Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase α subunit

The Na⁺/K⁺-ATPase mediates electrogenic transport by exporting three Na⁺ ions in exchange for two K⁺ ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb⁺ ions in the crystal structures of the Na⁺/K⁺-ATPase has defined two “common” cation binding sites, I and II, which accommodate Na⁺ or K⁺ ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this “unique” Na⁺ binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na⁺/K⁺-ATPase α₂ subunit decreases the affinity for extracellular and intracellular Na⁺, in agreement with previous biochemical studies. Apparently, the ΔYY deletion changes Na⁺ affinity at site III but leaves the common sites unaffected, whereas the more extensive ΔKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K⁺, the ΔYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na⁺ dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na⁺/Na⁺ exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na⁺ release/binding mechanism. In analogy to the mechanism proposed for the H⁺ leak currents of the wild-type Na⁺/K⁺-ATPase, we suggest that the ΔYY deletion lowers the energy barrier for the intracellular Na⁺ occlusion reaction, thus destabilizing the Na⁺-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na⁺/K⁺-ATPase α subunit represents a structural and functional relay between Na⁺ binding site III and the intracellular cation occlusion gate.     

Airway Surface Liquid Volume Regulation Determines Different Airway Phenotypes in Liddle Compared with βENaC-overexpressing Mice

Studies in cystic fibrosis patients and mice overexpressing the epithelial Na⁺ channel β-subunit (βENaC-Tg) suggest that raised airway Na⁺ transport and airway surface liquid (ASL) depletion are central to the pathogenesis of cystic fibrosis lung disease. However, patients or mice with Liddle gain-of-function βENaC mutations exhibit hypertension but no lung disease. To investigate this apparent paradox, we compared the airway phenotype (nasal versus tracheal) of Liddle with CFTR-null, βENaC-Tg, and double mutant mice. In mouse nasal epithelium, the region that functionally mimics human airways, high levels of CFTR expression inhibited Liddle epithelial Nat channel (ENaC) hyperfunction. Conversely, in mouse trachea, low levels of CFTR failed to suppress Liddle ENaC hyperfunction. Indeed, Na⁺ transport measured in Ussing chambers (“flooded” conditions) was raised in both Liddle and βENaC-Tg mice. Because enhanced Na⁺ transport did not correlate with lung disease in these mutant mice, measurements in tracheal cultures under physiologic “thin film” conditions and in vivo were performed. Regulation of ASL volume and ENaC-mediated Na⁺ absorption were intact in Liddle but defective in βENaC-Tg mice. We conclude that the capacity to regulate Na⁺ transport and ASL volume, not absolute Na⁺ transport rates in Ussing chambers, is the key physiologic function protecting airways from dehydration-induced lung disease.     

Dietary cardenolides enhance growth and change the direction of the fecundity‐longevity trade‐off in milkweed bugs (Heteroptera: Lygaeinae)

Sequestration, that is, the accumulation of plant toxins into body tissues for defense, was predicted to incur physiological costs and may require resistance traits different from those of non‐sequestering insects. Alternatively, sequestering species could experience a cost in the absence of toxins due to selection on physiological homeostasis under permanent exposure of sequestered toxins in body tissues. Milkweed bugs (Heteroptera: Lygaeinae) sequester high amounts of plant‐derived cardenolides. Although being potent inhibitors of the ubiquitous animal enzyme Na⁺/K⁺‐ATPase, milkweed bugs can tolerate cardenolides by means of resistant Na⁺/K⁺‐ATPases. Both adaptations, resistance and sequestration, are ancestral traits of the Lygaeinae. Using four milkweed bug species (Heteroptera: Lygaeidae: Lygaeinae) and the related European firebug (Heteroptera: Pyrrhocoridae: Pyrrhocoris apterus) showing different combinations of the traits “cardenolide resistance” and “cardenolide sequestration,” we tested how the two traits affect larval growth upon exposure to dietary cardenolides in an artificial diet system. While cardenolides impaired the growth of P. apterus nymphs neither possessing a resistant Na⁺/K⁺‐ATPase nor sequestering cardenolides, growth was not affected in the non‐sequestering milkweed bug Arocatus longiceps, which possesses a resistant Na⁺/K⁺‐ATPase. Remarkably, cardenolides increased growth in the sequestering dietary specialists Caenocoris nerii and Oncopeltus fasciatus but not in the sequestering dietary generalist Spilostethus pandurus, which all possess a resistant Na⁺/K⁺‐ATPase. We furthermore assessed the effect of dietary cardenolides on additional life history parameters, including developmental speed, longevity of adults, and reproductive success in O. fasciatus. Unexpectedly, nymphs under cardenolide exposure developed substantially faster and lived longer as adults. However, fecundity of adults was reduced when maintained on cardenolide‐containing diet for their entire lifetime but not when adults were transferred to non‐toxic sunflower seeds. We speculate that the resistant Na⁺/K⁺‐ATPase of milkweed bugs is selected for working optimally in a “toxic environment,” that is, when sequestered cardenolides are stored in the body.     

Relationship between Intracellular Na+ Concentration and Reduced Na+ Affinity in Na+,K+-ATPase Mutants Causing Neurological Disease

The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are caused by mutations of Na⁺,K⁺-ATPase α₂ and α₃ isoforms, expressed in glial and neuronal cells, respectively. Although these disorders are distinct, they overlap in phenotypical presentation. Two Na⁺,K⁺-ATPase mutations, extending the C terminus by either 28 residues (“+28” mutation) or an extra tyrosine (“+Y”), are associated with FHM2 and RDP, respectively. We describe here functional consequences of these and other neurological disease mutations as well as an extension of the C terminus only by a single alanine. The dependence of the mutational effects on the specific α isoform in which the mutation is introduced was furthermore studied. At the cellular level we have characterized the C-terminal extension mutants and other mutants, addressing the question to what extent they cause a change of the intracellular Na⁺ and K⁺ concentrations ([Na⁺]_i and [K⁺]_i) in COS cells. C-terminal extension mutants generally showed dramatically reduced Na⁺ affinity without disturbance of K⁺ binding, as did other RDP mutants. No phosphorylation from ATP was observed for the +28 mutation of α₂ despite a high expression level. A significant rise of [Na⁺]_i and reduction of [K⁺]_i was detected in cells expressing mutants with reduced Na⁺ affinity and did not require a concomitant reduction of the maximal catalytic turnover rate or expression level. Moreover, two mutations that increase Na⁺ affinity were found to reduce [Na⁺]_i. It is concluded that the Na⁺ affinity of the Na⁺,K⁺-ATPase is an important determinant of [Na⁺]_i.     

Nuclear Magnetic Resonance of Tissue 23Na: I. 23Na Signal and Na+ Activity in Homogenate

The ability to depress the resonance intensity of ²³Na in rat liver tissue was not found in the supernatant fraction. It was exclusively localized in particulate fractions. The intensity and saturation behavior of the ²³Na signal was examined in suspensions containing various amounts of the particulate fraction of rat liver homogenate. The results strongly suggest that the ²³Na signal of tissue reflects quadrupole interactions and does not result from a slow exchange between the free and bound fractions of Na⁺. The activity coefficient of Na⁺ in rat liver homogenate (no medium was added) was 0.59, about 20% less than that in the isotonic saline. Available evidences and discussion indicate that the bound Na⁺ in the homogenate is much less than the so-called “NMR-invisible” fraction of Na⁺.     

ATP Dependence of Na+/H+ Exchange : Nucleotide Specificity and Assessment of the Role of Phospholipids

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na⁺/H⁺ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pH_i) by microfluorimetry. Na⁺/H⁺ exchange activity was measured as the Na⁺-driven pH_i recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na⁺/H⁺ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-d-glucose and oligomycin. In cells dialyzed in the presence of ATP, no “run-down” was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at ∼5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na⁺/H⁺ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca²⁺ exchanger, this requirement has been attributed to an aminophospholipid translocase, or “flippase.” The involvement of this enzyme in Na⁺/H⁺ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na⁺/H⁺ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATPγS were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTPγS was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg²⁺ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the γ-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.     

Activation of Na+-permeant Cation Channel by Stretch and Cyclic AMP-dependent Phosphorylation in Renal Epithelial A6 Cells

It is currently believed that a nonselective cation (NSC) channel, which responds to arginine vasotocin (an antidiuretic hormone) and stretch, regulates Na⁺ absorption in the distal nephron. However, the mechanisms of regulation of this channel remain incompletely characterized. To study the mechanisms of regulation of this channel, we used renal epithelial cells (A6) cultured on permeable supports. The apical membrane of confluent monolayers of A6 cells expressed a 29-pS channel, which was activated by stretch or by 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase. This channel had an identical selectivity for Na⁺, K⁺, Li⁺, and Cs⁺, but little selectivity for Ca²⁺ (P_Ca/P_Na < 0.005) or Cl⁻ (P_Cl/P_Na < 0.01), identifying it as an NSC channel. Stretch had no additional effects on the open probability (P _o) of the IBMX-activated channel. This channel had one open (“O”) and two closed (short “C _S” and long “C _L”) states under basal, stretch-, or IBMX-stimulated conditions. Both stretch and IBMX increased the P _o of the channel without any detectable changes in the mean open or closed times. These observations led us to the conclusion that a kinetic model “C _L ↔ C _S ↔ O” was the most suitable among three possible linear models. According to this model, IBMX or stretch would decrease the leaving rate of the channel for C _L from C _S, resulting in an increase in P _o. Cytochalasin D pretreatment abolished the response to stretch or IBMX without altering the basal activity. H89 (an inhibitor of cAMP-dependent protein kinase) completely abolished the response to both stretch and IBMX, but, unlike cytochalasin D, also diminished the basal activity. We conclude that: (a) the functional properties of the cAMP-activated NSC channel are similar to those of the stretch-activated one, (b) the actin cytoskeleton plays a crucial role in the activation of the NSC channel induced by stretch and cAMP, and (c) the basal activity of the NSC channel is maintained by PKA-dependent phosphorylation but is not dependent on actin microfilaments.     

Pre‐steady‐state kinetics and solvent isotope effects support the “billiard‐type” transport mechanism in Na +‐translocating pyrophosphatase

Membrane‐bound pyrophosphatase (mPPase) found in microbes and plants is a membrane H⁺ pump that transports the H⁺ ion generated in coupled pyrophosphate hydrolysis out of the cytoplasm. Certain bacterial and archaeal mPPases can in parallel transport Na⁺ via a hypothetical “billiard‐type” mechanism, also involving the hydrolysis‐generated proton. Here, we present the functional evidence supporting this coupling mechanism. Rapid‐quench and pulse‐chase measurements with [³²P]pyrophosphate indicated that the chemical step (pyrophosphate hydrolysis) is rate‐limiting in mPPase catalysis and is preceded by a fast isomerization of the enzyme‐substrate complex. Na⁺, whose binding is a prerequisite for the hydrolysis step, is not required for substrate binding. Replacement of H₂O with D₂O decreased the rates of pyrophosphate hydrolysis by both Na⁺‐ and H⁺‐transporting bacterial mPPases, the effect being more significant than with a non‐transporting soluble pyrophosphatase. We also show that the Na⁺‐pumping mPPase of Thermotoga maritima resembles other dimeric mPPases in demonstrating negative kinetic cooperativity and the requirement for general acid catalysis. The findings point to a crucial role for the hydrolysis‐generated proton both in H⁺‐pumping and Na⁺‐pumping by mPPases.     

Non-Equilibrium Dynamics Contribute to Ion Selectivity in the KcsA Channel

The ability of biological ion channels to conduct selected ions across cell membranes is critical for the survival of both animal and bacterial cells. Numerous investigations of ion selectivity have been conducted over more than 50 years, yet the mechanisms whereby the channels select certain ions and reject others are not well understood. Here we report a new application of Jarzynski’s Equality to investigate the mechanism of ion selectivity using non-equilibrium molecular dynamics simulations of Na⁺ and K⁺ ions moving through the KcsA channel. The simulations show that the selectivity filter of KcsA adapts and responds to the presence of the ions with structural rearrangements that are different for Na⁺ and K⁺. These structural rearrangements facilitate entry of K⁺ ions into the selectivity filter and permeation through the channel, and rejection of Na⁺ ions. A mechanistic model of ion selectivity by this channel based on the results of the simulations relates the structural rearrangement of the selectivity filter to the differential dehydration of ions and multiple-ion occupancy and describes a mechanism to efficiently select and conduct K⁺. Estimates of the K⁺/Na⁺ selectivity ratio and steady state ion conductance for KcsA from the simulations are in good quantitative agreement with experimental measurements. This model also accurately describes experimental observations of channel block by cytoplasmic Na⁺ ions, the “punch through” relief of channel block by cytoplasmic positive voltages, and is consistent with the knock-on mechanism of ion permeation.     

Skeletal Muscle-specific Calpain Is an Intracellular Na+-dependent Protease

Because intracellular [Na⁺] is kept low by Na⁺/K⁺-ATPase, Na⁺ dependence is generally considered a property of extracellular enzymes. However, we found that p94/calpain 3, a skeletal-muscle-specific member of the Ca²⁺-activated intracellular “modulator proteases” that is responsible for a limb-girdle muscular dystrophy (“calpainopathy”), underwent Na⁺-dependent, but not Cs⁺-dependent, autolysis in the absence of Ca²⁺. Furthermore, Na⁺ and Ca²⁺ complementarily activated autolysis of p94 at physiological concentrations. By blocking Na⁺/K⁺-ATPase, we confirmed intracellular autolysis of p94 in cultured cells. This was further confirmed using inactive p94:C129S knock-in (p94CS-KI) mice as negative controls. Mutagenesis studies showed that much of the p94 molecule contributed to its Na⁺/Ca²⁺-dependent autolysis, which is consistent with the scattered location of calpainopathy-associated mutations, and that a conserved Ca²⁺-binding sequence in the protease acted as a Na⁺ sensor. Proteomic analyses using Cs⁺/Mg²⁺ and p94CS-KI mice as negative controls revealed that Na⁺ and Ca²⁺ direct p94 to proteolyze different substrates. We propose three roles for Na⁺ dependence of p94; 1) to increase sensitivity of p94 to changes in physiological [Ca²⁺], 2) to regulate substrate specificity of p94, and 3) to regulate contribution of p94 as a structural component in muscle cells. Finally, this is the first example of an intracellular Na⁺-dependent enzyme.     

On the functional use of the membrane compartmentalized pool of ATP by the Na+ and Ca++ pumps in human red blood cell ghosts

Previous evidence established that a sequestered form of adenosine triphosphate (ATP pools) resides in the membrane/cytoskeletal complex of red cell porous ghosts. Here, we further characterize the roles these ATP pools can perform in the operation of the membrane''s Na⁺ and Ca²⁺ pumps. The formation of the Na⁺- and Ca²⁺-dependent phosphointermediates of both types of pumps (E_Na-P and E_Ca-P) that conventionally can be labeled with trace amounts of [γ-³P]ATP cannot occur when the pools contain unlabeled ATP, presumably because of dilution of the [γ-³P]ATP in the pool. Running the pumps forward with either Na⁺ or Ca²⁺ removes pool ATP and allows the normal formation of labeled E_Na-P or E_Ca-P, indicating that both types of pumps can share the same pools of ATP. We also show that the halftime for loading the pools with bulk ATP is 10–15 minutes. We observed that when unlabeled “caged ATP” is entrapped in the membrane pools, it is inactive until nascent ATP is photoreleased, thereby blocking the labeled formation of E_Na-P. We also demonstrate that ATP generated by the membrane-bound pyruvate kinase fills the membrane pools. Other results show that pool ATP alone, like bulk ATP, can promote the binding of ouabain to the membrane. In addition, we found that pool ATP alone functions together with bulk Na⁺ (without Mg²⁺) to release prebound ouabain. Curiously, ouabain was found to block bulk ATP from entering the pools. Finally, we show, with red cell inside-outside vesicles, that pool ATP alone supports the uptake of ⁴⁵Ca by the Ca²⁺ pump, analogous to the Na⁺ pump uptake of ²²Na in this circumstance. Although the membrane locus of the ATP pools within the membrane/cytoskeletal complex is unknown, it appears that pool ATP functions as the proximate energy source for the Na⁺ and Ca²⁺ pumps.     

Altered Ionic Selectivity of the Sodium Channel Revealed by Cysteine Mutations within the Pore

To explore the role of pore-lining amino acids in Na⁺ channel ion-selectivity, pore residues were  replaced serially with cysteine in cloned rat skeletal muscle Na⁺ channels. Ionic selectivity was determined by measuring permeability and ionic current ratios of whole-cell currents in Xenopus oocytes. The rSkM1 channels displayed an ionic selectivity sequence Na⁺>Li⁺>NH₄ ⁺>>K⁺>>Cs⁺ and were impermeable to divalent cations.  Replacement of residues in domain IV showed significantly enhanced current and permeability ratios of NH₄ ⁺ and K⁺, and negative shifts in the reversal potentials recorded in the presence of external Na⁺ solutions when compared to cysteine mutants in domains I, II, and III (except K1237C). Mutants in domain IV showed altered selectivity sequences: W1531C (NH₄ ⁺>K⁺>Na⁺≥Li⁺≈Cs⁺), D1532C, and G1533C (Na⁺>Li⁺≥NH₄ ⁺>K⁺>Cs⁺). Conservative replacement of the aromatic residue in domain IV (W1531) with phenylalanine or tyrosine retained Na⁺ selectivity of the channel while the alanine mutant (W1531A) reduced ion selectivity. A single mutation within the third pore forming region (K1237C) dramatically altered the selectivity sequence of the rSkM1 channel (NH₄ ⁺>K⁺>Na⁺≥Li⁺≈Cs⁺) and was permeable to divalent cations having the selectivity sequence Ca²⁺≥Sr²⁺>Mg²⁺>Ba²⁺. Sulfhydryl modification of K1237C, W1531C or D1532C with methanethiosulfonate derivatives that introduce a positively charged ammonium group, large trimethylammonium moiety, or a negatively charged sulfonate group within the pore was ineffective in restoring Na⁺ selectivity to these channels. Selectivity of D1532C mutants could be largely restored by increasing extracellular pH suggesting altering the ionized state at this position influences selectivity. These data suggest that K1237 in domain III and W1531, D1532, and G1533 in domain IV play a critical role in determining the ionic selectivity of the Na⁺ channel.     

Intragenic Suppressor of a Plug Deletion Nonmotility Mutation in PotB,a Chimeric Stator Protein of Sodium-Driven Flagella

The torque of bacterial flagellar motors is generated by interactions between the rotor and the stator and is coupled to the influx of H⁺ or Na⁺ through the stator. A chimeric protein, PotB, in which the N-terminal region of Vibrio alginolyticus PomB was fused to the C-terminal region of Escherichia coli MotB, can function with PomA as a Na⁺-driven stator in E. coli. Here, we constructed a deletion variant of PotB (with a deletion of residues 41 to 91 [Δ41–91], called PotBΔL), which lacks the periplasmic linker region including the segment that works as a “plug” to inhibit premature ion influx. This variant did not confer motile ability, but we isolated a Na⁺-driven, spontaneous suppressor mutant, which has a point mutation (R109P) in the MotB/PomB-specific α-helix that connects the transmembrane and peptidoglycan binding domains of PotBΔL in the region of MotB. Overproduction of the PomA/PotBΔL(R109P) stator inhibited the growth of E. coli cells, suggesting that this stator has high Na⁺-conducting activity. Mutational analyses of Arg109 and nearby residues suggest that the structural alteration in this α-helix optimizes PotBΔL conformation and restores the proper arrangement of transmembrane helices to form a functional channel pore. We speculate that this α-helix plays a key role in assembly-coupled stator activation.     

Evidence for Na-Independent HCO(3) Uptake by the Cyanobacterium Synechococcus leopoliensis

At low levels of dissolved inorganic carbon (DIC) and alkaline pH the rate of photosynthesis by air-grown cells of Synechococcus leopoliensis (UTEX 625) was enhanced 7- to 10-fold by 20 millimolar Na⁺. The rate of photosynthesis greatly exceeded the CO₂ supply rate and indicated that HCO₃⁻ was taken up by a Na⁺-dependent mechanism. In contrast, photosynthesis by Synechococcus grown in standing culture proceeded rapidly in the absence of Na⁺ and exceeded the CO₂ supply rate by 8 to 45 times. The apparent photosynthetic affinity (K_½) for DIC was high (6-40 micromolar) and was not markedly affected by Na⁺ concentration, whereas with air-grown cells K_½ (DIC) decreased by more than an order of magnitude in the presence of Na⁺. Lithium, which inhibited Na⁺-dependent HCO₃⁻ uptake in air-grown cells, had little effect on Na⁺-independent HCO₃⁻ uptake by standing culture cells. A component of total HCO₃⁻ uptake in standing culture cells was also Na⁺-dependent with a K_½ (Na⁺) of 4.8 millimolar and was inhibited by lithium. Analysis of ¹⁴C-fixation during isotopic disequilibrium indicated that standing culture cells also possessed a Na⁺-independent CO₂ transport system. The conversion from Na⁺-independent to Na⁺-dependent HCO₃⁻ uptake was readily accomplished by transferring cells grown in standing to growth in cultures bubbled with air. These results demonstrated that the conditions experienced during growth influenced the mode by which Ssynechococcus acquired HCO₃⁻ for subsequent photosynthetic fixation.     

Actions of Ca2+ on an Early Stage in Phototransduction Revealed by the Dynamic Fall in Ca2+ Concentration during the Bright Flash Response

To study the actions of Ca²⁺ on “early” stages of the transduction cascade, changes in cytoplasmic calcium concentration (Ca²⁺ _i) were opposed by manipulating Ca²⁺ fluxes across the rod outer segment membrane immediately following a bright flash. If the outer segment was exposed to 0 Ca²⁺/0 Na⁺ solution for a brief period immediately after the flash, then the period of response saturation was prolonged in comparison with that in Ringer solution. But if the exposure to 0 Ca²⁺/0 Na⁺ solution instead came before or was delayed until 1 s after the flash then it had little effect. The degree of response prolongation increased with the duration of the exposure to 0 Ca²⁺/0 Na⁺ solution, revealing a time constant of 0.49 ± 0.03 s. By the time the response begins to recover from saturation, Ca²⁺ _i seems likely to have fallen to a similar level in each case. Therefore the prolongation of the response when Ca²⁺ _i was prevented from changing immediately after the flash seems likely to reflect the abolition of actions of the usual dynamic fall in Ca²⁺ _i on an early stage in the transduction cascade at a site which is available for only a brief period after the flash. One possibility is that the observed time constant corresponds to the phosphorylation of photoisomerized rhodopsin.     

Ion Conduction through C-Type Inactivated Shaker Channels

C-type inactivation of Shaker potassium channels involves entry into a state (or states) in which the inactivated channels appear nonconducting in physiological solutions. However, when Shaker channels, from which fast N-type inactivation has been removed by NH₂-terminal deletions, are expressed in Xenopus oocytes and evaluated in inside-out patches, complete removal of K⁺ ions from the internal solution exposes conduction of Na⁺ and Li⁺ in C-type inactivated conformational states. The present paper uses this observation to investigate the properties of ion conduction through C-type inactivated channel states, and demonstrates that both activation and deactivation can occur in C-type states, although with slower than normal kinetics. Channels in the C-type states appear “inactivated” (i.e., nonconducting) in physiological solutions due to the summation of two separate effects: first, internal K⁺ ions prevent Na⁺ ions from permeating through the channel; second, C-type inactivation greatly reduces the permeability of K⁺ relative to the permeability of Na⁺, thus altering the ion selectivity of the channel.     

Transport of lithium and rectification by frog skin

The isolated frog skin, bathed with Li⁺-Ringer (Na⁺-free) on the outside and Na⁺-Ringer on the inside, can maintain a normal potential difference (PD) and short-circuit current (s.c.c.) for more than 6. h. The s.c.c. correspondended to the Li⁺ influx. The Na⁺ efflux was 4% of the s.c.c. 10⁻⁵ M ouabain depressed Li⁺ influx and s.c.c. 10¹⁰⁻⁵ M amiloride abolished the Li⁺ s.c.c., while 0.1 unit/ml oxytocin stimulated it. When the inside of the skin was bathed with Li⁺-Ringer, PD and s.c.c. fell to zero within 2 h. The oxygen consumption of skin slices bathed in Li⁺-Ringer was 29% lower than controls bathed in Na⁺-Ringer.When the isolated frog skin is bathed in Na₂SO₄-Ringer it shows electrical rectification which has been correlated with the active transport of Na⁺. In skins transporting Li⁺, rectification characteristics are similar to those of skins transporting Na⁺. When the inner face of the skin is bathed with Li⁺-Ringer, rectification, PD and s.c.c. decline in a parallel fashion.It is concluded that: (1) Li⁺ can be transported when Na⁺ is present at the inner face. (2) Amiloride, ouabain and oxytocin affect Li⁺ and Na⁺ transport in a similar manner. (3) Li⁺ transport, like Na⁺ transport, is associated with rectification. (4) Active transport of Na⁺ and Li⁺ seems to depend on two different but associated proceses; one taking place at the external barrier (where rectification occurs) as shown by the effect of amiloride; and the other of an inner site related to energy requirements and affected by ouabain and Li⁺. (5) The cation being transported is not necessarily activating the (Na⁺-K⁺-ATPase.     

K+ Block Is the Mechanism of Functional Asymmetry in Bacterial Nav Channels

Crystal structures of several bacterial Na_v channels have been recently published and molecular dynamics simulations of ion permeation through these channels are consistent with many electrophysiological properties of eukaryotic channels. Bacterial Na_v channels have been characterized as functionally asymmetric, and the mechanism of this asymmetry has not been clearly understood. To address this question, we combined non-equilibrium simulation data with two-dimensional equilibrium unperturbed landscapes generated by umbrella sampling and Weighted Histogram Analysis Methods for multiple ions traversing the selectivity filter of bacterial Na_vAb channel. This approach provided new insight into the mechanism of selective ion permeation in bacterial Na_v channels. The non-equilibrium simulations indicate that two or three extracellular K⁺ ions can block the entrance to the selectivity filter of Na_vAb in the presence of applied forces in the inward direction, but not in the outward direction. The block state occurs in an unstable local minimum of the equilibrium unperturbed free-energy landscape of two K⁺ ions that can be ‘locked’ in place by modest applied forces. In contrast to K⁺, three Na⁺ ions move favorably through the selectivity filter together as a unit in a loose “knock-on” mechanism of permeation in both inward and outward directions, and there is no similar local minimum in the two-dimensional free-energy landscape of two Na⁺ ions for a block state. The useful work predicted by the non-equilibrium simulations that is required to break the K⁺ block is equivalent to large applied potentials experimentally measured for two bacterial Na_v channels to induce inward currents of K⁺ ions. These results illustrate how inclusion of non-equilibrium factors in the simulations can provide detailed information about mechanisms of ion selectivity that is missing from mechanisms derived from either crystal structures or equilibrium unperturbed free-energy landscapes.     

Ionic selectivity in L-type calcium channels by electrostatics and hard-core repulsion

A physical model of selective “ion binding” in the L-type calcium channel is constructed, and consequences of the model are compared with experimental data. This reduced model treats only ions and the carboxylate oxygens of the EEEE locus explicitly and restricts interactions to hard-core repulsion and ion–ion and ion–dielectric electrostatic forces. The structural atoms provide a flexible environment for passing cations, thus resulting in a self-organized induced-fit model of the selectivity filter. Experimental conditions involving binary mixtures of alkali and/or alkaline earth metal ions are computed using equilibrium Monte Carlo simulations in the grand canonical ensemble. The model pore rejects alkali metal ions in the presence of biological concentrations of Ca²⁺ and predicts the blockade of alkali metal ion currents by micromolar Ca²⁺. Conductance patterns observed in varied mixtures containing Na⁺ and Li⁺, or Ba²⁺ and Ca²⁺, are predicted. Ca²⁺ is substantially more potent in blocking Na⁺ current than Ba²⁺. In apparent contrast to experiments using buffered Ca²⁺ solutions, the predicted potency of Ca²⁺ in blocking alkali metal ion currents depends on the species and concentration of the alkali metal ion, as is expected if these ions compete with Ca²⁺ for the pore. These experiments depend on the problematic estimation of Ca²⁺ activity in solutions buffered for Ca²⁺ and pH in a varying background of bulk salt. Simulations of Ca²⁺ distribution with the model pore bathed in solutions containing a varied amount of Li⁺ reveal a “barrier and well” pattern. The entry/exit barrier for Ca²⁺ is strongly modulated by the Li⁺ concentration of the bath, suggesting a physical explanation for observed kinetic phenomena. Our simulations show that the selectivity of L-type calcium channels can arise from an interplay of electrostatic and hard-core repulsion forces among ions and a few crucial channel atoms. The reduced system selects for the cation that delivers the largest charge in the smallest ion volume.     

Rescue of Na+ Affinity in Aspartate 928 Mutants of Na+,K+-ATPase by Secondary Mutation of Glutamate 314

The Na⁺,K⁺-ATPase binds Na⁺ at three transport sites denoted I, II, and III, of which site III is Na⁺-specific and suggested to be the first occupied in the cooperative binding process activating phosphorylation from ATP. Here we demonstrate that the asparagine substitution of the aspartate associated with site III found in patients with rapid-onset dystonia parkinsonism or alternating hemiplegia of childhood causes a dramatic reduction of Na⁺ affinity in the α1-, α2-, and α3-isoforms of Na⁺,K⁺-ATPase, whereas other substitutions of this aspartate are much less disruptive. This is likely due to interference by the amide function of the asparagine side chain with Na⁺-coordinating residues in site III. Remarkably, the Na⁺ affinity of site III aspartate to asparagine and alanine mutants is rescued by second-site mutation of a glutamate in the extracellular part of the fourth transmembrane helix, distant to site III. This gain-of-function mutation works without recovery of the lost cooperativity and selectivity of Na⁺ binding and does not affect the E₁-E₂ conformational equilibrium or the maximum phosphorylation rate. Hence, the rescue of Na⁺ affinity is likely intrinsic to the Na⁺ binding pocket, and the underlying mechanism could be a tightening of Na⁺ binding at Na⁺ site II, possibly via movement of transmembrane helix four. The second-site mutation also improves Na⁺,K⁺ pump function in intact cells. Rescue of Na⁺ affinity and Na⁺ and K⁺ transport by second-site mutation is unique in the history of Na⁺,K⁺-ATPase and points to new possibilities for treatment of neurological patients carrying Na⁺,K⁺-ATPase mutations.