Study of the volt-ampere characteristics of ion channels by transmembrane current harmonics

A method is proposed for measuring the coefficient of non--linearity beta of current--voltage characteristics of the class i (U) approximately U (1 + beta U2) of ionic channels formed by grammicidine A (Gra) and amphotericine B by the 3rd harmonics of the membrane current. For Gra A beta depends on the concentration of electrolyte c increasing lg c from -17 B-2 at 0.03 M to 8 B-2 at 3.4 M KCl turning to 0 at c0 = 0.3 divided by 1 M. The membranes of egg lecithin and glycerylmonooleate (GMO) differ in c0 value. Substitution of K+ ion for Li+, of the membrane solvent (n-heptane for n-hexadecane) and freezing of the GMO membrane do not affect beta.     

On measurements of higher harmonics of transmembrane current

Measurements of higher harmonics of transmembrane current in bilayer lipid membranes from diphytanoyl phosphatidylcholine (DPhPC) in n-decane and n-tetradecane, caused by alternating voltage applied to the membrane, have been conducted. A universal relation between the amplitudes of harmonics was suggested and experimentally checked. This allowed one to calculate the coefficients of expansion of membrane capacity in series with even powers of membrane voltage and to compare the inhomogeneity of membranes from diphytanoyl phosphatidylcholine in n-decane and n-tetradecane with respect to thickness.     

Dynamics of active transmembrane ion transfer in transport enzymes

It has been shown that recent investigations of the electron density distribution and high resolution (approximately 0.5 nm) spatial structure of transport ATPases open new possibilities in the development of general models for the mechanisms of energy of ATP hydrolysis and its use for active transmembrane ion transfer.     

Molecular dynamics study of ion transport in transmembrane protein channels

Ion transport through biological membranes often takes place via pore-like protein channels. The elementary process of this transport can be described as a motion of the ion in a quasi-periodic multi-well potential. In this study molecular dynamics simulations of ion transport in a model channel were performed in order to test the validity of reaction-rate theory for this process. The channel is modelled as a hexagonal helix of infinite length, and the ligand groups interacting with the ion are represented by dipoles lining the central hole of the channel. The dipoles interact electrostatically with each other and are allowed to oscillate around an equilibrium orientation. The coupled equations of motion for the ion and the dipoles were solved simultaneously with the aid of a numerical integration procedure. From the calculated ion trajectories it is seen that, particularly at low temperatures, the ion oscillates back and forth in the trapping site many times before it leaves the site and jumps over the barrier. The observed oscillation frequency was found to be virtually temperature-independent (nu 0 approximately equal to 2 X 10(12) s-1) so that the strong increase of transport rate with temperature results almost exclusively from the Arrhenius-type exponential dependence of jump probability w on 1/T. At higher temperatures simultaneous jumps over several barriers occasionally occur. Although the exponential form of w(T) was in agreement with the predictions of rate theory, the activation energy Ea as determined from w(T) was different from the barrier height which was calculated from the static potential of the ion in the channel; the actual transport rate was 1 X 10(3) times higher than the rate predicted from the calculated barrier height. This observation was interpreted by the notion that ion transport in the channel is strongly influenced by thermal fluctuations in the conformation of the ligand system which in turn give rise to fluctuations of barrier height.     

Development of models of active ion transport for whole-cell modelling: cardiac sodium-potassium pump as a case study

This study presents a method for the reduction of biophysically-based kinetic models for the active transport of ions. A lumping scheme is presented which exploits the differences in timescales associated with fast and slow transitions between model states, while maintaining the thermodynamic properties of the model. The goal of this approach is to contribute to modelling of the effects of disturbances to metabolism, associated with ischaemic heart disease, on cardiac cell function.

The approach is illustrated for the sodium-potassium pump in the myocyte. The lumping scheme is applied to produce a 4-state representation from the detailed 15-state model of Läuger and Apell, Eur. Biophys. J. 13 (1986) 309, for which the principles of free energy transduction are used to link the free energy released from ATP hydrolysis (ΔG_ATP) to the transition rates between states of the model. An iterative minimisation algorithm is implemented to determine the transition rate parameters based on the model fit to experimental data. Finally, the relationship between ΔG_ATP and pump cycling direction is investigated and compared with recent experimental findings.     

Regulation of ion transport in mitochondria by respiratory chain enzymes and ATPase

The effects of the respiratory chain inhibitors as well as those of the inhibitors and substrates of ATP-synthetase in Ca2+ and K+ transport induced in the mitochondria upon the medium acidification in the presence of phosphate or arsenate, were investigated. Evidence has been obtained suggesting that under the experimental conditions used the transmembrane fluxes of K+ and Ca2+ are paralleled with H+ leakage through the proton channel of ATPase. It was found also that the system inducing cation fluxes at low pH values included peroxidation and hydrolysis of phospholipids. A scheme of regulation of ion transport in the mitochondria involving oxidative phosphorylation and oxidation and hydrolysis of lipids is proposed.     

Distribution of sodium-potassium ATPase in the rat testis and epididymis.

Sodium-potassium ATPase (Na+K(+)-ATPase) is a ubiquitous plasma membrane enzyme which uses the hydrolysis of ATP to regulate cellular Na+ and K+ levels and fluid volume. This ion pumping action is also thought to be involved in fluid movement across certain epithelia. There are several different genes for this enzyme, some of which are tissue specific. Using an antibody specific for the catalytic subunit of canine kidney Na+K(+)-ATPase, we have localized immunoreactivity in the seminiferous and epididymal epithelium of rats of various ages. There was no specific staining of 10-day-old rat testis. Faint staining was detected at 13 days and appeared to be associated with the borders of Sertoli cells. At 16 days prominent apical and lateral staining but no basal staining of Sertoli cell membranes was observed. This type of distribution continued until spermatids were present in the epithelium. In the adult rat testis, specific staining was detected in Sertoli cell crypts associated with elongating spermatids, and on the apical and lateral Sertoli cell membrane. In some instances immunoreactivity was concentrated at presumed sites of junctional specializations. In the excurrent ducts of immature and mature rats, Na+K(+)-ATPase staining was heavy in the efferent ducts and somewhat lighter in the epididymis. In all regions, the staining was basolateral although there were variations in intensity among the different parts of the epididymis. These results show 1) that rat testis and epididymal Na+K(+)-ATPase share some immunological determinants with the canine enzyme; 2) that the epididymal enzyme is located in the conventional basolateral position; and 3) that the distribution of Sertoli cell Na+K(+)-ATPase is probably apical and lateral rather than basal.     

Single amino acid substitution in the putative transmembrane helix V in KdpB of the KdpFABC complex of Escherichia coli uncouples ATPase activity and ion transport

The KdpFABC complex, found in a variety of prokaryotes, is an emergency potassium uptake system which belongs to the family of P-type ATPases. Site-directed mutagenesis of the charged residues aspartate 583 and lysine 586 in the putative transmembrane helix V of subunit KdpB revealed that these charges are involved in the coupling of ATP hydrolysis and ion translocation. Phenotypic characterization of KdpFABC derivatives carrying alterations at either D583 or K586 demonstrated that only restoration of charges at these positions allowed growth on low potassium concentrations. Substitutions, which eliminated the negative charge at position 583, did not allow growth below 15 mM potassium on solid media. In contrast, substitutions of the positive charge at position 586 allowed growth down to 0.3 mM potassium. Purified KdpFABC complexes carrying these substitutions exhibited ATPase activity, which was, however, found to be comparatively resistant to o-vanadate. Furthermore, elimination of the charges led to a complete loss of ion-stimulated ATPase activity, though the rate of hydrolysis was comparable to wild-type activity, indicating an uncoupling between ATP hydrolysis and ion translocation. This fact was substantiated by reconstitution experiments, in which the D583A complex was unable to facilitate ion translocation, whereas the D583E mutant complex still exhibited such activity. On the basis of these results, a new transport model for the Kdp-ATPase is presented here, in which the amino acids D583 and K586 are supposed to play a role in the gating mechanism of the complex. Furthermore, movement of the charged side chains could have a direct influence on the free energy profile within the potassium transporting subunit KdpA, thereby facilitating ion transport against the concentration gradient into the cytosol.     

Immunohistochemical localization of sodium-potassium ATPase in human normal stomach and gastric adenocarcinomas

We studied the expression pattern of Na, K-ATPase beta 1 subunit in human normal stomachs and in gastric adenocarcinomas by using anti-Na, K-ATPase beta 1 subunit-specific monoclonal antibody. Tissue samples were processed in formalin solution or in a cold acetic acid-ethanol solution, routinely processed, embedded in paraffin, and an immunoperoxidase method for Na, K-ATPase beta 1 subunit was performed. After antigen retrieval using a steamer in citrate buffer (pH 6.0), tissue sections initially fixed in cold acetic acid-ethanol showed intense immunoreactivity with the antibody at the lateral or basolateral cytoplasmic membrane of normal gastric epithelial cells, at the cytoplasmic membrane of gastric carcinoma cells according to the level of differentiation, and at the cytoplasmic membrane and in the cytoplasm of Schwann cells and neurons in the mesenteric plexus of the gastric wall. Acetic acid-ethanol and paraffin embedding is a useful method for the investigation of the immunohistochemical localization of Na, K-ATPase in normal and diseased tissues.     

A study of inhomogeneity of bilayer lipid membranes by measuring the higher harmonics of the transmembrane current

The higher harmonics of the alternating current in bilayer lipid membranes induced by a sinusoidal voltage applied to the membrane were measured. The bilayer lipid membranes were prepared from diphytanoylphosphatidylcholine in decane and tetradecane; a 16-bit analog-to-digital converter was used for the measurements. A sinusoidal voltage was formed with a 16-bit digital-to-analog converter. The dynamic measurement range reached 90 dB. The coefficients α and β of the expansion of capacitance C in terms of the membrane voltage U—C = C ₀(1 + αU ² + βU ⁴)—were determined from the measurement results. It was shown within the framework of the electrostriction model that a certain ratio of the coefficients α and β characterizes the inhomogeneity of the membrane with respect to its thickness and Young’s modulus of elasticity.     

Characteristics of the generation of transmembrane current higher harmonics in lipid bilayers in glycerol and 1,2-propane diol solutions

It is shown that structural rearrangements of bilayer lipid membrane induced by glycerol and 1,2-propane diol result in noncompensated second harmonic of transmembrane current having quadratic dependence on input voltage and a weak dependence on frequency. The third harmonic has cubic dependence on input voltage and decreases with an increase in frequency. The mechanism is discussed according to which generation of non-compensated second harmonic is due to the formation of the intermediate lipid phase with viscoelastic properties differing from the initial bilayer in the process of structural rearrangements. The presence of this phase conditions the possibility of appearance of noncompensated second harmonic.     

The effects of carbon monoxide and hydrogen sulfide on transmembrane ion transport

The activity of electroneutral ion transport in response to the effect of the gasotransmitters carbon monoxide and hydrogen sulfide was investigated. It was shown that phenylephrine, an activator of receptorregulated calcium uptake, enhanced the relaxing action of carbon monoxide and hydrogen sulfide. In contrast, inhibition of the membrane potassium conductance, especially its voltage-dependent component, decreased the myogenic effects of carbon monoxide in the smooth muscles. The effects of hydrogen sulfide depended on its concentration and the means of activation of the cell transport systems. Furthermore, sodium-dependent components of the membrane conductivity are also involved in the effects of this gasotransmitter on ion transport systems in addition to the calcium and potassium conductance. This expands the range of the potential gasotransmitter-affected targets of signaling pathways, which may result in either activation or inhibition of cell functions. The consequences of such impacts on the functionally significant responses of cells, organs, and systems should be taken into account in various physiological and pathological states.     

Interaction of reactive oxygen species with ion transport mechanisms

The use ofelectrophysiological and molecular biology techniques has shed light onreactive oxygen species (ROS)-induced impairment of surface andinternal membranes that control cellular signaling. These deleteriouseffects of ROS are due to their interaction with various ion transportproteins underlying the transmembrane signal transduction, namely,1) ion channels, such asCa²⁺ channels (includingvoltage-sensitive L-type Ca²⁺currents, dihydropyridine receptor voltage sensors, ryanodine receptorCa²⁺-release channels, andD-myo-inositol1,4,5-trisphosphate receptor Ca²⁺-release channels),K⁺ channels (such asCa²⁺-activatedK⁺ channels, inward and outwardK⁺ currents, and ATP-sensitiveK⁺ channels),Na⁺ channels, andCl channels;2) ion pumps, such as sarcoplasmicreticulum and sarcolemmal Ca²⁺pumps,Na⁺-K⁺-ATPase(Na⁺ pump), andH⁺-ATPase(H⁺ pump);3) ion exchangers such as theNa⁺/Ca²⁺exchanger andNa⁺/H⁺exchanger; and 4) ion cotransporterssuch asK⁺-Cl,Na⁺-K⁺-Cl,andP_i-Na⁺cotransporters. The mechanism of ROS-induced modificationsin ion transport pathways involves1) oxidation of sulfhydryl groups located on the ion transport proteins,2) peroxidation of membrane phospholipids, and 3) inhibition ofmembrane-bound regulatory enzymes and modification of the oxidativephosphorylation and ATP levels. Alterations in the ion transportmechanisms lead to changes in a second messenger system, primarilyCa²⁺ homeostasis, which furtheraugment the abnormal electrical activity and distortion of signaltransduction, causing cell dysfunction, which underlies pathologicalconditions.

     

Modern views on the mechanisms of sodium ion transport from the external environment in freshwater hydrobionts

On the basis of kinetic characteristics of the ion exchange between freshwater aquatic organisms and external medium, the existing concepts on the action mechanisms of the ion carriers located in cell membranes of skin ionocytes of embryos and in the gill epithelium of fishes are analyzed. It is shown that the main mechanism compensating for the Na⁺ loss by the organism of freshwater hydrobionts into the external environment is the Na⁺-Cl^– cotransporter and, to a certain degree, the Na⁺-K⁺-2Cl^– cotransporter. A supplementary role in the Na⁺ transport from the water under extreme conditions may play the Na⁺/H⁺ and Na⁺/NH₄ ⁺ exchangers.     

Use of digitonin fractionation to determine mitochondrial transmembrane ion distribution in cells during anoxia

A method to determine intracellular distribution of ions and metabolites under conditions of low oxygen concentration was developed. The technique involves rapid digitonin fractionation and addition of cyanide to inhibit reoxygenation. Applicability of the procedure was examined by studying the distribution of the lipophilic ion triphenylmethylphosphonium, the weak acid 5,5-dimethyloxazolidine-2,4-dione, and adenine nucleotides.     

Components of depolarization-induced transmembrane ion current in isolated smooth muscle cells of the guinea pigtaenia coli

Transmembrane ion currents in isolated single smooth muscle cells (SMC) from the guinea pigtaenia coli were investigated using a whole-cell mode of the patch-clamp technique. Currents induced by depolarizing shifts in the membrane potential from its holding level of −60 mV contained an initial inward phase (Ca²⁺ current), which in 30–40 msec was followed by an outward phase. It was shown that outward current was carried by K ions and consisted at least of three components: one Ca²⁺-independent K⁺ current of delayed rectifier (KV) and two Ca²⁺-dependent K⁺ currents. The latter can be further divided into the apamin-sensitive (SK) and charybdotoxin-sensitive (BK) currents. It was found that relative contributions of these three components in total outward current at 0 mV were 35–45%, 5–15%, and 45–55% for KV, SK, and BK currents, respectively. A potential-dependent current carried by Ci ions was also found. This Cl⁻ current had inward direction within the range of potentials below the chloride equilibrium potential (E _Cl) and outward direction above theE _Cl. The magnitude of Cl⁻ current was significantly lower than the magnitude of total K⁺ current.     

Dual mechanisms of ion absorption in relation to long distance transport in plants

The characteristics of ion transport to the shoots of young corn seedlings were studied with respect to the nature of the isotherm through a wide concentration range, the competitive influence of closely related ions upon the transport of a given ion, and the influence of the counter-ion. Both with respect to ³⁶Cl and ⁸⁶Rb transport, the characteristics of the process in every way resemble uptake by non-vacuolate root tips wherein the plasma membrane is the only membrane involved in absorption, and where system 1 — of the 2 systems which can be shown to participate in absorption by vacuolate tissue — is the only system operative. Net ion uptake by the roots per se was shown to display both the high affinity (system 1) and low affinity (system 2) mechanisms. It is concluded that the symplastic theory of ion movement to the xylem is valid, and that the contention that system 1 operates at the plasma membrane while system 2 functions at the tonoplast is strengthened.     

Hydrogen ion modulation of Ca channel current in cardiac ventricular cells. Evidence for multiple mechanisms

We have investigated the effects of H ions on (L-type) Ca channel current in isolated ventricular cells. We find that the current amplitude is enhanced in solutions that are alkaline relative to pH 7.4 and reduced in solutions acidic to this pH. We measured pH0-induced shifts in channel gating and analyzed our results in terms of surface potential theory. The shifts are well described by changes in surface potential caused by the binding of H ions to negative charges on the cell surface. The theory predicts a pK of 5.8 for this binding. Gating shifts alone cannot explain all of our observations on modulation of current amplitude. Our results suggest that an additional mechanism contributes to modification of the current amplitude.     

Changes in electrical potential difference and sodium-potassium ATPase of canine mucosa after jejunal-ileal bypass surgery

Changes in electrical potential difference and sodium-potassium ATPase activity of rectal mucosa in dogs were examined before and after jejunal-ileal bypass surgery. The potential difference in the postoperative period was significantly higher (P less than 0.05) than the preoperative value. The potential difference increased by 160% at the 3rd day after the surgery, and then gradually declined with prolonged recovery periods. Ouabain-sensitive sodium-potassium ATPase activity also increased at the 3rd day (160%) and at the 10th day (86%) after the surgery. Fourteen days after the surgery the sodium-potassium ATPase activity returned almost to the preoperative value. These results indicated a close correlation between changes in transmucosal potential difference and sodium-potassium ATPase activity of rectal mucosa, suggesting a significant participation of sodium-potassium ATPase in changes of potential difference induced by jejunal-ileal bypass surgery.