натрия Na+,K+-ATP-азой в модельной системе, состоящей из фрагментов мембран с белком, адсорбированных на бислойной липидной мембране (БЛМ). Кинетические параметры транспорта определяли, регистрируя ток короткого замыкания при быстром освобождении ATP из Caged-ATP под действием вспышки ультрафиолетового света. Температурные зависимости постоянной времени экспоненты τ2, описывающей спадающую фазу тока короткого замыкания, позволили оценить энергию активации (Еа)самой медленной стадии транспорта, представляющей собой корформационный переход Na+,K+-ATP-азы, и предэкспоненциальный фактор (А) в уравнении Аррениуса для скорости процесса 1/τ2=A exp(-Ea/RT). Иодиды или бромиды холина уменьшают значения параметров Еа и А. Несмотря на то, что изменения этих параметров оказывают противоположный эффект на скорость, уменьшение А преобладает, в результате чего скорость процесса снижается. Эффект солей холина зависит от того, с какой стороны мембранного фрагмента с Na+,K+-ATP-азой они находятся. Если иодид или бромид холина присутствовали с обеих (внеклеточной и цитоплазматической) сторон белка, изменение температурной зависимости было сильнее по сравнению с ситуацией, когда эти соли были только с цитоплазматической стороны. Это свидетельствует в пользу того, что данные соли значительно эффективнее влияют на работу Na+,K+-ATP-азы, когда они находятся с внеклеточной стороны белка. Полученные результаты можно объяснить влиянием анионов холина на структуру воды на поверхности белка с внеклеточной стороны, где открывается глубокий канал, соединяющий внутреннюю область Na+,K+-ATP-азы с раствором. Хлорид холина тоже вызывал замедление электрогенного транспорта, однако в отличие от иодида и бромида он не влиял на значение Еа и был более эффективен при введении с цитоплазматической стороны белка. Этот эффект вызван, по-видимому, асимметричным экранированием отрицательно заряженного фрагмента мембраны с Na+,K+-ATP-азой, что приводит к появлению трансмембранного потенциала, влияющего на перенос ионов натрия белком

Influence of sodium concentration on changes of membrane capacitance associated with the electrogenic ion transport by the Na,K-ATPase

Electrogenic ion transport by the Na,K-ATPase was investigated in a model system of protein-containing membrane fragments adsorbed to a lipid bilayer. Transient Na+ currents were induced by photorelease of ATP from inactive caged ATP. This process was accompanied by a capacitance change of the membrane system. Two methods were applied to measure capacitances in the frequency range 1 to 6000 Hz. It was, however, dependent on Na+ concentration and on the membrane potential which was generated by the charge transferred. Below 5 mM Na+ a negative capacitance change was detected which may be assigned to electrogenic Na+ binding to cytoplasmic sites. It could be shown that the results obtained by experiments with the presented alternating current method contain the information which is determined by current-relaxation experiments with cell membranes

Effect of chaotropic anions on the sodium transport by the Na,K-ATPase

The effect of choline iodide, bromide and chloride on the kinetics of the electrogenic sodium transport by the Na,K-ATPase was investigated in a model system of ATPase-containing membrane fragments adsorbed on the lipid bilayer membrane. The kinetic parameters of Na+ transport were determined from short circuit currents after fast release of ATP from its caged precursor. The falling phase of the current transients could be fitted by a single exponential with the time constant, s2. Its temperature dependence allowed an estimation of the activation energy of the rate-limiting reaction step, the conformation transition E1/E2. Choline iodide and bromide caused a decrease of the activation energy as well as the overall rate of the process expressed as the pre-exponential factor A of the Arrhenius equation. If choline iodide or bromide were present on the cytoplasmic and extracellular sides of the protein, the temperature dependent changes were more pronounced than when present on the cytoplasmic side only. These results can be explained by an effect of the anions on water structure on the extracellular surface of the protein, where a deep access channel connects the ion-binding sites with the solution. Chloride ions also caused a deceleration of the electrogenic transport, however, in contrast to iodide or bromide, they did not affect the activation energy, and were more effective when added on the cytoplasmic side. This effect can be explained by asymmetric screening of the negative surface charges which leads to a transmembrane electric potential that modifies the ion transfer

Fast Transient Currents in Na,K-ATPase Induced by ATP Concentration Jumps from the P3-[1-(3′,5′-Dimethoxyphenyl)-2-Phenyl-2-Oxo]ethyl Ester of ATP

AbstractElectrogenic ion transport by Na,K-ATPase was investigated by analysis of transient currents in a model system of protein-containing membrane fragments adsorbed to planar lipid bilayers. Sodium transport was triggered by ATP concentration jumps in which ATP was released from an inactive precursor by an intense near-UV light flash. The method has been used previously with the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP), from which the relatively slow rate of ATP release limits analysis of processes in the pump mechanism controlled by rate constants greater than 100s−1 at physiological pH. Here Na,K-ATPase was reinvestigated using the P3-[1-(3,5-dimethoxyphenyl)-2-phenyl-2-oxo]ethyl ester of ATP (DMB-caged ATP), which has an ATP release rate of >105s−1. Under otherwise identical conditions, photorelease of ATP from DMB-caged ATP showed faster kinetics of the transient current compared to that from NPE-caged ATP. With DMB-caged ATP, transient currents had rate profiles that were relatively insensitive to pH and the concentration of caged compound. Rate constants of ATP binding and of the E1 to E2 conformational change were compatible with earlier studies. Rate constants of enzyme phosphorylation and ADP-dependent dephosphorylation were 600s−1 and 1.5×106M−1s−1, respectively, at pH 7.2 and 22°C

Involvement of protons in the ion transport cycle of Na+ ,K+-ATPase

The effect of pH on electrogenic sodium transport by the Na+,K+-ATPase has been studied. Experiments were carried out by admittance recording in a model system consisting of a bilayer lipid membrane with adsorbed membrane fragments containing purified Na+,K+-ATPase. Changes in the membrane admittance (capacitance and conductance increments in response to photo-induced release of ATP from caged ATP) were measured as function of AC voltage frequency, sodium ion concentration, and pH. In solutions containing 150 mM Na+, the frequency dependence of capacitance increments was not significantly dependent on pH in the range between 6 and 8. At a low NaCl concentration (3 mM), the capacitance increments at low frequencies decreased with the increasing pH. In the absence of NaCl, the frequency-dependent capacitance increment at low frequencies was similar to that measured in the presence of 3 mM NaCl. These results may be explained by involvement of protons in the Na+,K+-ATPase pump cycle, i.e., electroneutral exchange of sodium ions for protons under physiological conditions, electrogenic transport of sodium ions at high pH, and electrogenic transport of protons at low concentrations (and in the absence) of sodium ions