Molecular dynamics of a protein surface: ion-residues interactions

Time-resolved measurements indicated that protons could propagate on the surface of a protein or a membrane by a special mechanism that enhanced the shuttle of the proton toward a specific site. It was proposed that a suitable location of residues on the surface contributes to the proton shuttling function. In this study, this notion was further investigated by the use of molecular dynamics simulations, where Na(+) and Cl(-) are the ions under study, thus avoiding the necessity for quantum mechanical calculations. Molecular dynamics simulations were carried out using as a model a few Na(+) and Cl(-) ions enclosed in a fully hydrated simulation box with a small globular protein (the S6 of the bacterial ribosome). Three independent 10-ns-long simulations indicated that the ions and the protein's surface were in equilibrium, with rapid passage of the ions between the protein's surface and the bulk. However, it was noted that close to some domains the ions extended their duration near the surface, thus suggesting that the local electrostatic potential hindered their diffusion to the bulk. During the time frame in which the ions were detained next to the surface, they could rapidly shuttle between various attractor sites located under the electrostatic umbrella. Statistical analysis of the molecular dynamics and electrostatic potential/entropy consideration indicated that the detainment state is an energetic compromise between attractive forces and entropy of dilution. The similarity between the motion of free ions next to a protein and the proton transfer on the protein's surface are discussed.     

Zwitterionic polymers functionalised nanoporous graphene for water desalination: a molecular dynamics study

In this paper, one nanoporous graphene grafting several zwitterionic polymer chains was designed as the osmosis membrane for seawater desalination. Using molecular dynamics simulation, the efficiency and mechanism of salt rejection were discussed. The simulated results showed that the zwitterionic polymer chains on nanoporous graphene can form the charge channel to block Na⁺ and Cl^? ions pass through, and the slat rejection efficiency of functionalised graphene can reach to about 90%. In the simulation, the steric hindrance and electrostatic interaction are the main factors for the salt rejection. With time evolution, the charge channel formed by the soft polymer chains can decrease the effective pore area of membrane, leading to the increase of steric hindrance; the positive and negative centres of polymer chains can adsorb Na⁺ and Cl^? ions under electrostatic interaction in the solution, contributing into the increase of charge density above the membrane. These conclusions are consistent with experimental report. Our designed osmosis membrane about the graphene is helpful for improving the potential application of defect graphene in water desalination and reducing the trouble of obtaining appropriate graphene sheet with small aperture.     

Interfacial interactions of glutamate, water and ions with carbon nanopore evaluated by molecular dynamics simulations

Molecular dynamics simulations were used to assess the transport of glutamate, water and ions (Na⁺ and Cl⁻) in a single wall carbon nanopore. The spatial profiles of Na⁺ and Cl⁻ ions are largely determined by the pore wall charges. Co-ions are repelled whereas the counter-ions are attracted by the pore charges, but this ‘rule’ breaks down when the water concentration is set to a level significantly below that in the physiological bulk solution. In such cases water is less able to counteract the ion-wall interactions (electrostatic or non-electrostatic), co-ions are layered near the counter-ions attracted by the wall charges and are thus layered as counter-ions. Glutamate is concentrated near the pore wall even at physiological water concentration, and irrespective of whether the pore wall is neutral or charged (positively or negatively), and its peak levels are up to 40 times above mean values. The glutamate is thus always layered as a counter-ion. Layering of water near the wall is independent of charges on the pore wall, but its peak levels near the wall are ‘only’ 6-8 times above the pore mean values. However, if the mean concentration of water is significantly below the level in the physiological bulk solution, its layering is enhanced, whereas its concentration in the pore center diminishes to very low levels. Reasons for such a ‘paradoxical’ behavior of molecules (glutamate and water) are that the non-electrostatic interactions are (except at very short distances) attractive, and electrostatic interactions (between the charged atoms of the glutamate or water and the pore wall) are also attractive overall. Repulsive interactions (between equally charged atoms) exist, and they order the molecules near the wall, whereas in the pore center the glutamate (and water) angles are largely randomly distributed, except in the presence of an external electric field. Diffusion of molecules and ions is complex. The translational diffusion is in general both inhomogeneous and anisotropic. Non-electrostatic interactions (ion-wall, glutamate-wall or water-wall) powerfully influence diffusion. In the neutral nanopore the effective axial diffusion constants of glutamate, water and Na⁺ and Cl⁻ ions are all < 10% of their values in the bulk, and the electrostatic interactions can reduce them further. Diffusion of molecules and ions is further reduced if the water concentration in the pore is low. Glutamate⁻ is slowed more than water, and ions are reduced the most especially co-ions. In conclusion the interfacial interactions influence the spatial distribution of glutamate, water and ions, and regulate powerfully, in a complex manner and over a very wide range their transport through nanosize pores.     

Na+-K+-Cl− cotransport system in brain capillary endothelial cells: Response to endothelin and hypoxia

Effect of endothelin-1 and chemically induced hypoxia on Na⁺−K⁺−Cl⁻ cotransport activity in cultured rat brain capillary endothelial cells was examined by using⁸⁶Rb⁺ as a tracer for K⁺; bumetanide-sensitive K⁺ uptake was defined as Na⁺−K⁺−Cl⁻ cotransport activity. Endothelin-1, phorbol 12-myristate 13-acetate (PMA), or thapsigargin increased Na⁺−K⁺−Cl⁻ cotransport activity. A protein kinase C inhibitor, bisindolylmaleimide, inhibited PMA- and endothelin-1- (but not thapsigargin-) induced Na⁺−K⁺−Cl⁻ cotransport activity, indicating the presence of both protein kinase C-dependent regulatory mechanisms and protein kinase C-independent mechanisms which involve intracellular Ca²⁺. Oligomycin, sodium azide, or antimycin A increased Na⁺−K⁺−Cl⁻ cotransport activity by 80–200%. Oligomycin-induced Na⁺−K⁺−Cl⁻ cotransport activity was reduced by an intracellular Ca²⁺ chelator (BAPTA/AM) but not affected by bisindolylmaleimide, suggesting the involvement of intracellular Ca²⁺, and not protein kinase C, in hypoxia-induced Na⁺−K⁺−Cl⁻ cotransport activity. Portions were presented at “27th Annual Meeting, The American Society for Neurochemistry” Philadelphia, Pennsylvania, March 2–6, 1996.     

Specific Binding of Chloride Ions to Lipid Vesicles and Implications at?Molecular Scale

Biological membranes composed of lipids and proteins are in contact with electrolytes like aqueous NaCl solutions. Based on molecular dynamics studies it is widely believed that Na⁺ ions specifically bind to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes, whereas Cl⁻ ions stay in solution. Here, we present a careful comparison of recent data from electrophoresis and isothermal titration calorimetry experiments as well as molecular dynamics simulations suggesting that in fact both ions show very similar affinities. The corresponding binding constants are 0.44(±0.05) M⁻¹ for Na⁺ and 0.40(±0.04) M⁻¹ for Cl⁻ ions. This is highlighted by our observation that a widely used simulation setup showing asymmetric affinities of Na⁺ and Cl⁻ for POPC bilayers overestimates the effect of NaCl on the electrophoretic mobility of a POPC membrane by an order of magnitude. Implications for previous simulation results on the effect of NaCl on polarization of interfacial water, transmembrane potentials, and mechanisms for ion transport through bilayers are discussed. Our findings suggest that a range of published simulations results on the interaction of NaCl with phosphocholine bilayers have to be reconsidered and revised and that force field refinements are necessary for reliable simulation studies of membranes at physiological conditions on a molecular level.     

Transport and accumulation of ions in two spring wheat lines differing in salt tolerance

Fifty-two-day old plants of a salt tolerant line, S24 and a salt sensitive, Yecora Rojo were subjected for 15 days to 125 mol·m⁻³ NaCl in Hoagland’s nutrient solution under glass-house conditions. The dry matter of shoots and roots of the salt tolerant line was significantly greater over all time intervals in saline substrate than the salt sensitive line, Yecora Rojo. In the leaves of salt-treated former line concentration of Na⁺ and Cl⁻ was lower as compared to the latter line. The lower Na⁺ and Cl⁻ concentrations in the leaves of S24 were found to be associated with lower transport of these ions to the shoots whereas the reverse was true for Yecora Rojo. The lines did not differ in accumulation of either ion in roots. It is concluded that salt tolerance in these two genotypes of spring wheat is associated with restricted accumulation of toxic Na⁺ and Cl⁻ ions to the shoots or with restricted transport.     

ATP-Induced Inhibition of Na+, K+, Cl− Cotransport in Madin-Darby Canine Kidney Cells: Lack of Involvement of Known Purinoceptor-Coupled Signaling Pathways

P_2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca²⁺] _i , activation of phospholipases C (PLC) and A₂ (PLA₂), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na⁺,K⁺,Cl⁻ cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier (∼50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca²⁺]_i that was abolished by a chelator of Ca²⁺ _i , BAPTA. However, neither BAPTA nor the Ca²⁺ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca²⁺-pump, thapsigargin, modified ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4,5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na⁺,K⁺,Cl⁻ cotransport activity. Inhibitors of PLA₂ (AACOCF₃), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na⁺ channels (amiloride), Cl⁻ channels (NPPB) and Na⁺/H⁺ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport and suggest the presence of a novel P₂-receptor-coupled signaling mechanism. Received: 29 July 1998/Revised: 19 October     

Molecular Dynamics Simulation of Limiting Conductance for Na2+, Cl2−, Na°, and Cl° in Supercritical Water

Abstract

We report results of molecular dynamics simulations of the limiting conductance of Na²⁺, Cl^2?, Na°, and Cl° in supercritical water using the SPC/E model for water in conjuction with our previous study (Lee et al., Chem. Phys. Lett. 293, 289 (1998)). The behavior of the limiting conductances of Na²⁺ and Cl^2? in the whole range of water density shows almost the same trend as those of Na⁺ and Cl^?, but the deviation from the assumed linear dependence of limiting conductances of Na²⁺ and Cl^2? on the water density is smaller than that of Na⁺ and Cl^?. The ratio of the limiting conductance of the divalentions to that of the corresponding monovalentions over the whole range of water density is almost constant. In the cases of Na²⁺ and Cl^2?, the dominating factor of the number of hydration water molecules around ions in the higher-density region and the dominating factor of the interaction strength between the ions and the hydration water molecules in the lower-density region are also found as was the cases for Na⁺ and Cl^?. These factors, however, are not so strong as for the corresponding monovalent ions because the change in the energetics, structure, and dynamics are very small mainly due to the strong Coulomb interaction of the divalent ions with the hydration water molecules. The diffusion coefficient of Na° and Cl° monotonically increases with decreasing water density over the whole range of water density. The increase of the diffusion coefficient with decreasing water density is attributed only to the dramatic decrease of the hydration number of water in the first solvation shell around the uncharged species. Among the two important competing factors in the limiting conductance of Na⁺ and Cl^?, the effect of the number of hydration water molecules around the uncharged species is the only existing factor over the whole range of water density since the interaction strength between the uncharged species and the hydration water molecules very small through the LJ interaction. This result has confirmed the dominating factor of the number of hydration water molecules around ions in the higher-density region in the explanation of the limiting conductance of Na⁺ and Cl^? in supercritical water at 673 K.     

Functional Characterization of a ClC-2-Like Cl− Conductance in Surface Epithelial Cells of Rat Rectal Colon

ClC-2, a member of the voltage-gated Cl⁻ channel family, is expressed in the distal colonic surface epithelial cells of various species, but its functional significance remains unclear. Here, by means of electrophysiological and molecular biological techniques, we have identified and characterized a ClC-2-like conductance naturally expressed by surface epithelial cells acutely dissociated from rectal colon of rats fed a standard diet. Whole-cell patch-clamp experiments showed that the surface cells, whether an amiloride-sensitive Na⁺ conductance was present or not, displayed a strong hyperpolarization-activated, inwardly rectifying Cl⁻ current. Analysis both by in situ hybridization and immunohistochemistry confirmed the expression of ClC-2 in the rectal surface epithelium. The native Cl⁻ current shared common electrophysiological properties including voltage-dependent activation, anion selectivity sequence, and Zn²⁺ sensitivity with that recorded from HEK293 cells transfected with ClC-2 cloned from rat rectal colon (rClC-2). Cell-attached patch recordings on the surface cells revealed that native ClC-2-like currents activated only at potentials at least 40 mV more negative than resting membrane potentials. In Ussing chamber experiments with rat rectal mucosa, either basolateral or apical application of Zn²⁺ (0.1 mM), which inhibited both native ClC-2-like currents and recombinant rClC-2 currents, had little, if any, effects on basal amiloride-sensitive short-circuit current. Collectively, these results not only demonstrate that a functional ClC-2-type Cl⁻ channel is expressed in rat rectal surface epithelium, but also suggest that the channel activity may be negligible and thus nonessential for controlling electrogenic Na⁺ transport in this surface epithelium under basal physiological conditions.     

Control of ion selectivity in LeuT: two Na+ binding sites with two different mechanisms

The x-ray structure of LeuT, a bacterial homologue of Na⁺/Cl⁻-dependent neurotransmitter transporters, provides a great opportunity to better understand the molecular basis of monovalent cation selectivity in ion-coupled transporters. LeuT possesses two ion binding sites, NA1 and NA2, which are highly selective for Na⁺. Extensive all-atom free-energy molecular dynamics simulations of LeuT embedded in an explicit membrane are performed at different temperatures and various occupancy states of the binding sites to dissect the molecular mechanism of ion selectivity. The results show that the two binding sites display robust selectivity for Na⁺ over K⁺ or Li⁺, the competing ions of most similar radii. Of particular interest, the mechanism primarily responsible for selectivity for each of the two binding sites appears to be different. In NA1, selectivity for Na⁺ over K⁺ arises predominantly from the strong electrostatic field arising from the negatively charged carboxylate group of the leucine substrate coordinating the ion directly. In NA2, which comprises only neutral ligands, selectivity for Na⁺ is enforced by the local structural restraints arising from the hydrogen-bonding network and the covalent connectivity of the polypeptide chain surrounding the ion according to a “snug-fit” mechanism.     

Ion transport and energy transduction of P-type ATPases: Implications from electrostatic calculations

This paper summarizes our present electrostatic calculations on P-type ATPases and their contribution to understand the molecular details of the reaction mechanisms. One focus was set on analyzing the proton countertransport of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a). Protonation of acidic residues was calculated in dependence of pH for different enzyme states in the reaction cycle of the Ca²⁺-ATPase. We proposed that the acidic Ca²⁺ ligands Glu 771, Asp 800 and Glu 908 participate in the proton countertransport whereas Glu 309 is more likely to serve as a proton shuttle between binding site I and the cytoplasm. Complementary to infrared measurements, we assigned infrared bands to specific Ca²⁺ ligands that are hydrogen bonded. Ion pathways were proposed based on the calculations and structural data. Another focus was set on analyzing the energy transduction mechanism of P-type ATPases. In accordance to electrophysiological experiments, we simulated an electric field across the membrane. The impact of the electric field was studied by an accumulated number of residue conformational and ionization changes on specific transmembrane helices. Our calculations on the Ca²⁺-ATPase and the Na⁺/K⁺-ATPase indicated that the highly conserved transmembrane helix M5 is one structural element that is likely to act as energy transduction element in P-type ATPases. Perspectives and limitations of the electrostatic calculations for future computational studies are pointed out.     

Competitive Binding of Mg2+ and Na+ Ions to Nucleic Acids: From Helices to Tertiary Structures

Nucleic acids generally reside in cellular aqueous solutions with mixed divalent/monovalent ions, and the competitive binding of divalent and monovalent ions is critical to the structures of nucleic acids because of their polyanionic nature. In this work, we first proposed a general and effective method for simulating a nucleic acid in mixed divalent/monovalent ion solutions with desired bulk ion concentrations via molecular dynamics (MD) simulations and investigated the competitive binding of Mg²⁺/Na⁺ ions to various nucleic acids by all-atom MD simulations. The extensive MD-based examinations show that single MD simulations conducted using the proposed method can yield desired bulk divalent/monovalent ion concentrations for various nucleic acids, including RNA tertiary structures. Our comprehensive analyses show that the global binding of Mg²⁺/Na⁺ to a nucleic acid is mainly dependent on its structure compactness, as well as Mg²⁺/Na⁺ concentrations, rather than the specific structure of the nucleic acid. Specifically, the relative global binding of Mg²⁺ over Na⁺ is stronger for a nucleic acid with higher effective surface charge density and higher relative Mg²⁺/Na⁺ concentrations. Furthermore, the local binding of Mg²⁺/Na⁺ to a phosphate of a nucleic acid mainly depends on the local phosphate density in addition to Mg²⁺/Na⁺ concentrations.     

Sodium plays a more important role than potassium and chloride in growth of <Emphasis Type="Italic">Salicornia europaea</Emphasis>

Salicornia europaea is a succulent euhalophyte that belongs to the Chenopodiaceae family. It is found that moderate concentration of NaCl can dramatically stimulate the growth of S. europaea plants. To elucidate the mechanism underlying the phenomenon, morphological and physiological changes of S. europaea in response to different ions, including cations (Na⁺, K⁺, Li⁺, Cs⁺) and anions (Cl⁻, NO₃ ⁻, CH₃COO⁻) were investigated, and the effects of Na⁺, Cl⁻ and K⁺ on the growth of S. europaea were also studied. Na⁺ was more effective than K⁺ and Cl⁻ in stimulating shoot succulence, cell expansion, and stomatal opening. Plants treated with Na⁺ (including NaCl, Na⁺, NaNO₃) showed better plant growth, increased photosynthesis and less cell membrane damage than those untreated and treated with 200 mM of Cl⁻ and K⁺ (including KCl and KNO₃). Both SEM-X-Ray microanalysis and flame emission results revealed that well developed S. europaea plants had a higher content of sodium but lower potassium and chlorine. It is concluded that sodium plays a more important role in the growth and development of S. europaea than potassium and chloride.     

Solvation structures of sodium halides in dimethyl sulfoxide (DMSO)–methanol (MeOH) mixtures

A constrained molecular dynamics technique has been used to study the structures and dynamics of the solvation shells of three sodium halides, namely sodium chloride (Na⁺–Cl^?), sodium bromide (Na⁺–Br^?) and sodium iodide (Na⁺–I^?) in DMSO–MeOH mixtures. In the case of Na⁺–Cl^? and Na⁺–Br^?, Na⁺ is preferentially solvated by DMSO and Cl^? and Br^? are preferentially solvated by methanol in the contact ion pair (CIP) state. In the solvent-assisted ion pair (SAIP) configuration, Na⁺ ions of Na⁺–Cl^? and Na⁺–Br^? are preferentially solvated by methanol and Cl^? and Br^? also show preferential solvation by methanol over DMSO. In the case of Na⁺–I^?, the only preferential solvation is in the SAIP state for I^? ion by methanol. These observations are supported by the calculated excess coordination numbers and spatial density maps. The heights of the transition states barriers for CIPs and SAIPs/solvent-shared ion pairs (SSHIPs) are significantly affected when the mole fraction of methanol (x_MeOH) changes from 0.0 to 0.25 because of a significant increase in the methanol density around halides. From the analysis of angular distribution functions of DMSO and methanol around the cations and anions, it is seen that DMSO and methanol molecules are present in parallel dipolar orientations (with respect to cation–solvent vector) in the first coordination shell of these three ion pairs at the CIP and SAIP states. Methanol molecules are nearly in an antiparallel (with respect to ion–solvent vector) orientation around the three halide ions.     

Water-Transporting Proteins

Transport through lipids and aquaporins is osmotic and entirely driven by the difference in osmotic pressure. Water transport in cotransporters and uniporters is different: Water can be cotransported, energized by coupling to the substrate flux by a mechanism closely associated with protein. In the K⁺/Cl⁻ and the Na⁺/K⁺/2Cl⁻ cotransporters, water is entirely cotransported, while water transport in glucose uniporters and Na⁺-coupled transporters of nutrients and neurotransmitters takes place by both osmosis and cotransport. The molecular mechanism behind cotransport of water is not clear. It is associated with the substrate movements in aqueous pathways within the protein; a conventional unstirred layer mechanism can be ruled out, due to high rates of diffusion in the cytoplasm. The physiological roles of the various modes of water transport are reviewed in relation to epithelial transport. Epithelial water transport is energized by the movements of ions, but how the coupling takes place is uncertain. All epithelia can transport water uphill against an osmotic gradient, which is hard to explain by simple osmosis. Furthermore, genetic removal of aquaporins has not given support to osmosis as the exclusive mode of transport. Water cotransport can explain the coupling between ion and water transport, a major fraction of transepithelial water transport and uphill water transport. Aquaporins enhance water transport by utilizing osmotic gradients and cause the osmolarity of the transportate to approach isotonicity.     

Calculations of K+, Na+, and Cl? fluxes across cell membrane with Na+/K+ pump, NKCC, NC cotransport and ionic channels with non-Goldman rectification in K+-channels: Normal and apoptotic cells

The balance of K⁺, Na⁺, and Cl⁻ fluxes across the cell membrane with the Na⁺/K⁺ pump, ion channels, and Na⁺K⁺2Cl⁻ (NKCC) and Na⁺-Cl⁻ (NC) cotransport was calculated to determine the mechanism of cell shrinkage in apoptosis. It is shown that all unidirectional K⁺, Na⁺, and Cl⁻ fluxes; the ion channel permeability; and the membrane potential can be found using the principle of the flux balance if the following experimental data are known: K⁺, Na⁺, and Cl⁻ concentrations in cell water; total Cl⁻ flux; total K⁺ influx; and the ouabain-inhibited pump component of the Rb⁺(K⁺) influx. The change in different ionic pathways during apoptosis was estimated by calculations based on the data reported in the preceded paper (Yurinskaya et al., 2010). It is found that cell shrinkage and the shift in ion balance in U937 cells induced to apoptosis with 1 μM staurosporine occur due to the coupling of reduced pump activity with a decrease in the integral permeability of Na⁺ channels, whereas K⁺ and Cl⁻ channel permeability remains almost unchanged. Calculations show that only a small part of the total fluxes of K⁺, Na⁺, and Cl⁻ account for the fluxes mediated by NKCC and NC cotransporters. Despite the importance of cotransport fluxes for maintaining the nonequilibrium steady-state distribution of Cl⁻, they cannot play a significant role in apoptotic cell shrinkage because of their minority and cannot be revealed by inhibitors.     

Changes in K<Superscript>+</Superscript>, Na<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> balance and K<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> fluxes in U937 cells induced to apoptosis by staurosporine: On cell dehydration in apoptosis

The K⁺, Na⁺, and Cl⁻ balance and K⁺ (Rb⁺) and ³⁶Cl⁻ fluxes in U937 cells induced to apoptosis by 0.2 or 1 μM staurosporine were studied using flame emission and radioisotope techniques. It is found that two-thirds of the total decrease in the amount of intracellular osmolytes in apoptotic cells is accounted for by monovalent ions and one-third consists of other intracellular osmolytes. A decrease in the amount of monovalent ions results from a decrease in the amount of K⁺ and Cl⁻ and an increase in the Na⁺ content. The rate of ³⁶Cl⁻, Rb⁺ (K⁺), and ²²Na⁺ equilibration between cells and the medium was found to significantly exceed the rate of apoptotic change in the cellular ion content, which indicates that unidirectional influxes and effluxes during apoptosis may be considered as being in near balance. The drift of the ion flux balance in apoptosis caused by 0.2 μM staurosporine was found to be associated with the increased ouabain-resistant Rb⁺ (K⁺) channel influx and insignificantly altered the ouabain-sensitive pump influx. Severe apoptosis induced by 1 μM staurosporine is associated with reduced pump fluxes and slightly changed channel Rb⁺ (K⁺) fluxes. In apoptotic cells, the 1.4–1.8-fold decreased Cl⁻ level is accompanied by a 1.2–1.6-fold decreased flux.     

The ionic hemolymph composition of the terrestrial isopod Porcellio scaber Latr. during molt

We analyzed the ionic composition of the hemolymph of Porcellio scaber in four different stages of the molt cycle using capillary electrophoresis and calcium selective mini- and microelectrodes. The main ions in the hemolymph were K⁺, Ca²⁺, Na⁺, Mg⁺, and Cl⁻. The values for total calcium obtained by means of capillary electrophoresis and calcium selective minielectrodes did not differ significantly from each other. In situ measurements of the free Ca²⁺ concentration ([Ca²⁺]) by means of calcium-selective microelectrodes indicated that Ca²⁺ is not bound in the hemolymph. During molt the [Ca²⁺] is significantly larger than during intermolt. The [Ca²⁺] increased by 13%, 19% and 18% during premolt, intramolt, and postmolt, respectively. The concentration of the other cations and of Cl⁻ decreased significantly between premolt and intramolt. Thus, the rise of the [Ca²⁺] in the hemolymph is not due to a general increase in all ions, but rather to the resorption of cuticular calcium. Furthermore, the results suggest that K⁺, Na⁺, Mg⁺, and Cl⁻are extruded from the hemolymph during and/or after posterior ecdysis. Accepted: 5 August 1997     

The role of protons in fast and slow gating of the Torpedo chloride channel ClC-0

Transmembrane proton transport is of fundamental importance for life. The list of H⁺ transporting proteins has been recently expanded with the discovery that some members of the CLC gene family are stoichiometrically coupled Cl⁻/H⁺ antiporters. Other CLC proteins are instead passive Cl⁻ selective anion channels. The gating of these CLC channels is, however, strongly regulated by pH, likely reflecting the evolutionary relationship with CLC Cl⁻/H⁺ antiporters. The role of protons in the gating of the model Torpedo channel ClC-0 is best understood. ClC-0 is a homodimer with separate pores in each subunit. Each protopore can be opened and closed independently from the other pore by a “fast gate”. A common, slow gate acts on both pores simultaneously. The opening of the fast gate is controlled by a critical glutamate (E166), whose protonation state determines the fast gate’s pH dependence. Extracellular protons likely can arrive directly at E166. In contrast, protonation of E166 from the inside has been proposed to be mediated by the dissociation of an intrapore water molecule. The OH⁻ anion resulting from the water dissociation is stabilized in one of the anion binding sites of the channel, competing with intracellular Cl⁻ ions. The pH dependence of the slow gate is less well understood. It has been shown that proton translocation drives irreversible gating transitions associated with the slow gate. However, the relationship of the fast gate’s pH dependence on the proton translocation and the molecular basis of the slow gate remain to be discovered.