Mitochondrial potassium and chloride channels

Channels selective for potassium or chloride ions are present in inner mitochondrial membranes. They probably play an important role in mitochondrial events such as the formation of delta pH and regulation of mitochondrial volume changes. Mitochondrial potassium and chloride channels could also be the targets for pharmacologically active compounds such as potassium channel openers and antidiabetic sulfonylureas. This review describes the properties, pharmacology, and current observations concerning the functional role of mitochondrial potassium and chloride channels.     

Bead-like passage of chloride ions through ClC chloride channels

The ClC chloride channels control the ionic composition of the cytoplasm and the volume of cells, and regulate electrical excitability. Recently, it has been proposed that prokaryotic ClC channels are H+-Cl- exchange transporter. Although X-ray and molecular dynamics (MD) studies of bacterial ClC channels have investigated the filter open-close and ion permeation mechanism of channels, details have remained unclear. We performed MD simulations of ClC channels involving H+, Na+, K+, or H3O+ in the intracellular region to elucidate the open-close mechanism, and to clarify the role of H+ ion an H+-Cl- exchange transporter. Our simulations revealed that H+ and Na+ caused channel opening and the passage of Cl- ions. Na+ induced a bead-like string of Cl- -Na+-Cl--Na+-Cl- ions to form and permeate through ClC channels to the intracellular side with the widening of the channel pathway.     

Insolubilization of potassium chloride crystals in Tradescantia pallida

Summary. Calcium oxalate crystals are by far the most prevalent and widely distributed mineral deposits in higher plants. In Tradescantia pallida, an evergreen perennial plant widely used as an ornamental plant, calcium oxalate crystals occur in the parenchymal tissues of stem, leaf, and root, as well as in flower organs, in the form of either raphides or tetragonal prismatic crystals or both. Energy-dispersive X-ray analysis revealed that C, O, and Ca were the main elements; and K, Cl, and Si, the minor elements. Infrared and X-ray analyses of crystals collected from these tissues detected the coexistence of two calcium oxalate chemical forms, i.e., whewellite and weddellite, as well as calcite, opal, and sylvite. Here, we show for the first time the occurrence of epitaxy in mineral crystals of plants. Epitaxy, which involves the oriented overgrowth of one crystal onto a second crystalline substrate, might explain how potassium chloride (sylvite) – one of the most water-soluble salts – stays insoluble in crystal form when coated with a calcium oxalate epilayer. The results indicate the potential role of crystals in regulating the ionic equilibrium of both calcium and potassium ions. Correspondence and reprints: Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EGA, Ciudad de Buenos Aires, Argentina.     

Coupling of water and potassium ions in K+ channels of the tonoplast of Chara

Electrokinetic measurements, of streaming potential, were carried out on an excised inside-out patch of the vacuolar membrane of Chara corallina. A water activity gradient was imposed across the patch membrane containing a single K⁺ channel by addition of sorbitol to one side. Two different K⁺ channels were found in the tonoplast. Their open channel conductance was investigated as a function of KCl concentration. They had a maximal open channel conductance of 247 and 173 pS, and an apparent affinity (K_M) of 116 and 92 mM, respectively. Single-channel zero-current potentials were determined in the presence of an osmotic gradient, and dilution artifacts were corrected for by addition of valinomycin to the bath. Our results suggest that 29 water molecules were coupled to the transport of one K⁺ ion in the large conductance K⁺ channel which has a pore radius of ~1.5 nm.     

Conduction mechanisms of chloride ions in ClC-type channels

The conduction properties of ClC-0 and ClC-1 chloride channels are examined using electrostatic calculations and three-dimensional Brownian dynamics simulations. We create an open-state configuration of the prokaryotic ClC Cl(-) channel using its known crystallographic structure as a basis. Two residues that are occluding the channel are slowly pushed outward with molecular dynamics to create a continuous ion-conducting path with the minimum radius of 2.5 A. Then, retaining the same pore shape, the prokaryotic ClC channel is converted to either ClC-0 or ClC-1 by replacing all the nonconserved dipole-containing and charged amino acid residues. Employing open-state ClC-0 and ClC-1 channel models, current-voltage curves consistent with experimental measurements are obtained. We find that conduction in these pores involves three ions. We locate the binding sites, as well as pinpointing the rate-limiting steps in conduction, and make testable predictions about how the single channel current across ClC-0 and ClC-1 will vary as the ionic concentrations are increased. Finally, we demonstrate that a ClC-0 homology model created from an alternative sequence alignment fails to replicate any of the experimental observations.     

Intracellular potassium and chloride channels: an update.

Channels selective for potassium or chloride ions are present in all intracellular membranes such as mitochondrial membranes, sarcoplasmic/endoplasmic reticulum, nuclear membrane and chromaffin granule membranes. They probably play an important role in events such as acidification of intracellular compartments and regulation of organelle volume. Additionally, intracellular ion channels are targets for pharmacologically active compounds, e.g. mitochondrial potassium channels interact with potassium channel openers such as diazoxide. This review describes current observations concerning the properties and functional roles of intracellular potassium and chloride channels.     

Diffusion of lysozyme chloride in water and aqueous potassium chloride solutions.

The diffusion of lysozyme chloride in aqueous solution has been studied at 25 degrees C using the Goüy interferometric technique. The concentration dependence of the diffusion coefficient in water has been measured over the concentration range 1.1599-9.1556 gcm-3 and the results suggest a value of D 25, w at infinite dilution of 5.838 x 10(-6) cm2s-1. The variation in diffusion coefficient with ionic strength has also been considered by following the diffusion of 0.45% lysozyme chloride in a series of potassium chloride solutions. The value of D in 0.15 M KCl has been found to be approximately one quarter of that in water alone an the diffusion coefficient has been shown to increase markedly as the KCl concentration is reduced below 0.05 M. Interpretation of these observations involves consideration of solution electrostatic effects.     

Molecular dynamics estimates of ion diffusion in model hydrophobic and KcsA potassium channels

Molecular dynamics simulations are carried out to obtain estimates of diffusion coefficients of biologically important Na+, K+, Ca2+ and Cl- ions in hydrophobic cylindrical channels with varying radii and large reservoirs. Calculations for the cylindrical channels are compared to those for the KcsA potassium channel, for which the protein structure has recently been determined from X-ray diffraction experiments. Our results show that ion diffusion is maintained at reasonably high levels even within narrow channels, and does not support the very small diffusion coefficients used in some continuum models in order to fit experimental data. The present estimates of ion diffusion coefficients are useful in the calculation of channel conductance using the Poisson-Nernst-Planck theory, or Brownian dynamics.     

Intracellular chloride and potassium ions in relation to excitability ofChara membrane

Summary Internodal cells ofChara australis were made tonoplast-free by replacing the cell sap with EGTA-containing media; then the involvement of internal Cl^– and K⁺ in the excitation of the plasmalemma was studied.[Cl^–] _i was drastically decreased by perfusing the cell interior twice with a medium lacking Cl^–. The lowered [Cl^–] _i was about 0.01mm. Cells with this low [Cl^–] _i generated action potential and showed anN-shapedV–I curve under voltage clamped depolarization like Cl^–-rich cells containing 13 or 29mm Cl^–.E _m at the peak of the action potential was constant at [Cl^–] _i between 0.01 and 29mm. The possibility that the plasmalemma becomes as permeable to other anions as to Cl^– during excitation is discussed.At [Cl^–] _i higher than 48mm, cells were inexcitable. When anions were added to the perfusion medium to bring the K⁺ concentration to 100mm, NO ₃ ^– , F^–, SO ₄ ^2– , acetate, and propionate inhibited the generation of action potentials like Cl^–, while methane sulfonate, PIPES, and phosphate did not inhibit excitability.The duration of the action potential depended strongly on the intracellular K⁺ concentration. It decreased as [K⁺] _i (K-methane sulfonate) increased. Increase in [Na⁺] _i (Na-methane sulfonate) also caused its decrease, although this effect was weaker than that of K⁺. The action of these monovalent cations on the duration of the action potential is the opposite of their action on the membrane from the outside (cf. Shimmen, Kikuyama & Tazawa, 1976,J. Membrane Biol. 30:249).     

Repulsion between tetraethylammonium ions in cloned voltage-gated potassium channels.

Tetraethylammonium ion (TEA+) blocks voltage-gated K+ channels by acting at two sites located at opposite ends of the aqueous pore. This allowed us to test two predictions made by models of ion permeation, namely that K+ channels can be simultaneously occupied by multiple ions and that the ions repel each other. We show that externally applied TEA+ antagonize block by internal TEA+ and vice versa. The antagonism is less than predicted for competitive binding, hence TEA+ may occupy both sites simultaneously. External TEA+ and internal TEA+ reduce each others affinity 4- to 5-fold. In addition, K+ antagonizes block by TEA+ at the opposite side of the membrane, and external TEA+ antagonizes is block by internal Ba2+. The antagonism between ions applied at opposite sides of the membrane may be common to all cations binding to K+ channels.     

The influence of hydrophobic ions and dipolar molecules on the electrostatic barrier in biomembranes

To calculate the electric field inside a membrane the aqueous phase can be approximated by a conductor since the dielectric constant of water is much larger than that of the membrane. Then, using the method of image charges, ions adsorbed inside the membrane can be considered as dipoles and dipolar molecules adsorbed inside the membrane may similarly be regarded as sets of two similarly oriented dipoles. The microscopic interactions and, therefore, the spatial correlations of the adsorbed species can then be obtained. Together with the Gouy theory for the diffuse double layer these results allow the determination of the adsorbed phase—aqueous phase equilibrium. From the densities and spatial correlations of the adsorbed ions and dipolar species, their influence upon the electrostatic barrier as experienced by an ion translocating the membrane can be calculated. Changes observed in the relaxation time and initial conductance of translocating hydrophobic ions in voltage-pulse experiments on bilayer membranes are predicted using this model of the electrostatic barrier. In addition, an equation giving the surface tension as a function of the (non-ideal) adsorption of hydrophobic ions and dipoles is derived.     

Effect of calcium ions on potassium conductivity of latrotoxin channels

It has been shown that inhibition of potassium current through latrotoxin channels by calcium ions is followed by electrostatic interaction of these ions with a total charge on the mouth of the channel.     

Block of neuronal fast chloride channels by internal tetraethylammonium ions

The classical potassium-selective ion channel blocker tetraethylammonium ion (TEA) was shown to block chloride-selective ion channels from excised surface membranes of acutely dissociated rat cortical neurons when applied to the formerly intracellular membrane surface. The patch voltage clamp method was used to record single channel currents from fast Cl channels in the presence of TEAi. At the filtering cut-off frequencies used (3-12.4 kHz, -3 dB) the TEAi-induced block appeared as a reduction in single channel current amplitude, which was interpreted as the result of extremely fast on the off rates for the blocking reaction. Under the conditions of these experiments, the magnitude of TEAi block was independent of membrane potential. Analysis of dose-response experimental results suggests that TEA binding resulted in a partial block of these channels with an equilibrium dissociation constant of approximately 12-15 mM. Analysis of amplitude distributions in the absence and presence of TEAi using the method of Yellen (1994. Journal of General Physiology. 84:157-186.) produced a similar equilibrium dissociation constant and provided a blocking rate constant of approximately 16,000 mM-1.s-1 and an unblocking rate constant of approximately 200,000 s-1. The distributions of open and closed interval durations were fit with a blocking scheme where TEAi binds to the open kinetic state with the constraint that the channel must reenter the open state before TEA can dissociate. The increase in the mean lifetime of the open state could be well fit by this model, but the distribution of closed interval durations could not, suggesting a more complex underlying blocking mechanism.     

Networks of water molecules in a proflavine deoxydinucleoside phosphate complex

Using previously reported ab-initio atom-atom potentials for the interactions of a water molecule with phosphates, sugars and bases and newly computed ab-initio atom-atom potentials for the interaction between a proflavine ion and water, we have analyzed with the Monte-Carlo Metropolis method networks of water molecules hydrating a 2:2 complex of proflavine and deoxycytidylyl-3',5'-guanosine, recently studied with X-ray crystallography. From our simulations we have i) verified the quality of our atom-atom potentials by obtaining patterns of oxygen atoms in very good agreement with the X-ray patterns for the minor groove and in reasonable agreement in the major groove, ii) predicted the water's hydrogen atoms positions and iii) preliminarily predicted the number of water molecules not reported in the X-ray study but present in the major groove. The above data, even if preliminary, and the analyses on the energetics of the water-water, water-proflavine and water-dCpG interactions indicate that very detailed accounts on the water filaments in the above crystal can be obtained optimally by merging computer and X-ray experiments.     

Substrate regulation of single potassium and chloride ion channels in Arabidopsis plasma membrane

Patch clamp measurements of excised inside-out patches of Arabidopsis thaliana plasma membrane reveal at least two ion channels which conduct either potassium or chloride. The conductance of the potassium channel ranged from 5 to 70 picosiemens depending on KCl concentration. The conductance increased linearly with increasing cytoplasmic-side [KCl]; the extent of this dependence declined as extracytoplasmic-side [KCl] was increased. This indicates that substrate regulation of the potassium channel is a consequence of the molecular architecture of the channel: in particular, multi-ion binding sites within the channel pore. The chloride channel conductance (ranging from 5-40 picosiemens) was independent of cytoplasmic-side [KCl] until a threshold concentration of about 300 millimolar was reached. Such behavior is expected only if the channel is allosterically regulated by cytoplasmic-side K⁺ and/or Cl⁻. The median open times of either channel (about 200 milliseconds for the potassium channel and 20 milliseconds for the chloride channel) were unaffected by substrate concentrations.     

Small conductance potassium channels drive ATP-activated chloride secretion in the oviduct

The molecular mechanisms controlling fluid secretion within the oviduct have yet to be determined. As in other epithelia, both secretory and absorptive pathways are likely to work in tandem to drive appropriate ionic movement to support fluid movement across the oviduct epithelium. This study explored the role of potassium channels in basolateral extracellular ATP (ATP(e))-stimulated ion transport in bovine oviduct epithelium using the Ussing chamber short-circuit current (I(SC)) technique. Basal I(SC) in bovine oviduct epithelium comprises both chloride secretion and sodium absorption and was inhibited by treatment with basolateral K(+) channel inhibitors tetrapentlyammonium chloride (TPeA) or BaCl(2). Similarly, ATP-stimulated chloride secretion was significantly attenuated by pretreatment with BaCl(2,) tetraethylammonium (TEA), tolbutamide, and TPeA. Basolateral K(+) current, isolated using nystatin-perforation technique, was rapidly activated by ATP(e), and pretreatment of monolayers with thapsigargin or TPeA abolished this ATP-stimulated K(+) current. To further investigate the type of K(+) channel involved in the ATP response in the bovine oviduct, a number of specific Ca(2+)-activated K(+) channel inhibitors were tested on the ATP-induced ΔI(SC) in intact monolayers. Charbydotoxin, (high conductance and intermediate conductance inhibitor), or paxilline, (high conductance inhibitor) did not significantly alter the ATP(e) response. However, pretreatment with the small conductance inhibitor apamin resulted in a 60% reduction in the response to ATP(e). The presence of small conductance family member KCNN3 was confirmed by RT-PCR and immunohistochemistry. Measurements of intracellular calcium using Fura-2 spectrofluorescence imaging revealed the ability of ATP(e) to increase intracellular calcium in a phospholipase C-inositol 1,4,5-trisphosphate pathway-sensitive manner. In conclusion, these results provide strong evidence that purinergic activation of a calcium-dependent, apamin-sensitive potassium conductance is essential to promote chloride secretion and thus fluid formation in the oviduct.     

Combined transport of water and ions through membrane channels

The coupling of ion and water flow through membrane channels is under dispute. Among all human aquaporins only aquaporin-6 exhibits ion channel activity. Whether aquaporin-6 functions also as a water channel cannot yet be determined with confidence. Similarly, a comparison of single-channel water permeabilities from ion channels and aquaporins suggests that ion channels may play a secondary role as water channels. However, the fraction of absorbed fluid that crosses epithelial ion channels still remains to be determined.     

Heat capacity effects of water molecules and ions at a protein-DNA interface

The interaction of the TATA-box binding protein from the thermophilic and halophilic archaea Pyrococcus woesei (PwTBP) with an oligonucleotide containing a specific binding site is stable over a very broad range of temperatures and ionic strengths, and is consequently an outstanding system for characterising general features of protein-DNA thermodynamics. In common with other specific protein-DNA recognition events, the PwTBP-TATA box interaction is accompanied by a large negative change in heat capacity (ΔC_p) arising from the total change in solvation that occurs upon binding, which in this case involves a net uptake of cations. Contrary to previous hypotheses, we find no overall effect of ionic strength on this heat capacity change. We investigate the local contributions of site-specific ion and water binding to the overall change in heat capacity by means of a series of site-directed mutations of PwTBP. We find that although changes in the local ion binding capacity affect the enthalpic and entropic contributions to the free energy of the interaction, they do not affect the change in heat capacity. In contrast, we find remarkably large heat capacity effects arising from two particular symmetry-related mutations. The great magnitude of these effects is not explicable in terms of current semi-empirical models of heat capacity change. Previously reported X-ray crystal structures show that these mutated residues are at the centre of an evolutionarily conserved network of water-mediated hydrogen bonds between the protein and the DNA backbone. Consequently, we conclude that, in addition to water molecules buried in the protein-DNA interface that have been previously shown to influence heat capacity, bridging water molecules in a highly polar surface environment can also contribute substantially to negative heat capacity change on formation of a protein-DNA complex.     

Intracellular activities of chloride, potassium and sodium ions in rabbit corneal epithelium

The mechanism of ion transport in the epithelium of rabbit cornea was studied by determining the intracellular ion activity of Cl-, Na+ and K+ under various conditions. Ionic activities were measured by means of microelectrodes containing liquid ion-exchangers selective for Cl-, Na+ or K+. The Cl- activity in basal cells of the epithelium in Na+ containing bathing solutions amounts to 28 +/- 2 mM (n = 11). This value is 1.9-times greater than expected on the basis of passive distribution across the tear side membrane. This finding suggests the existence of a Cl- accumulating process. Replacement of Na+ in the aqueous bathing solution by choline or tetraethylammonium results in a reversible decrease in Cl- activity to 22 +/- 1 mM (n = 11, P less than 0.025). The ratio of observed and predicted Cl- activity decreased significantly from 1.9 to 1.4 (P less than 0.05). The decrease in Cl- activity due to Na+ replacement was rather slow. In contrast, after readmittance of Na+ to the aqueous bathing solution, Cl- activity rose to a stable level within 30 min. These results indicate involvement of Na+ in Cl- accumulation into the basal cells of the epithelium. The K+ and Na+ activities of the basal cells of rabbit corneal epithelium in control bathing solutions were 75 +/- 4 mM (n = 13) and 24 +/- 3 mM (n = 12), respectively. The results can be summarized in the following model for Cl- transport across corneal epithelium. Cl- is accumulated in the basal cells across the aqueous side membrane, energized by a favourable Na+ gradient. Cl- will subsequently leak out across the tear side membranes. Na+ is extruded again across the aqueous side membrane of the epithelium by the (Na+ + K+)-ATPase.