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.     

P-Loop Residues Critical for Selectivity in K+ Channels Fail to Confer Selectivity to Rabbit HCN4 Channels

HCN channels are thought to be structurally similar to K_v channels, but show much lower selectivity for K⁺. The ∼3.3 Å selectivity filter of K⁺ channels is formed by the pore-lining sequence XT(V/I)GYG, with X usually T, and is held stable by key residues in the P-loop. Differences in the P-loop sequence of HCN channels (eg. the pore-lining sequence L₄₇₈C₄₇₉IGYG) suggest these residues could account for differences in selectivity between these channel families. Despite being expressed, L478T/C479T HCN4 channels did not produce current. Since threonine in the second position is highly conserved in K⁺ channels, we also studied C479T channels. Based on permeability ratios (P_X/P_K), C479T HCN4 channels (K⁺(1)>Rb⁺(0.85)>Cs⁺(0.59)>Li⁺(0.50)≥Na⁺(0.49)) were less selective than WT rabbit HCN4 (K⁺(1)>Rb⁺(0.48)>Cs⁺(0.31)≥Na⁺(0.29)>Li⁺(0.03)), indicating that the TIGYG sequence is insufficient to confer K⁺ selectivity to HCN channels. C479T HCN4 channels had an increased permeability to large organic cations than WT HCN4 channels, as well as increased unitary K⁺ conductance, and altered channel gating. Collectively, these results suggest that HCN4 channels have larger pores than K⁺ channels and replacement of the cysteine at position 479 with threonine further increases pore size. Furthermore, selected mutations in other regions linked previously to pore stability in K⁺ channels (ie. S475D, S475E and F471W/K472W) were also unable to confer K⁺ selectivity to C479T HCN4 channels. Our findings establish the presence of the TIGYG pore-lining sequence does not confer K⁺ selectivity to rabbit HCN4 channels, and suggests that differences in selectivity of HCN4 versus K⁺ channels originate from differences outside the P-loop region.     

Selectivity Changes during Activation of Mutant Shaker Potassium Channels

Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics. We studied mutations at this position in the backgrounds of NH₂-terminal–truncated Shaker H4 and a Shaker -NGK2 chimeric channel having high conductance (Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1994. Nature (Lond.). 367: 179–182). While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel. Activation time courses at depolarized potentials were unaffected by the mutations, as were first-latency distributions in the T442S chimeric channel. The mutant channels show two subconductance levels, 37 and 70% of full conductance. From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state. The smaller subconductance state is traversed in ∼40% of activation time courses. These states apparently represent kinetic intermediates in channel gating having voltage-dependent transitions with apparent charge movements of ∼1.6 e₀. The fully open T442S chimeric channel has the conductance sequence Rb⁺ > NH₄ ⁺ > K⁺. The opposite conductance sequence, K⁺ > NH₄ ⁺ > Rb⁺, is observed in each of the subconductance states, with the smaller subconductance state discriminating most strongly against Rb⁺.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

Characterization of a k-stimulated adenosine triphosphatase associated with the plasma membrane of red beet

A membrane fraction enriched with a magnesium-dependent, monovalent cation-stimulated ATPase was isolated from red beet (Beta vulgaris L.) storage roots by a combination of differential centrifugation, extraction with KI, and sucrose density gradient centrifugation. This fraction was distinct from endoplasmic reticulum, Golgi, mitochondrial, and possibly tonoplast membranes as determined from an analysis of marker enzymes. The ATPase activity associated with this fraction was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was substrate specific for ATP and had a temperature optimum near 40°C. Kinetics with Mg:ATP followed a simple Michaelis-Menten relationship. However the kinetics of K⁺-stimulation were complex and suggestive of negative cooperativity. When monovalent cations were present at 2.5 millimolarity, ATPase was stimulated in the sequence K⁺ > Rb⁺ > Na⁺ > Li⁺ but when the concentration was raised to 50 millimolarity, the sequence changed to K⁺ ≥ Na⁺ ≥ Rb⁺ > Li. The activity was not synergistically stimulated by combinations of Na⁺ and K⁺. The enzyme was insensitive to NaN₃, oligomycin, ouabain, and sodium molybdate but sensitive to N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and sodium vanadate. Based on the similarity between the properties of this ATPase activity and those from other well characterized plant tissues, it has been concluded that this membrane fraction is enriched with plasma membrane vesicles.     

Ion conduction and selectivity in acid-sensing ion channel 1

The ability of acid-sensing ion channels (ASICs) to discriminate among cations was assessed based on changes in conductance and reversal potential with ion substitution. Human ASIC1a was expressed in Xenopus laevis oocytes, and acid-induced currents were measured using two-electrode voltage clamp. Replacement of extracellular Na⁺ with Li⁺, K⁺, Rb⁺, or Cs⁺ altered inward conductance and shifted the reversal potentials consistent with a selectivity sequence of Li ∼ Na > K > Rb > Cs. Permeability decreased more rapidly than conductance as a function of atomic size, with P_K/P_Na = 0.1 and G_K/G_Na = 0.7 and P_Rb/P_Na = 0.03 and G_Rb/G_Na = 0.3. Stimulation of Cl⁻ currents when Na⁺ was replaced with Ca²⁺, Sr²⁺, or Ba²⁺ indicated a finite permeability to divalent cations. Inward conductance increased with extracellular Na⁺ in a hyperbolic manner, consistent with an apparent affinity (K_m) for Na⁺ conduction of 25 mM. Nitrogen-containing cations, including NH₄⁺, NH₃OH⁺, and guanidinium, were also permeant. In addition to passing through the channels, guanidinium blocked Na⁺ currents, implying competition for a site within the pore. The role of negative charges in an external vestibule of the pore was evaluated using the point mutation D434N. The mutant channel had a decreased single-channel conductance, measured in excised outside-out patches, and a macroscopic slope conductance that increased with hyperpolarization. It had a weakened interaction with Na⁺ (K_m = 72 mM) and a selectivity that was shifted toward larger atomic sizes. We conclude that the selectivity of ASIC1 is based at least in part on interactions with binding sites both within and internal to the outer vestibule.     

Adenosine Triphosphate Activates a Noninactivating K+ Current in Adrenal Cortical Cells through Nonhydrolytic Binding

Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K⁺ current (I_AC) that is inhibited by adrenocorticotropic hormone and angiotensin II at subnanomolar concentrations. Since I_AC appears to set the membrane potential of AZF cells, these channels may function critically in coupling peptide receptors to membrane depolarization, Ca²⁺ entry, and cortisol secretion. I_AC channel activity may be tightly linked to the metabolic state of the cell. In whole cell patch clamp recordings, MgATP applied intracellularly through the patch electrode at concentrations above 1 mM dramatically enhanced the expression of I_AC K⁺ current. The maximum I_AC current density varied from a low of 8.45 ± 2.74 pA/pF (n = 17) to a high of 109.2 ± 26.3 pA/pF (n = 6) at pipette MgATP concentrations of 0.1 and 10 mM, respectively. In the presence of 5 mM MgATP, I_AC K⁺ channels were tonically active over a wide range of membrane potentials, and voltage-dependent open probability increased by only ∼30% between −40 and +40 mV. ATP (5 mM) in the absence of Mg²⁺ and the nonhydrolyzable ATP analog AMP-PNP (5 mM) were also effective at enhancing the expression of I_AC, from a control value of 3.7 ± 0.1 pA/pF (n = 3) to maximum values of 48.5 ± 9.8 pA/pF (n = 11) and 67.3 ± 23.2 pA/pF (n = 6), respectively. At the single channel level, the unitary I_AC current amplitude did not vary with the ATP concentration or substitution with AMP-PNP. In addition to ATP and AMP-PNP, a number of other nucleotides including GTP, UTP, GDP, and UDP all increased the outwardly rectifying I_AC current with an apparent order of effectiveness: MgATP > ATP = AMP-PNP > GTP = UTP > ADP >> GDP > AMP and ATP-γ-S. Although ATP, GTP, and UTP all enhanced I_AC amplitude with similar effectiveness, inhibition of I_AC by ACTH (200 pM) occurred only in the presence of ATP. As little as 50 μM MgATP restored complete inhibition of I_AC, which had been activated by 5 mM UTP. Although the opening of I_AC channels may require only ATP binding, its inhibition by ACTH appears to involve a mechanism other than hydrolysis of this nucleotide. These findings describe a novel form of K⁺ channel modulation by which I_AC channels are activated through the nonhydrolytic binding of ATP. Because they are activated rather than inhibited by ATP binding, I_AC K⁺ channels may represent a distinctive new variety of K⁺ channel. The combined features of I_AC channels that allow it to sense and respond to changing ATP levels and to set the resting potential of AZF cells, suggest a mechanism where membrane potential, Ca²⁺ entry, and cortisol secretion could be tightly coupled to the metabolic state of the cell through the activity of I_AC K⁺ channels.     

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.     

Stimulation of plasmalemma adenosine triphosphatase from oat roots by inorganic and organic cations: concentration-dependence, selectivity, and sites

K⁺-stimulated ATPase activity of a plasmalemma-enriched fraction from excised roots of oat was triphasic in the range 5 to 80 millimolar KCl. The phases obeyed Michaelis-Menten kinetics and were separated from each other by jumps or sharp breaks at about 10 and 20 millimolar. Stimulation by alkali cations was in the order K⁺ > Rb⁺ > Na⁺ > Cs⁺ > Li⁺ or in a closely related sequence. The specificity reflected differences in V_max, not in affinity (K_m⁻¹). Stimulation by the organic cations ethanolamine and choline in the interval 11 to 80 millimolar appeared monophasic rather than biphasic. Substitution on the quaternary nitrogen of the amino alcohols decreased their effectiveness, as did extension and branching of the chain. Stimulation was maximal at about pH 7 both for K⁺ and choline.     

Adenosine triphosphatase from soybean callus and root cells

The ATPase activity of a membrane fraction from soybean (Glycine max L.) root and callus cells, presumed to be enriched in plasma membrane, has been characterized with respect to ion stimulation, pH requirement, and nucleotide specificity. The enzyme from both sources was activated by divalent cations (Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺ > Sr²⁺) and further stimulated by monovalent salts. Preparations from root cells were stimulated by monovalent ions according to the sequence: K⁺ > Rb⁺ > Choline⁺ > Na⁺ > Li⁺ > NH₄⁺ > Cs⁺ > tris⁺. Membrane preparations from callus cells showed similar stimulatory patterns except for a slight preference for Na⁺ over K⁺. No synergism between K⁺ and Na⁺ was found with preparations from either cell source.     

Sarcoplasmic Reticulum K+ (TRIC) Channel Does Not Carry Essential Countercurrent during Ca2+ Release

The charge translocation associated with sarcoplasmic reticulum (SR) Ca²⁺ efflux is compensated for by a simultaneous SR K⁺ influx. This influx is essential because, with no countercurrent, the SR membrane potential (V_m) would quickly (<1 ms) reach the Ca²⁺ equilibrium potential and SR Ca²⁺ release would cease. The SR K⁺ trimeric intracellular cation (TRIC) channel has been proposed to carry the essential countercurrent. However, the ryanodine receptor (RyR) itself also carries a substantial K⁺ countercurrent during release. To better define the physiological role of the SR K⁺ channel, we compared SR Ca²⁺ transport in saponin-permeabilized cardiomyocytes before and after limiting SR K⁺ channel function. Specifically, we reduced SR K⁺ channel conduction 35 and 88% by replacing cytosolic K⁺ for Na⁺ or Cs⁺ (respectively), changes that have little effect on RyR function. Calcium sparks, SR Ca²⁺ reloading, and caffeine-evoked Ca²⁺ release amplitude (and rate) were unaffected by these ionic changes. Our results show that countercurrent carried by SR K⁺ (TRIC) channels is not required to support SR Ca²⁺ release (or uptake). Because K⁺ enters the SR through RyRs during release, the SR K⁺ (TRIC) channel most likely is needed to restore trans-SR K⁺ balance after RyRs close, assuring SR V_m stays near 0 mV.     

Control of voltage-gated K+ channel permeability to NMDG+ by a residue at the outer pore

Crystal structures of potassium (K⁺) channels reveal that the selectivity filter, the narrow portion of the pore, is only ∼3-Å wide and buttressed from behind, so that its ability to expand is highly constrained, and the permeation of molecules larger than Rb⁺ (2.96 Å in diameter) is prevented. N-methyl-d-glucamine (NMDG⁺), an organic monovalent cation, is thought to be a blocker of Kv channels, as it is much larger (∼7.3 Å in mean diameter) than K⁺ (2.66 Å in diameter). However, in the absence of K⁺, significant NMDG⁺ currents could be recorded from human embryonic kidney cells expressing Kv3.1 or Kv3.2b channels and Kv1.5 R487Y/V, but not wild-type channels. Inward currents were much larger than outward currents due to the presence of intracellular Mg²⁺ (1 mM), which blocked the outward NMDG⁺ current, resulting in a strong inward rectification. The NMDG⁺ current was inhibited by extracellular 4-aminopyridine (5 mM) or tetraethylammonium (10 mM), and largely eliminated in Kv3.2b by an S6 mutation that prevents the channel from opening (P468W) and by a pore helix mutation in Kv1.5 R487Y (W472F) that inactivates the channel at rest. These data indicate that NMDG⁺ passes through the open ion-conducting pore and suggest a very flexible nature of the selectivity filter itself. 0.3 or 1 mM K⁺ added to the external NMDG⁺ solution positively shifted the reversal potential by ∼16 or 31 mV, respectively, giving a permeability ratio for K⁺ over NMDG⁺ (P_K⁺/P_NMDG⁺) of ∼240. Reversal potential shifts in mixtures of K⁺ and NMDG⁺ are in accordance with P_K⁺/P_NMDG⁺, indicating that the ions compete for permeation and suggesting that NMDG⁺ passes through the open state. Comparison of the outer pore regions of Kv3 and Kv1.5 channels identified an Arg residue in Kv1.5 that is replaced by a Tyr in Kv3 channels. Substituting R with Y or V allowed Kv1.5 channels to conduct NMDG⁺, suggesting a regulation by this outer pore residue of Kv channel flexibility and, as a result, permeability.     

Ionic Regulation for Cytokinin-dependent Betacyanin Synthesis in Amaranthus Seedlings

Potassium ions at low concentrations stimulate cytokinin-dependent betacyanin synthesis in Amaranthus tricolor seedlings more than other alkali metal ions when tested as the chloride salts. The sequence of relative stimulation is K⁺ > Rb⁺ > (Na⁺ = Li⁺). Calcium and Mg²⁺ ions are inhibitory at concentrations > 1 millimolar when tested as chlorides. Anions also have an effect on the degree of alkali metal stimulation in the order PO₄³⁻ > NO₃⁻ > Cl⁻. The high activity of phosphate may be partly due to its chelating effect on inhibitory Ca²⁺ ions, or to effects on K⁺ uptake. A mixture of Na⁺ and K⁺ in the presence of phosphate is more effective than either cation alone. This result may be due either to effects on tyrosine transport or on the potassium uptake system. Phytochrome-dependent betacyanin synthesis shows the same stimulation by Na⁺ plus K⁺. The effect of a number of inhibitors of transport systems on betacyanin accumulation is reported. The possible role of the ionic environment of cells in their metabolic regulation is discussed, particularly in relation to cytokinin action.     

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.     

Functional reconstitution of the mitochondrial Ca2+/H+ antiporter Letm1

The leucine zipper, EF hand–containing transmembrane protein 1 (Letm1) gene encodes a mitochondrial inner membrane protein, whose depletion severely perturbs mitochondrial Ca²⁺ and K⁺ homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca²⁺ fluorophore and ⁴⁵Ca²⁺-based assays, we demonstrate directly that Letm1 is a Ca²⁺ transporter, with apparent affinities of cations in the sequence of Ca²⁺ ≈ Mn²⁺ > Gd³⁺ ≈ La³⁺ > Sr²⁺ >> Ba²⁺, Mg²⁺, K⁺, Na⁺. Kinetic analysis yields a Letm1 turnover rate of 2 Ca²⁺/s and a K_m of ∼25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca²⁺/2 H⁺ antiport. Letm1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na⁺/Ca²⁺ exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H⁺-dependent Ca²⁺ transport mechanism identified in intact mitochondria.     

Transport of Ammonium and Methylammonium Ions by Azotobacter vinelandii

Ammonium and methylammonium are rapidly taken up by cultures of Azotobacter vinelandii respiring in the presence of succinate. The rate of methylamine uptake increased with external pH from 5.5 to 7.5 but increasing the pH further to 8.5 had little effect on activity, indicating that methylammonium cation rather than uncharged methylamine is the permeant species. The kinetics of methylammonium entry followed the Michaelis-Menten relationship, yielding a K_m of 25 μM and a V_max of 3.8 nmol/min per mg of cell protein. At saturating concentrations ammonium was taken up at rates 30-fold higher than those for methylammonium. Ammonium was a competitive inhibitor of methylammonium uptake and gave an inhibition constant of 1 μM. Ammonium derivatives were inhibitors of methylammonium entry in order of effectiveness: hydrazine > methylhydrazine > formamidine > guanidine > dimethylamine > ethylamine; amides and amino acids did not block uptake. Likewise, metal cations inhibited in the order Tl⁺ > Cs⁺ > Rb⁺, whereas Na⁺, K⁺, and Li⁺ produced no significant effect. Methylammonium uptake was blocked in cells exposed to an uncoupler, p-trifluorome-thoxycarbonyl cyanide-phenyl hydrazone or gramicidin D, but not with dicyclo-hexylcarbodiimide or arsenate. Valinomycin stimulated methylammonium entry into cells in a K⁺-free medium but prevented entry in the presence of 10 mM K⁺. Monensin and nigericin had little effect on transport. These results indicate that methylammonium and ammonium ions enter A. vinelandii electrogenically via a specific transporter.     

Characterization of nigericin-stimulated ATPase from sealed microsomal vesicles of tobacco callus

To understand the function and membrane origin of ionophore-stimulated ATPases, the activity of nigericin-stimulated ATPase was characterized from a low-density microsomal fraction containing sealed vesicles of autonomous tobacco (Nicotiana tabacum Linnaeous cv. Wisconsin no. 38) callus. The properties of KCl-stimulated, Mg-requiring ATPases (KCl-Mg,ATPase) were similar in the absence or presence of nigericin. Nigericin (or gramicidin) stimulation of a KCl-Mg,ATPase activity was optimum at pH 6.5 to 7.0. The enzyme was inhibited completely by N,N′-dicyclohexylcarbodiimide (10 μm), tributyltin (5 μm), and partially by vanadate (200 μm), but it was insensitive to fusicoccin and mitochondrial ATPase inhibitors, such as azide (1 mm) and oligomycin (5 μg/ml). The ATPase was more sensitive to anions than cations. Cations stimulated ATPase activity with a selectivity sequence of NH₄⁺ > K⁺, Rb⁺, Cs⁺, Na⁺, Li⁺ > Tris⁺. Anions stimulated Mg, ATPase activity with a decreasing sequence of Cl⁻ = acetate > SO₄²⁻ > benzene sulfonate > NO₃⁻. The anion stimulation was caused partly by dissipation of the electrical potential (interior positive) by permeant anions and partly by a specific ionic effect. Plant membranes had at least two classes of nigericin-stimulated ATPases: one sensitive and one insensitive to vanadate. Many of the properties of the nigericin-sensitive, salt-stimulated Mg,ATPase were similar to a vanadate-sensitive plasma membrane ATPase of plant tissues, yet other properties (anion stimulation and vanadate insensitivity) resembled those of a tonoplast ATPase. These results support the idea that nigericin-stimulated ATPases are mainly electrogenic H⁺ pumps originated in part from the plasma membrane and in part from other nonmitochondrial membranes, such as the tonoplast.     

Patch clamp studies on root cell vacuoles of a salt-tolerant and a salt-sensitive plantago species : regulation of channel activity by salt stress

Plantago media L. and Plantago maritima L. differ in their strategy toward salt stress, a major difference being the uptake and distribution of ions. Patch clamp techniques were applied to root cell vacuoles to study the tonoplast channel characteristics. In both species the major channel found was a 60 to 70 picosiemens channel with a low ion selectivity. The conductance of this channel for Na⁺ was the same as for K⁺, P_K⁺/P_Na⁺ = 1, whereas the cation/anion selectivity (P_K⁺/P_c1⁻) was about 5. Gating characteristics were voltage and calcium dependent. An additional smaller channel of 25 picosiemens was present in P. maritima. In the whole vacuole configuration, the summation of the single channel currents resulted in slowly activated inward currents (t^½ = 1.2 second). Inwardly directed, ATP-dependent currents could be measured against a ΔpH gradient of 1.5 units over the tonoplast. This observation strongly indicated the physiological intactness of the used vacuoles. The open probability of the tonoplast channels dramatically decreased when plants were grown on NaCl, although single channel conductance and selectivity were not altered.     

Cation Penetration through Isolated Leaf Cuticles

The rates of penetration of various cations through isolated apricot Prunus armeniaca L. leaf cuticles were determined. Steady state rates were measured by using a specially constructed flow-through diffusion cell. The penetration rates of the monovalent cations in group IA followed a normal lyotropic series, i.e., CS⁺ ≥ Rb⁺ > K⁺ > Na⁺ > Li⁺. The divalent cations all penetrated through the cuticle more slowly than the monovalent cations. Comparison of the relative values of k (permeability coefficient) and D (diffusion coefficient) indicates that the penetration of ions through isolated cuticles took place by diffusion and was impeded by charge interactions between the solute and charge sites in the penetration pathway. Cuticular penetration rates of K⁺ and H₂O at pH above 9 were of similar magnitude. At pH 5.5 H₂O penetration was not affected but that of K⁺ was greatly reduced. From this observation and from data on cuticle titration and ion adsorption studies, we hypothesize that cuticular pores are lined with a substance (perhaps a protein) which has exposed positively charged sites.     

The Interaction of Na+ and K+ in Voltage-gated Potassium Channels : Evidence for Cation Binding Sites of Different Affinity

Voltage-gated potassium (K⁺) channels are multi-ion pores. Recent studies suggest that, similar to calcium channels, competition between ionic species for intrapore binding sites may contribute to ionic selectivity in at least some K⁺ channels. Molecular studies suggest that a putative constricted region of the pore, which is presumably the site of selectivity, may be as short as one ionic diameter in length. Taken together, these results suggest that selectivity may occur at just a single binding site in the pore. We are studying a chimeric K⁺ channel that is highly selective for K⁺ over Na⁺ in physiological solutions, but conducts Na⁺ in the absence of K⁺. Na⁺ and K⁺ currents both display slow (C-type) inactivation, but had markedly different inactivation and deactivation kinetics; Na⁺ currents inactivated more rapidly and deactivated more slowly than K⁺ currents. Currents carried by 160 mM Na⁺ were inhibited by external K⁺ with an apparent IC₅₀ <30 μM. K⁺ also altered both inactivation and deactivation kinetics of Na⁺ currents at these low concentrations. In the complementary experiment, currents carried by 3 mM K⁺ were inhibited by external Na⁺, with an apparent IC₅₀ of ∼100 mM. In contrast to the effects of low [K⁺] on Na⁺ current kinetics, Na⁺ did not affect K⁺ current kinetics, even at concentrations that inhibited K⁺ currents by 40–50%. These data suggest that Na⁺ block of K⁺ currents did not involve displacement of K⁺ from the high affinity site involved in gating kinetics. We present a model that describes the permeation pathway as a single high affinity, cation-selective binding site, flanked by low affinity, nonselective sites. This model quantitatively predicts the anomalous mole fraction behavior observed in two different K⁺ channels, differential K⁺ and Na⁺ conductance, and the concentration dependence of K⁺ block of Na⁺ currents and Na⁺ block of K⁺ currents. Based on our results, we hypothesize that the permeation pathway contains a single high affinity binding site, where selectivity and ionic modulation of gating occur.