Ion Selectivity of the KcsA Channel: A Perspective from Multi-Ion Free Energy Landscapes

Potassium (K⁺) channels are specialized membrane proteins that are able to facilitate and regulate the conduction of K⁺ through cell membranes. Comprising five specific cation binding sites (S₀-S₄) formed by the backbone carbonyl groups of conserved residues common to all K⁺ channels, the narrow selectivity filter allows fast conduction of K⁺ while being highly selective for K⁺ over Na⁺. To extend our knowledge of the microscopic mechanism underlying selectivity in K⁺ channels, we characterize the free energy landscapes governing the entry and translocation of a Na⁺ or a K⁺ from the extracellular side into the selectivity filter of KcsA. The entry process of an extracellular ion is examined in the presence of two additional K⁺ in the pore, and the three-ion potential of mean force is computed using extensive all-atom umbrella sampling molecular dynamics simulations. A comparison of the potentials of mean force yields a number of important results. First, the free energy minima corresponding to configurations with extracellular K⁺ or Na⁺ in binding site S₀ or S₁ are similar in depth, suggesting that the thermodynamic selectivity governed by the free energy minima for those two binding sites is insignificant. Second, the free energy barriers between stable multi-ion configurations are generally higher for Na⁺ than for K⁺, implying that the kinetics of ion conduction is slower when a Na⁺ enters the pore. Third, the region corresponding to binding site S₂ near the center of the narrow pore emerges as the most selective for K⁺ over Na⁺. In particular, while there is a stable minimum for K⁺ in site S₂, Na⁺ faces a steep free energy increase with no local free energy well in this region. Lastly, analysis shows that selectivity is not correlated with the overall coordination number of the ion entering the pore, but is predominantly affected by changes in the type of coordinating ligands (carbonyls versus water molecules). These results further highlight the importance of the central region near binding site S₂ in the selectivity filter of K⁺ channels.     

An Energy-Barrier Model for the Permeation of Monovalent and Divalent Cations Through the Maxi Cation Channel in the Plasma Membrane of Rye Roots

The depolarization-activated, high-conductance ``maxi' cation channel in the plasma membrane of rye (Secale cereale L.) roots is permeable to a wide variety of monovalent and divalent cations. The permeation of K⁺, Na⁺, Ca²⁺ and Ba²⁺ through the pore could be simulated using a model composed of three energy barriers and two ion binding sites (a 3B2S model), which assumed single-file permeation and the possibility of double cation occupancy. The model had an asymmetrical free energy profile. Differences in permeation between cations were attributed primarily to differences in their free energy profiles in the regions of the pore adjacent to the extracellular solution. In particular, the height of the central free energy peak differed between cations, and cations differed in their affinities for ion binding sites. Significant ion repulsion occurred within the pore, and the mouths of the pore had considerable surface charge. The model adequately described the diverse current vs. voltage (I/V) relationships obtained over a wide variety of experimental conditions. It described the phenomena of non-Michaelian unitary conductance vs. activity relationships for K⁺, Na⁺ and Ca²⁺, differences in selectivity sequences obtained from measurements of conductance and permeability ratios, changes in relative cation permeabilities with solution composition, and the complex effects of Ba²⁺ and Ca²⁺ on K⁺ currents through the channel. The model enabled the prediction of unitary currents and ion fluxes through the maxi cation channel under physiological conditions. It could be used, in combination with data on the kinetics of the channel, as input to electrocoupling models allowing the relationships between membrane voltage, Ca²⁺ influx and Ca²⁺ signaling to be studied theoretically. Received: 29 April 1998/Revised: 20 November 1998     

Temperature-activity relationships of cation activation and ouabain inhibition of (Na+ + K+)-ATPase.

The nonlinear temperature-activity relationship of membrane preparations of (Na⁺ + K⁺)-ATPase gives rise to discontinuities in Arrhenius plots of this enzyme. The different apparent energies of activation of (Na⁺ + K⁺) — ATPase which are observed above and below the critical temperature of the system have been considered to result from different conformational forms of the enzyme protein. Because both activation of (Na⁺ + K⁺)-ATPase by cations, and its specific inhibition by cardiac glycosides may be influenced by the conformational form of the enzyme protein, we have reexamined the effect of temperature upon the activation energy of the system under the different experimental conditions of cation activation and ouabain inhibition.Our results indicate that the activation of (Na⁺ + K⁺)-ATPase by cations, is less influenced by change in temperature than is inhibition of the enzyme by ouabain. In addition, mild lipolysis by phospholipase-A had a marked effect upon the ouabain-dependent response of the enzyme to temperature, but not upon the cation-dependent response. The effect of phospholipase-A can be overcome by reincubation of the treated preparation with phosphatidyl serine.We conclude that the ouabain-dependent temperature effects of (Na⁺ + K⁺)-ATPase are more dependent upon the integrity and nature of the membrane lipids than are the cation-dependent responses. It is possible that phosphatidyl serine plays a unique role in this regard.     

Quantum mechanics study on the selectivity of alkali metal cations by a novel fluorescent chemosensor

The selectivity of alkali metal cations (Na⁺ and K⁺) by a novel fluorescent chemosensor was investigated by quantum mechanics calculations. The binding energy calculations of the cationic complexes indicate that both of the aza-18-crown-6 rings bind Na⁺ more strongly than K⁺ and ring A favors both of Na⁺ and K⁺ comparing to ring B. The order of the stability implied by Gibbs free energy study agrees with that by binding energy calculation. Solvent effect was further investigated. Due to the large difference of solvation energies, the order of the stability of complexes changes in water.     

Cholesterol affects physical properties and (Na+,K+)-ATPase in basolateral membranes of renal and intestinal epithelia from thermally acclimated rainbow trout

Previous work has shown that cholesterol levels are modulated in plasma membranes from some but not all tissues of poikilotherms over the course of temperature change. To gain a better understanding of tissue and membrane domain-specific cholesterol function during thermal adaptation we examined effects of cholesterol on membrane physical properties and (Na⁺,K⁺)-ATPase in native and cholesterol-enriched basolateral membranes from kidney and intestine of thermally acclimated trout (Oncorhynchus mykiss). Membrane order (as indicated by fluorescence depolarization studies) is increased, whereas its thermal sensitivity is decreased by elevated cholesterol levels in mem branes with relatively low endogenous amounts of cholesterol (intestinal membranes and renal membranes from cold-acclimated fish). Thermal sensitivities of membrane order in kidney are 1.5-fold higher in native compared with cholesterol-enriched basolateral membranes. For renal plasma membranes, (Na⁺,K⁺)- ATPase activity is lowest near the transition between native and surpraphysiological cholesterol levels. Endogenous cholesterol levels (relative to phospholipid contents) in intestinal basolateral membranes from cold-acclimated fish vary more than 1.5-fold; membranes with cholesterol/phospholipid molar ratios of 0.3 have activities of (Na⁺,K⁺)-ATPase that are twofold lower than native membranes having a ratio of 0.2. These results suggests that maintenance of cholesterol levels in intestinal basolateral membranes during thermal acclimation may ensure sufficient activity of (Na⁺,K⁺)-ATPase. Membrane function in kidney, with its high native cholesterol content, is less likely to be affected by temperature change. Accepted: 21 January 1997     

A mutant of Paramecium with increased relative resting potassium permeability

Fast-2, a membrane mutant of Paramecium aurelia, is due to a single-gene mutation and has behavioral abnormalities. Intracellular recordings through changes of external solutions were made. The mutant membrane hyperpolarized when it encountered solutions with low K⁺ concentration. This hyperpolarization and other associated activities were best observed in Ca- or Na-solutions devoid of K⁺. Membrane potential was plotted against the concentration of K⁺ (0.5 to 16 mM) in solutions of fixed Na⁺ or Ca⁺⁺ concentration. The slopes of the curves for the mutant membrane were steeper than those for the wild type at the lower concentrations of K⁺. Inclusion of 2 mM tetraethylammonium chloride (TEA-Cl) counteracted the mutational effects. Spontaneous action potentials in Ba-solution and the electrically evoked action potentials in various solutions are normal in this mutant. We conclude that the resting permeability to K⁺ relative to the permeabilities to Na⁺ and Ca⁺⁺ has been increased by the mutation.     

Ouabain-induced Internalization and Lysosomal Degradation of the Na+/K+-ATPase

Internalization of the Na⁺/K⁺-ATPase (the Na⁺ pump) has been studied in the human lung carcinoma cell line H1299 that expresses YFP-tagged α1 from its normal genomic localization. Both real-time imaging and surface biotinylation have demonstrated internalization of α1 induced by ≥100 nm ouabain which occurs in a time scale of hours. Unlike previous studies in other systems, the ouabain-induced internalization was insensitive to Src or PI3K inhibitors. Accumulation of α1 in the cells could be augmented by inhibition of lysosomal degradation but not by proteosomal inhibitors. In agreement, the internalized α1 could be colocalized with the lysosomal marker LAMP1 but not with Golgi or nuclear markers. In principle, internalization could be triggered by a conformational change of the ouabain-bound Na⁺/K⁺-ATPase molecule or more generally by the disruption of cation homeostasis (Na⁺, K⁺, Ca²⁺) due to the partial inhibition of active Na⁺ and K⁺ transport. Overexpression of ouabain-insensitive rat α1 failed to inhibit internalization of human α1 expressed in the same cells. In addition, incubating cells in a K⁺-free medium did not induce internalization of the pump or affect the response to ouabain. Thus, internalization is not the result of changes in the cellular cation balance but is likely to be triggered by a conformational change of the protein itself. In physiological conditions, internalization may serve to eliminate pumps that have been blocked by endogenous ouabain or other cardiac glycosides. This mechanism may be required due to the very slow dissociation of the ouabain·Na⁺/K⁺-ATPase complex.     

Charge translocation by the Na,K-pump: II. Ion binding and release at the extracellular face

Summary In the first part of the paper, evidence has been presented that electrochromic styryl dyes, such as RH 421, incorporate into Na, K-ATPase membranes isolated from mammalian kidney and respond to changes of local electric field strength. In this second part of the paper, fluorescence studies with RH-421-labeled membranes are described, which were carried out to obtain information on the nature of charge-translocating reaction steps in the pumping cycle. Experiments with normal and chymotrypsin-modified membranes show that phosphorylation by ATP and occlusion of Na⁺ are electroneutral steps, and that release of Na⁺ from the occluded state to the extracellular side is associated with translocation of charge. Fluorescence signals observed in the presence of K⁺ indicate that binding and occlusion of K⁺ at the extracellular face of the pump is another major electrogenic reaction step. The finding that the fluorescence signals are insensitive to changes of ionic strength leads to the conclusion that the binding pocket accommodating Na⁺ or K⁺ is buried in the membrane dielectric. This corresponds to the notion that the binding sites are connected with the extracellular medium by a narrow access channel (ion well). This notion is further supported by experiments with lipophilic ions, such as tetraphenylphosphonium (TPP⁺) or tetraphenylborate (TPB^–), which are known to bind to lipid bilayers and to change the electrostatic potential inside the membrane. Addition of TPP⁺ leads to a decrease of binding affinity for Na⁺ and K⁺, which is thought to result from the TPP^–-induced change of electric field strength in the access channel.Deceased (September 13, 1990).     

Frog striated muscle is permeable to hydroxide and buffer anions

Hydroxide, bicarbonate and buffer anion permeabilities in semitendinosus muscle fibers of Rana pipiens were measured. In all experiments, the fibers were initially equilibrated in isotonic, high K₂SO₄ solutions at pH _o =7.2 buffered with phosphate. Two different methods were used to estimate permeabilities: (i) membrane potential changes were recorded in response to changes in external ion concentrations, and (ii) intracellular pH changes were recorded in response to changes in external concentrations of ions that alter intracellular pH. Constant field equations were used to calculate relative or absolute permeabilities.In the first method, to increase the size of the membrane potential change produced by a sudden change in anion entry, external K⁺ was replaced by Cs⁺ prior to changes of the anion under study. At constant external Cs⁺ activity, a hyperpolarization results from increasing external pH from 7.2 to 10.0 or higher, using either CAPS (3-[cyclohexylamino]-1-propanesulfonic acid) or CHES (2-[N-cyclohexylamino]-ethanesulfonic acid) as buffer. For each buffer, the protonated form is a zwitterion of zero net charge and the nonprotonated form is an anion. Using reported values of H⁺ permeability, calculations show that the reduction in [H⁺] _o cannot account for the hyperpolarizations produced by alkaline solutions. Membrane hyperpolarization increases with increasing total external buffer concentration at constant external pH, and with increasing external pH at constant external buffer anion concentration. Taken together, these observations indicate that both OH^– and buffer anions permeate the surface membrane. The following relative permeabilities were obtained at pH_o, 10.0± 0.3: (P_OH/P_K) = 890 ± 150, (P_CAPS/P_K) = 12 ± 2 (P_CHIES/P_K) = 5.3 ± 0.9, and (P_NO3/P_K) = 4.7 ± 0.5 P_NO/P_K was independent of pH _o up to 10.75. At pH_o = 9.6, (P_HCO3/P_K) = 0.49 ± 0.03; at pH _o = 8.9, (P_Cl/P_K) = 18± 2 and at pH _o = 7.1, (P_HEPES/P_K) = 20 ± 2.In the second method, on increasing external pH from 7.2 to 10.0, using 2.5 mm CAPS (total buffer concentration), the internal pH increases linearly with time over the next 10 min. This alkalinization is due to the entry of OH^– and the absorption of internal H⁺ by entering CAPS^– anion. The rate of CAPS^– entry was determined in experiments in which the external CAPS concentration was increased at constant external pH. Such increases invariably produced an increase in the rate of internal alkalinization, which was reversed when the CAPS concentration was reduced to its initial value. From the internal buffer power, the diameter of the fiber under study and the rates of change of internal pH, the absolute permeability for both OH^– and CAPS^– were calculated. At external pH = 10.0, the average (±sem) permeabilities were: P_OH=1.68±0.19×10^–4 cm/sec and P_CAPS=2.10±0.74×10^–6cm/sec.We conclude that OH^– is about 50 times more permeable than Cl^– at alkaline pH and that the anionic forms of commonly used buffers have significant permeabilities.This research was supported by a grant from the National Institutes of Health (AR 31814). The authors wish to thank Dr. Peter G. Shrager and Dr. Bruce C. Spalding for reading an early draft of this report and for providing helpful suggestions.     

Identification of Electric-Field-Dependent Steps in the Na,K-Pump Cycle

The charge-transporting activity of the Na⁺,K⁺-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme’s reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na⁺,K⁺-ATPase’s transport sites in competition with Na⁺ and K⁺, but is not occluded within the protein. We find that only the occludable ions Na⁺, K⁺, Rb⁺, and Cs⁺ cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na⁺ or K⁺ binding in a high field access channel is a major electrogenic reaction of the Na⁺,K⁺-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.     

Identification of Electric-Field-Dependent Steps in the Na+,K+-Pump Cycle

The charge-transporting activity of the Na⁺,K⁺-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme’s reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na⁺,K⁺-ATPase’s transport sites in competition with Na⁺ and K⁺, but is not occluded within the protein. We find that only the occludable ions Na⁺, K⁺, Rb⁺, and Cs⁺ cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na⁺ or K⁺ binding in a high field access channel is a major electrogenic reaction of the Na⁺,K⁺-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.     

A study of the K+-site mutant of ascorbate peroxidase: mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side

A series of ferric and ferrous derivatives of wild-type ascorbate peroxidase (APX) and of an engineered K⁺-site mutant of APX that has had its potassium cation binding site removed have been examined by electronic absorption and magnetic circular dichroism (MCD) spectroscopy at 4??°C. Wild-type ferric APX has spectroscopic properties that are very similar to those of ferric cytochrome c peroxidase (CCP) and likely exists primarily as a five-coordinate high-spin heme ligated on the proximal side by a histidine at pH 7. There is also evidence for minority contributions from six-coordinate high- and low-spin species (histidine-water, histidine-hydroxide, and bis-histidine). The K⁺-site mutant of APX varies considerably in the electronic absorption and MCD spectra in both the ferric and ferrous states when compared with spectra of the wild-type APX. The electronic absorption and MCD spectra of the engineered K⁺-site APX mutant are essentially identical to those of cytochrome b ₅, a known bis-imidazole (histidine) ligated heme system. It therefore appears that the K⁺-site mutant of APX has undergone a conformational change to yield a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH. This conformational change is the result of mutagenesis of the protein to remove the K⁺-binding site which is located ～8?Å from the peroxide binding pocket. Thus, mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side.     

Loss of membrane cholesterol influences lysosomal permeability to potassium ions and protons

Cholesterol is an essential component of lysosomal membranes. In this study, we investigated the effects of membrane cholesterol on the permeability of rat liver lysosomes to K⁺ and H⁺, and the organelle stability. Through the measurements of lysosomal β-hexosaminidase free activity, membrane potential, membrane fluidity, intra-lysosomal pH, and lysosomal proton leakage, we established that methyl-β-cyclodextrin (MβCD)-produced loss of membrane cholesterol could increase the lysosomal permeability to both potassium ions and protons, and fluidize the lysosomal membranes. As a result, potassium ions entered the lysosomes through K⁺/H⁺ exchange, which produced osmotic imbalance across the membranes and osmotically destabilized the lysosomes. In addition, treatment of the lysosomes with MβCD caused leakage of the lysosomal protons and raised the intra-lysosomal pH. The results indicate that membrane cholesterol plays important roles in the maintenance of the lysosomal limited permeability to K⁺ and H⁺. Loss of this membrane sterol is critical for the organelle acidification and stability.     

The Permeability of the Sodium Channel to Metal Cations in Myelinated Nerve

The relative permeability of sodium channels to eight metal cations is studied in myelinated nerve fibers. Ionic currents under voltage-clamp conditions are measured in Na-free solutions containing the test ion. Measured reversal potentials and the Goldman equation are used to calculate the permeability sequence: Na⁺ ≈ Li⁺ > Tl⁺ > K⁺. The ratio P_K/P_Na is 1/12. The permeabilities to Rb⁺, Cs⁺, Ca⁺⁺, and Mg⁺⁺ are too small to measure. The permeability ratios agree with observations on the squid giant axon and show that the reversal potential E_Na differs significantly from the Nernst potential for Na⁺ in normal axons. Opening and closing rates for sodium channels are relatively insensitive to the ionic composition of the bathing medium, implying that gating is a structural property of the channel rather than a result of the movement or accumulation of particular ions around the channel. A previously proposed pore model of the channel accommodates the permeant metal cations in a partly hydrated form. The observed sequence of permeabilities follows the order expected for binding to a high field strength anion in Eisenman's theory of ion exchange equilibria.     

Ouabain-insensitive Na+-stimulated ATPase activity of basolateral plasma membranes from guinea-pig kidney cortex cells: II. Effect of Ca2+

The ouabain-insensitive, Mg²⁺-dependent, Na⁺-stimulated ATPase activity present in fresh basolateral plasma membranes from guinea-pig kidney cortex cells (prepared at pH 7.2) can be increased by the addition of micromolar concentrations of Ca²⁺ to the assay medium. The Ca²⁺ involved in this effect seems to be associated with the membranes in two different ways: as a labile component, which can be quickly and easily ‘deactivated’ by reducing the free Ca²⁺ concentration of the assay medium to values lower than 1 μM; and as a stable component, which can be ‘deactivated’ by preincubating the membranes for periods of 3–4 h with 2 mM EDTA or EGTA. Both components are easily activated by micromolar concentrations of Ca²⁺. The $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of the system for Na⁺ is the same, 8 mM, whether only the stable component or both components, stable and labile, are working. In other words, the activating effect of Ca²⁺ on the Na⁺-stimulated ATPase is on the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$, and not on the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of the system for Na⁺. The activating effect of Ca²⁺ may be related to some conformational change produced by the interaction of this ion with the membranes, since it can also be obtained by resuspending the membranes at pH 7.8 or by ageing the preparations. Changes in the Ca²⁺ concentration may modulate the ouabain-insensitive, Na⁺-stimulated ATPase activity. This modulation could regulate the magnitude of the extrusion of Na⁺ accompanied by Cl^? and water that these cells show, and to which the Na⁺-ATPase has been associated as being responsible for the energy supply of this mode of Na⁺ extrusion.     

Mycobacterium tuberculosis ESAT-6 Exhibits a Unique Membrane-interacting Activity That Is Not Found in Its Ortholog from Non-pathogenic Mycobacterium smegmatis

Mycobacterium tuberculosis ESAT-6 (MtbESAT-6) reportedly shows membrane/cell-lysis activity, and recently its biological roles in pathogenesis have been implicated in rupture of the phagosomes for bacterial cytosolic translocation. However, molecular mechanism of MtbESAT-6-mediated membrane interaction, particularly in relation with its biological functions in pathogenesis, is poorly understood. In this study, we investigated the pH-dependent membrane interaction of MtbESAT-6, MtbCFP-10, and the MtbESAT-6/CFP-10 heterodimer, by using liposomal model membranes that mimic phagosomal compartments. MtbESAT-6, but neither MtbCFP-10 nor the heterodimer, interacted with the liposomal membranes at acidic conditions, which was evidenced by release of K⁺ ions from the liposomes. Most importantly, the orthologous ESAT-6 from non-pathogenic Mycobacterium smegmatis (MsESAT-6) was essentially inactive in release of K⁺. The differential membrane interactions between MtbESAT-6 and MsESAT-6 were further confirmed in an independent membrane leakage assay using the dye/quencher pair, 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX). Finally, using intrinsic and extrinsic fluorescence approaches, we probed the pH-dependent conformational changes of MtbESAT-6 and MsESAT-6. At acidic pH conditions, MtbESAT-6 underwent a significant conformational change, which was featured by an increased solvent-exposed hydrophobicity, while MsESAT-6 showed little conformational change in response to acidification. In conclusion, we have demonstrated that MtbESAT-6 possesses a unique membrane-interacting activity that is not found in MsESAT-6 and established the utility of rigorous biochemical approaches in dissecting the virulence of M. tuberculosis.     

Ligand binding to a high‐energy partially unfolded protein

The conformational energy landscape of a protein determines populations of all possible conformations of the protein and also determines the kinetics of the conversion between the conformations. Interaction with ligands influences the conformational energy landscapes of proteins and shifts populations of proteins in different conformational states. To investigate the effect of ligand binding on partial unfolding of a protein, we use Escherichia coli dihydrofolate reductase (DHFR) and its functional ligand NADP⁺ as a model system. We previously identified a partially unfolded form of DHFR that is populated under native conditions. In this report, we determined the free energy for partial unfolding of DHFR at varying concentrations of NADP⁺ and found that NADP⁺ binds to the partially unfolded form as well as the native form. DHFR unfolds partially without releasing the ligand, though the binding affinity for NADP⁺ is diminished upon partial unfolding. Based on known crystallographic structures of NADP⁺‐bound DHFR and the model of the partially unfolded protein we previously determined, we propose that the adenosine‐binding domain of DHFR remains folded in the partially unfolded form and interacts with the adenosine moiety of NADP⁺. Our result demonstrates that ligand binding may affect the conformational free energy of not only native forms but also high‐energy non‐native forms.     

The action of androgens on Na+,K+-ATPase activity of erythrocyte membranes

The effect of androgens (testosterone, androsterone, dehydroepiandrosterone and dehydroepiandrosterone sulfate) on erythrocyte membrane during their nonspecific binding was investigated. The change in erythrocyte membrane Na⁺,K⁺-ATPase activity was measured at different hormone concentration in a suspension. It is shown that the dependence has dome-shaped character: at the elevated hormone concentration Na⁺,K⁺-ATPase activity starts to increase, reaches its maximum, and then decreases. The hypothesis is put forward that an increase in microscopists of erythrocyte membrane first intensifies Na⁺,K⁺-ATPase activity due to the growth of the maximum energy of membrane phonons, and then decreases it due to hindering conformational transitions in the enzyme molecule.     

Structural Changes in the Catalytic Cycle of the Na,K-ATPase Studied by Infrared Spectroscopy

Pig kidney Na⁺,K⁺-ATPase was studied by means of reaction-induced infrared difference spectroscopy. The reaction from E1Na₃⁺ to an E2P state was initiated by photolysis of P³-1-(2-nitrophenyl)ethyl ATP (NPE caged ATP) in samples that contained 3 mM free Mg²⁺ and 130 mM NaCl at pH 7.5. Release of ATP from caged ATP produced highly detailed infrared difference spectra indicating structural changes of the Na⁺,K⁺-ATPase. The observed transient state of the enzyme accumulated within seconds after ATP release and decayed on a timescale of minutes at 15°C. Several controls ensured that the observed difference signals were due to structural changes of the Na⁺,K⁺-ATPase. Samples that additionally contained 20 mM KCl showed similar spectra but less intense difference bands. The absorbance changes observed in the amide I region, reflecting conformational changes of the protein backbone, corresponded to only 0.3% of the maximum absorbance. Thus the net change of secondary structure was concluded to be very small, which is in line with movement of rigid protein segments during the catalytic cycle. Despite their small amplitude, the amide I signals unambiguously reveal the involvement of several secondary structure elements in the conformational change. Similarities and dissimilarities to corresponding spectra of the Ca²⁺-ATPase and H⁺,K⁺-ATPase are discussed, and suggest characteristic bands for the E1 and E2 conformations at 1641 and 1661 cm⁻¹, respectively, for αβ heterodimeric ATPases. The spectra further indicate the participation of protonated carboxyl groups or lipid carbonyl groups in the reaction from E1Na₃⁺ to an E2P state. A negative band at 1730 cm⁻¹ is in line with the presence of a protonated Asp or Glu residue that coordinates Na⁺ in E1Na₃⁺. Infrared signals were also detected in the absorption regions of ionized carboxyl groups.     

Cation binding to brain plasma membranes: An evaluation of the use of anionic fluorescent probes

Cation binding to brain plasma membranes has been studied using anionic sulfonate fluorescent probes. Ion affinity sequences follow the order Mg²⁺ > Ca²⁺ ? K⁺ > Cs⁺ > Na⁺ > Li⁺. The order of effectiveness, in increasing probe fluorescence, is the reverse of the affinity sequence for ions of the same charge. The affinity orders for erythrocyte membranes and dipalmitoyl lecithin are Mg²⁺ > Ca²⁺ ? Cs⁺ > K⁺ > Na⁺ > Li⁺ and Mg²⁺ > Ca²⁺ ? Li⁺ > Na⁺ > K⁺ > Cs⁺. These sequence variations are related to the differences in the nature of the ion binding sites. Heterogeneity in ion binding sites is demonstrated. Evidence is presented for the role of proteins in binding hydrophobic probes. The problem of separating specific conformational effects on ion binding from nonspecific charge neutralization effects is discussed. Pyrene excimer fluoresence rules out the possibility of extensive changes in mobility in the lipid phase on cation binding. Tetrodotoxin has been shown to inhibit Li⁺-, Na⁺-, and K⁺-induced fluorescence enancements of 1-anilino-8-naphthalene sulfonate bound to brain membranes.