Review. Peering into an ATPase ion pump with single-channel recordings

In principle, an ion channel needs no more than a single gate, but a pump requires at least two gates that open and close alternately to allow ion access from only one side of the membrane at a time. In the Na+,K+-ATPase pump, this alternating gating effects outward transport of three Na+ ions and inward transport of two K+ ions, for each ATP hydrolysed, up to a hundred times per second, generating a measurable current if assayed in millions of pumps. Under these assay conditions, voltage jumps elicit brief charge movements, consistent with displacement of ions along the ion pathway while one gate is open but the other closed. Binding of the marine toxin, palytoxin, to the Na+,K+-ATPase uncouples the two gates, so that although each gate still responds to its physiological ligand they are no longer constrained to open and close alternately, and the Na+,K+-ATPase is transformed into a gated cation channel. Millions of Na+ or K+ ions per second flow through such an open pump-channel, permitting assay of single molecules and allowing unprecedented access to the ion transport pathway through the Na+,K+-ATPase. Use of variously charged small hydrophilic thiol-specific reagents to probe cysteine targets introduced throughout the pump's transmembrane segments allows mapping and characterization of the route traversed by transported ions.     

KcsA通道对Na+、K+及Rb+离子选择性的统计热力学研究

钾离了的通透率至少比钠离子的通透率大10000倍,这个问题至今没有很好地解决,为了存分子水平阐释钾离子通道的选择性机制,以KcsA钾通道X射线衍射结构为基础,采用密度泛函理论计算了不同离子在离子通道中的位能．计算结果表明,Rb^ 离子具有与K^ 离子相类似的位能曲线,但是其在通透过程遇到的位垒要比K^ 离子的位垒高,因而所对应的通透率也就小十钾离子的通透率,而钠离子的的通透率仅仅足钾离子通透率的0．0067％．文中所涉及的系统仪仅包含269个原子,而用分子动力学虽然也可以得到相近的结果,但是它的系统火小为41000个原子．     

Distinct metal ion binding sites on Ca(2+)-activated K+ channels in inside-out patches of human erythrocytes.

Effects of Cd2+, Co2+, Pb2+, Fe2+ and Mg2+ (1-100 microM) on single-channel properties of the intermediate conductance Ca(2+)-activated K+ (CaK) channels were investigated in inside-out patches of human erythrocytes in a physiological K+ gradient. Cd2+, Co2+ and Pb2+, but not Fe2+ and Mg2+, were able to induce CaK channel openings. The potency of the metals to open CaK channels in human erythrocytes follows the sequence Pb2+, Cd2+ > Ca2+ > or = Co2+ > Mg2+, Fe2+. At higher concentrations Pb2+, Cd2+ and Co2+ block the CaK channel by reducing the opening frequency and the single-channel current amplitude. The potency of the metals to reduce CaK channel opening frequency follows the sequence Pb2+ > Cd2+, Co2+ > Ca2+, which differs from the potency sequence Cd2+ > Pb2+, Co2+ > Ca2+ to reduce the unitary single-channel current amplitude. Fe2+ reduced the channel opening frequency and enhanced the two open times of CaK channels activated by Ca2+, whereas up to 100 microM Mg2+ had no effect on any of the measured single-channel parameters. It is concluded that the activation of CaK channels of human erythrocytes by various metal ions occurs through an interaction with the same regulatory site at which Ca2+ activates these channels. The different potency orders for the activating and blocking effects suggest the presence of at least one activation and two blocking sites. A modulatory binding site for Fe2+ exists as well. In addition, the CaK channels in human erythrocytes are distinct from other subtypes of Ca(2+)-activated K+ channels in their sensitivity to the metal ions.     

Kinetic properties of Na+, K+ activated, Mg2+ -dependent ATP-hydrolysis of blood lymphocytes in patients with rheumatoid arthritis and ankylosing spondyloarthritis

The comparative analysis of the kinetic properties of ouabain-sensitive Na+, K+ -ATPase activity of saponin-perforated blood lymphocytes of donors and patients with rheumatoid arthritis (RA) and ankylosing spondyloarthritis (AS) was carried out. When analyzing the alterations in hydrolase activity of the examined enzyme it was shown that in the blood lymphocytes of patients with RA and AS the primary active transport of Na+ and K+ ions is less intensive in comparison with practically healthy donors, but it is characterized by almost the same capacity as in donors. The affinity constant of Na+, K+ -ATPase for ATP in the blood lymphocytes in patients with RA and AS is greater 3.1 and 2.5 times, respectively, in comparison with healthy donor. It was found that in conditions of rheumatic pathology in immunocompetent cells the inhibition of Na+, K+ -ATPase activity is not related to the reduction of maximum reaction rate, but is related to the decrease of Na+, K+ -ATPase affinity to ATP. However, Mg2+ -binding center of Na+, K+ -ATPase in patients with RA and AS remains native. It was identified that the affinity constant of Na+, K+ -ATPase to Na+ ions in the blood lymphocytes of patients with RA and AS is 2.75 times lower than its value in healthy donors. Na+, K+ -ATPase of the blood lymphocytes of patients with RA and AS retains its native receptor properties and sensitivity to ouabain does not change.     

Phenylalkylamine-sensitive calcium channels in osteoblast-like osteosarcoma cells. Characterization by ligand binding and single channel recordings

(-)-[3H]Desmethoxyverapamil ((-)-DMV) binds saturably to homogenates of the osteoblast-like cell lines UMR 106 and ROS 17/2.8 with KD values of 45 and 61 nM and Bmax values of 6.0 and 5 pmol/mg protein, respectively. Binding is stereoselective with (-)-DMV 8-10 times more potent than (+)-DMV. None of the dihydropyridine or benzothiazepine Ca2+ antagonists examined affect (-)-[3H]DMV binding. Monovalent cations such as Li+, Na+, and K+ inhibit (-)[3H]DMV binding in the 100-400 mM range. Divalent cations such as Ba2+, Sr2+, Ca2+, and Mg2+ are effective binding inhibitors in the 2-5 mM range. ROS 17/2.8 cells express a channel on the apical plasma membrane which conducts Ba2+ and Ca2+. With 110 mM BaCl2 or CaCl2 as charge carriers the single channel conductance is 3-5 picosiemens. In cell-excised patches the channel selects for Ba2+ over Na+ 3.3:1. In the absence of divalent ions the channel conducts Na+ ions with a single channel conductance of 13 picosiemens. This Na+ conductance decreases with physiological levels of Ca2+. The channel appears related to the (-)-[3H]DMV binding site, since its conductance is blocked by verapamil in a dose-dependent manner. Moreover, DMV blocks the channel stereoselectively with relative potencies of the isomers corresponding to their affinities for the binding site. The dihydropyridine drugs BAY K 8644 or (+)-202-791 do not affect channel opening. These binding and biophysical data indicate that osteoblast cells have a phenylalkylamine receptor associated with a Ca2+ channel.     

Tuning ion coordination architectures to enable selective partitioning

K+ ions seemingly permeate K-channels rapidly because channel binding sites mimic coordination of K+ ions in water. Highly selective ion discrimination should occur when binding sites form rigid cavities that match K+, but not the smaller Na+, ion size or when binding sites are composed of specific chemical groups. Although conceptually attractive, these views cannot account for critical observations: 1), K+ hydration structures differ markedly from channel binding sites; 2), channel thermal fluctuations can obscure sub-Angstr?m differences in ion sizes; and 3), chemically identical binding sites can exhibit diverse ion selectivities. Our quantum mechanical studies lead to a novel paradigm that reconciles these observations. We find that K-channels utilize a "phase-activated" mechanism where the local environment around the binding sites is tuned to sustain high coordination numbers (>6) around K+ ions, which otherwise are rarely observed in liquid water. When combined with the field strength of carbonyl ligands, such high coordinations create the electrical scenario necessary for rapid and selective K+ partitioning. Specific perturbations to the local binding site environment with respect to strongly selective K-channels result in altered K+/Na+ selectivities.     

Divalent ion trapping inside potassium channels of human T lymphocytes

Using the patch-clamp whole-cell recording technique, we investigated the influence of external Ca2+, Ba2+, K+, Rb+, and internal Ca2+ on the rate of K+ channel inactivation in the human T lymphocyte-derived cell line, Jurkat E6-1. Raising external Ca2+ or Ba2+, or reducing external K+, accelerated the rate of the K+ current decay during a depolarizing voltage pulse. External Ba2+ also produced a use-dependent block of the K+ channels by entering the open channel and becoming trapped inside. Raising internal Ca2+ accelerated inactivation at lower concentrations than external Ca2+, but increasing the Ca2+ buffering with BAPTA did not affect inactivation. Raising [K+]o or adding Rb+ slowed inactivation by competing with divalent ions. External Rb+ also produced a use-dependent removal of block of K+ channels loaded with Ba2+ or Ca2+. From the removal of this block we found that under normal conditions approximately 25% of the channels were loaded with Ca2+, whereas under conditions with 10 microM internal Ca2+ the proportion of channels loaded with Ca2+ increased to approximately 50%. Removing all the divalent cations from the external and internal solution resulted in the induction of a non-selective, voltage-independent conductance. We conclude that Ca2+ ions from the outside or the inside can bind to a site at the K+ channel and thereby block the channel or accelerate inactivation.     

Relief of Na+ block of Ca2+-activated K+ channels by external cations

The flickery block of single Ca2+-activated K+ channels that is produced by internally applied Na+ can be relieved by millimolar concentrations of external K+. This effect of K+ on the kinetics of Na+ block was studied by the method of amplitude distribution analysis described in the companion paper (Yellen, G., 1984b, J. Gen. Physiol., 84:157-186). It appears that K+ relieves block by increasing the exit rate of the blocking ion from the channel, not by competitively slowing its entrance rate. This suggests that a K ion that enters the channel from the outside can expel the blocking Na ion, which entered the channel from the inside. Cs+, which cannot carry current through the channel, and Rb+, which carries a reduced current through the channel, are just as effective as K+ in relieving the block by internal Na+. The kinetics of block by internal nonyltriethylammonium (C9) are unaffected by the presence of these ions in the external bathing solution.     

Permeation, selectivity, and blockade of the Ca2+-activated potassium channel of the guinea pig taenia coli myocyte

The permeation properties of the 147-pS Ca2+-activated K+ channel of the taenia coli myocytes are similar to those of the delayed rectifier channel in other excitable membranes. It has a selectivity sequence of K+ 1.0 greater than Rb+ 0.65 greater than NH4+ 0.50. Na+, Cs+, Li+, and TEA+ (tetraethylammonium) are impermeant. Internal Na+ blocks K+ channel in a strongly voltage-dependent manner with an equivalent valence (zd) of 1.20. Blockade by internal Cs+ and TEA+ is less voltage dependent, with d of 0.61 and 0.13, and half-blockage concentrations of 88 and 31 mM, respectively. External TEA+ is about 100 times more effective in blocking the K+ channel. All these findings suggest that the 147-pS Ca2+-activated K+ channel in the taenia myocytes, which functions physiologically like the delayed rectifier, is the single-channel basis of the repolarizing current in an action potential.     

Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle

The conductance and selectivity of the Ca-activated K channel in cultured rat muscle was studied. Shifts in the reversal potential of single channel currents when various cations were substituted for Ki+ were used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The selectivity was Tl+ greater than K+ greater than Rb+ greater than NH4+, with permeability ratios of 1.2, 1.0, 0.67, and 0.11. Na+, Li+, and Cs+ were not measurably permeant, with permeabilities less than 0.05 that of K+. Currents with the various ions were typically less than expected on the basis of the permeability ratios, which suggests that the movement of an ion through the channel was not independent of the other ions present. For a fixed activity of Ko+ (77 mM), plots of single channel conductance vs. activity of Ki+ were described by a two-barrier model with a single saturable site. This observation, plus the finding that the permeability ratios of Rb+ and NH+4 to K+ did not change with ion concentration, is consistent with a channel that can contain a maximum of one ion at any time. The empirically determined dissociation constant for the single saturable site was 100 mM, and the maximum calculated conductance for symmetrical solutions of K+ was 640 pS. TEAi+ (tetraethylammonium ion) reduced single channel current amplitude in a voltage-dependent manner. This effect was accounted for by assuming voltage-dependent block by TEA+ (apparent dissociation constant of 60 mM at 0 mV) at a site located 26% of the distance across the membrane potential, starting at the inner side. TEAo+ was much more effective in reducing single channel currents, with an apparent dissociation constant of approximately 0.3 mM.     

Tuning the voltage dependence of tetraethylammonium block with permeant ions in an inward-rectifier K+ channel.

To understand the role of permeating ions in determining blocking ion-induced rectification, we examined block of the ROMK1 inward-rectifier K+ channel by intracellular tetraethylammonium in the presence of various alkali metal ions in both the extra- and intracellular solutions. We found that the channel exhibits different degrees of rectification when different alkali metal ions (all at 100 mM) are present in the extra- and intracellular solution. A quantitative analysis shows that an external ion site in the ROMK1 pore binds various alkali metal ions (Na+, K+, Rb+, and Cs+) with different affinities, which can in turn be altered by the binding of different permeating ions at an internal site through a nonelectrostatic mechanism. Consequently, the external site is saturated to a different level under the various ionic conditions. Since rectification is determined by the movement of all energetically coupled ions in the transmembrane electrical field along the pore, different degrees of rectification are observed in various combinations of extra- and intracellular permeant ions. Furthermore, the external and internal ion-binding sites in the ROMK1 pore appear to have different ion selectivity: the external site selects strongly against the smaller Na+, but only modestly among the three larger ions, whereas the internal site interacts quite differently with the larger K+ and Rb+ ions.     

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.     

Survival of K+ permeability and gating currents in squid axons perfused with K+-free media

K+ currents were recorded in squid axons internally perfused with impermeant electrolyte. Total absence of permeant ions inside and out leads to an irreversible loss of potassium conductance with a time constant of approximately 11 min at 8 degrees C. Potassium channels can be protected against this effect by external K+, Cs+, NH4+, and Rb+ at concentrations of 100-440 mM. These experiments suggest that a K+ channel is normally occupied by one or more small cations, and becomes nonfunctional when these cations are removed. A large charge movement said to be related to K+ channel gating in frog skeletal muscle is absent in squid giant axons. However, deliberate destruction of K+ conductance by removal of permeant cations is accompanied by measurable loss in asymmetric charge movement. This missing charge component is large enough to contain a contribution from K+ gating charge movements of more than five elementary charges per channel.     

Permeation of Na+ through a delayed rectifier K+ channel in chick dorsal root ganglion neurons

In whole-cell patch clamp recordings from chick dorsal root ganglion neurons, removal of intracellular K+ resulted in the appearance of a large, voltage-dependent inward tail current (Icat). Icat was not Ca2+ dependent and was not blocked by Cd2+, but was blocked by Ba2+. The reversal potential for Icat shifted with the Nernst potential for [Na+]. The channel responsible for Icat had a cation permeability sequence of Na+ >> Li+ >> TMA+ > NMG+ (PX/PNa = 1:0.33:0.1:0) and was impermeable to Cl-. Addition of high intracellular concentrations of K+, Cs+, or Rb+ prevented the occurrence of Icat. Inhibition of Icat by intracellular K+ was voltage dependent, with an IC50 that ranged from 3.0-8.9 mM at membrane potentials between -50 and -110 mV. This voltage- dependent shift in IC50 (e-fold per 52 mV) is consistent with a single cation binding site approximately 50% of the distance into the membrane field. Icat displayed anomolous mole fraction behavior with respect to Na+ and K+; Icat was inhibited by 5 mM extracellular K+ in the presence of 160 mM Na+ and potentiated by equimolar substitution of 80 mM K+ for Na+. The percent inhibition produced by both extracellular and intracellular K+ at 5 mM was identical. Reversal potential measurements revealed that K+ was 65-105 times more permeant than Na+ through the Icat channel. Icat exhibited the same voltage and time dependence of inactivation, the same voltage dependence of activation, and the same macroscopic conductance as the delayed rectifier K+ current in these neurons. We conclude that Icat is a Na+ current that passes through a delayed rectifier K+ channel when intracellular K+ is reduced to below 30 mM. At intracellular K+ concentrations between 1 and 30 mM, PK/PNa remained constant while the conductance at -50 mV varied from 80 to 0% of maximum. These data suggest that the high selectivity of these channels for K+ over Na+ is due to the inability of Na+ to compete with K+ for an intracellular binding site, rather than a barrier that excludes Na+ from entry into the channel or a barrier such as a selectivity filter that prevents Na+ ions from passing through the channel.     

Fusicoccin- and IAA-induced elongation growth share the same pattern of K+ dependence

The dependence of growth induced by the fungal toxin fusicoccin (FC) on the K+ content of the incubation medium was investigated in abraded maize coleoptiles. If the divalent ion Ca2+ was included in the bathing medium, no FC-induced growth occurred in the absence of K+, whereas a strong response was detected in presence of K+. The optimal K+ concentration was in the range of 1-10 mM. With the exception of Rb+, none of the other alkali ions (Na+, Li+, Cs+) could replace for K+ in sustaining FC-induced growth. The potassium channel blocker tetraethylammonium (TEA) reversibly inhibited FC-induced growth. As shown earlier for auxin-induced growth, no strict potassium dependence of FC-triggered elongation was observed in Ca2+ -free media. However, TEA abolished this apparently K+ independent FC-induced growth. It is concluded that FC-induced growth, like auxin-induced growth, requires K+ uptake through K+ channels.     

Modulation of the activity of Ca(2+)-activated K+ channels by internal Mg2+ in cultured kidney cells vero.

The inside-out mode of the patch-clamp method was used to study the effects of internal Mg2+ on single large-conductance (193+/-7 pS) Ca(2+)-activated K+ channels in cultured kidney cells. In the absence of Ca2+, Mg2+ (1 to 10 mM) did not activate the channels but modified the activating effect of Ca2+ ions: it decreased the Hill coefficient (n), reduced the apparent dissociation constant (K0.5), and modified the channel open and closed times. K0.5 was found to be a voltage-dependent parameter. In the absence of Mg2+, it averaged 600 microM at -20 mV and 27 microM at +30 mV (22 degrees C, pH 6.8). Mg2+ at saturating concentrations (5 to 10 mM) decreased K0.5 to 50 microM at -20 mV and to 15 microM at +30 mV. Irrespective of the membrane potential, K0.5 tended to its limit value of about 12.6 microM. Thus, the effects of membrane depolarization and Mg2+ exhibited a non-additive, competitive relationship. Mg2+ perturbed the exponential shape of the voltage dependences of K0.5. The Hill coefficient characterizing the interaction of Ca2+ ions with the channels was found to be voltage-dependent. In the absence of Mg2+, it increased rather sharply from approx. 2 to 3.5 when the membrane potential was raised from -10 to 0 mV. Mg2+ increased n in a dose-dependent manner; however, about a twofold increase of n occurred within a narrow concentration range (2 to 3 mM). The action of Mg2+ on n was, apparently, voltage-independent, and the effects of Mg2+ and voltage on n were seemingly additive.     

Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength

Charybdotoxin (CTX), a small, basic protein from scorpion venom, strongly inhibits the conduction of K ions through high-conductance, Ca2+-activated K+ channels. The interaction of CTX with Ca2+-activated K+ channels from rat skeletal muscle plasma membranes was studied by inserting single channels into uncharged planar phospholipid bilayers. CTX blocks K+ conduction by binding to the external side of the channel, with an apparent dissociation constant of approximately 10 nM at physiological ionic strength. The dwell-time distributions of both blocked and unblocked states are single-exponential. The toxin association rate varies linearly with the CTX concentration, and the dissociation rate is independent of it. CTX is competent to block both open and closed channels; the association rate is sevenfold faster for the open channel, while the dissociation rate is the same for both channel conformations. Membrane depolarization enhances the CTX dissociation rate e-fold/28 mV; if the channel's open probability is maintained constant as voltage varies, then the toxin association rate is voltage independent. Increasing the external solution ionic strength from 20 to 300 mM (with K+, Na+, or arginine+) reduces the association rate by two orders of magnitude, with little effect on the dissociation rate. We conclude that CTX binding to the Ca2+-activated K+ channel is a bimolecular process, and that the CTX interaction senses both voltage and the channel's conformational state. We further propose that a region of fixed negative charge exists near the channel's CTX-binding site.     

Ion transport in the gramicidin channel: molecular dynamics study of single and double occupancy.

The structural and thermodynamic factors responsible for the singly and doubly occupied saturation states of the gramicidin channel are investigated with molecular dynamics simulations and free energy perturbation methods. The relative free energy of binding of all of the five common cations Li+, Na+, K+, Rb+, and Cs+ is calculated in the singly and doubly occupied channel and in bulk water. The atomic system, which includes the gramicidin channel, a model membrane made of neutral Lennard-Jones particles and 190 explicit water molecules to form the bulk region, is similar to the one used in previous work to calculate the free energy profile of a Na+ ion along the axis of the channel. In all of the calculations, the ions are positioned in the main binding sites located near the entrances of the channel. The calculations reveal that the doubly occupied state is relatively more favorable for the larger ions. Thermodynamic decomposition is used to show that the origin of the trend observed in the calculations is due to the loss of favorable interactions between the ion and the single file water molecules inside the channel. Small ions are better solvated by the internal water molecules in the singly occupied state than in the doubly occupied state; bigger ions are solvated almost as well in both occupation states. Water-channel interactions play a role in the channel response. The observed trends are related to general thermodynamical properties of electrolyte solutions.     

Conduction properties of the cloned Shaker K+ channel.

The conduction properties of the cloned Shaker K+ channel were studied using electrophysiological techniques. Single channel conductance increases in a sublinear manner with symmetric increases in K+ activity, reaching saturation by 0.6 M K+. The Shaker K+ channel is highly selective among monovalent cations; under bi-ionic conditions, its selectivity sequence is K+ > Rb+ > NH+4 > Cs+ > Na+, whereas, by relative conductance in symmetric solutions, it is K+ > NH+4 > Rb+ > Cs+. In Cs+ solutions, single channel currents were too small to be measured directly, so nonstationary fluctuation analysis was used to determine the unitary Cs+ conductance. The single channel conductance displays an anomalous molefraction effect in symmetric mixtures of K+ and NH+4, suggesting that the conducting pore is occupied by multiple ions simultaneously.     

A cation channel for K+ and Ca2+ from Tetrahymena cilia in planar lipid bilayers

The single-channel properties for monovalent and divalent cations of a voltage-independent cation channel from Tetrahymena cilia were studied in planar lipid bilayers. The single-channel conductance reached a maximum value as the K+ concentration was increased in symmetrical solutions of K+. The concentration dependence of the conductance was approximated to a simple saturation curve (a single-ion channel model) with an apparent Michaelis constant of 16.3 mM and a maximum conductance of 354 pS. Divalent cations (Ca2+, Ba2+, Sr2+, and Mg2+) also permeated this channel. The sequence of permeability determined by zero current potentials at high ionic concentrations was Ba2+ greater than or equal to K+ greater than or equal to Sr2+ greater than Mg2+ greater than Ca2+. Single-channel conductances for Ca2+ were nearly constant (13.9 pS-20.5 pS) in the concentrations between 0.5 mM and 50 mM Ca-gluconate. In the experiments with mixed solutions of K+ and Ca2+, a maximum conductance of Ca2+ (gamma Camax) and an apparent Michaelis constant of Ca2+ (K Cam) were obtained by assuming a simple competitive relation between the cations. Gamma Camax and K Cam were 14.0 pS and 0.160 mM, respectively. Single-channel conductances in mixed solutions were well-fitted to this competitive model supporting that this cation channel behaves as a single-ion channel. This channel had relatively high-affinity Ca2+-binding sites.