Stimulating effects of monovalent cations on activator-dissociated and activator-associated states of Ca2+-ATPase in human erythrocytes

The additional activation by monovalent cations of the $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-dependent}$ ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied.The Ca²⁺-ATPase occurs in two different states. In the A-state the enzyme is virtually free of protein activator and the kinetics of Ca²⁺ activation is characterized by low apparent Ca²⁺ affinity and low maximum activity. In the B-state the enzyme is associated with activator and the kinetics is characterized by high Ca²⁺ affinity and high maximum activity.At optimum concentrations of Ca²⁺ the additional activation of the B-state by K⁺, NH₄⁺, Na⁺ and Rb⁺ exceeded the corresponding activations of the A-state, and half-maximum activations by K⁺, NH₄⁺, and Na⁺ were achieved at lower concentrations in the B-state than in the A-state. Li⁺ and Cs⁺ activated the two states almost equally but maximum activation was obtained at lower cation concentrations in the B-state than in the A-state.The activation of the B-state by the various cations decreased in the order $\text{K}{}^{\mathrm{+}}\text{> NH}{}_4{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{= Rb}{}^{\mathrm{+}}\text{> Li}{}^{\mathrm{+}}\text{= Cs}{}^{\mathrm{+}}$. The A-state was activated almost equally by K⁺, Na⁺, NH₄⁺, and Rb⁺ and to a smaller extent by Li⁺ and Cs⁺.At sub-optimum concentrations of Ca²⁺ high concentrations of monovalent cations (100 mM) activated the Ca²⁺-ATPase equally in the A-state and the B-state. In the absence of Ca²⁺ the monovalent cations inhibited the Mg²⁺-dependent ATPase in both types of membranes. This dependence on Ca²⁺ indicates that the monovalent cations interact with the Ca²⁺ sites in the B-state.The results suggest that K⁺ or Na⁺, or both, contribute to the regulation of the Ca²⁺ pump in erythrocytes.     

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

In the E₁ state of the Na,K-ATPase all cations present in the cytoplasm compete for the ion binding sites. The mutual effects of mono-, di- and trivalent cations were investigated by experiments with the electrochromic fluorescent dye RH421. Three sites with significantly different properties could be identified. The most unspecific binding site is able to bind all cations, independent of their valence and size. The large organic cation Br₂-Titu³⁺ is bound with the highest affinity (<μm), among the tested divalent cations Ca²⁺ binds the strongest, and Na⁺ binds with about the same equilibrium dissociation constant as Mg²⁺ (∼0.8 mm). For alkali ions it exhibits binding affinities following the order of Rb⁺≃ K⁺ > Na⁺ > Cs⁺ > Li⁺. The second type of binding site is specific for monovalent cations, its binding affinity is higher than that of the first type, for Na⁺ ions the equilibrium dissociation constant is < 0.01 mm. Since binding to that site is not electrogenic it has to be close to the cytoplasmic surface. The third site is specific for Na⁺, no other ions were found to bind, the binding is electrogenic and the equilibrium dissociation constant is 0.2 mm. Received: 7 August 2000/Revised: 14 November 2000     

A study of Li+-selective permeation through lipid bilayer membranes mediated by a new ionophore (AS701)

The neutral, noncyclic, imide and ether containing ionophore AS701, has been developed as Li⁺-selective molecule, to be used potentially as an aid in the Li⁺-therapy of manic-depressive illness. The present report is a characterization of this molecule in neutral lipid bilayer membranes. This ionophore was found to the bilayers Li⁺-selective, acting as a selective carrier of monovalent cations. In addition, this molecule was found to be capable of acting as a selective carrier of monovalent anions. For both types of ions, the rate-limitting step in the process of permeation was found to be the diffusion of the carrier-ion complex through the membrane. The membrane-permeating species were found to be 2 : 1 carrier-ion complexes, carrying either a monovalent cation or a monovalent anion. The selectivity sequences among the ions studied being: Li⁺(1) > ClO₄^?(0.7) > Na⁺(0.07) > K⁺(0.016) > Rb⁺(0.0095) > Cs⁺(0.0083) > Cl^?(0.001). Mg²⁺ and SO₄^2? were found to be impermeant (under present experimental conditions). This sequence shows that the AS701 molecule has low selectivity for ions present in biological media, among those studied (i.e. Na⁺, K⁺, Mg²⁺, Cl^2? and SO₄^2?). This indicates that these ions will not interfere in the Li⁺ permeability induced by this carrier in vivo, and that the carrier will not interfere in the normal transport processes of these ions.     

Ion Permeation and Block of P2X2 Purinoceptors: Single Channel Recordings

P2X₂ purinoceptors are cation-selective channels activated by ATP and its analogues. Using single channel measurements we studied the channel's selectivity for the alkali metal ions and organic monovalent cations NMDG⁺, Tris⁺, TMA⁺, and TEA⁺. The selectivity sequence for currents carried by alkali metal ions is: K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺, which is Eisenman sequence IV. This is different from the mobility sequence of the ions in free solution suggesting there is weak interaction between the ions and the channel interior. The relative conductance for alkali ions increases linearly in relation to the Stokes radius. The organic ions NMDG⁺, Tris⁺, TMA⁺ and TEA⁺ were virtually impermeant. The divalent ions (Mn²⁺, Mg²⁺, Ca²⁺ and Ba²⁺) induced a fast block visible as a reduction in amplitude of the unitary currents. Using a single-site binding model, the divalent ions exhibited an equilibrium affinity sequence of Mn²⁺ > Mg²⁺ > Ca²⁺ > Ba²⁺. Received: 3 May 1999/Revised: 23 August 1999     

Effect of inotropic agents on the calcium binding to isolated cardiac sarcolemma

Ca²⁺ binding to fragmented sarcolemma isolated from canine heart was measured by an ultracentrifugation technique. Two classes of binding site with dissociation constants of 2.0 · 10^?5 and 1.2 · 10^?3 M were identified. The capacities of the high- and low-affinity sites were 15 and 452 nmol/mg, respectively. These sites were not affected by treatment with neuraminidase. The effects of various cations and drugs on Ca²⁺ binding were studied. All cations tested inhibited Ca²⁺ binding with the following order of potency: trivalent > divalent > monovalent cations. The order of potency for the monovalent ions was: Na⁺ > K⁺ > Li⁺ ? Cs⁺ and for the divalent and trivalent ions: La³⁺ ? Mn²⁺ > Sr²⁺ ? Ba²⁺ > Mg²⁺. 1 · 10^?3 M caffeine and 1 · 10^?8 M ouabain increased the capacity of the low-affinity sites to 1531 and 837 nmol/mg, respectively. 1 · 10^?7 M verapamil, acidosis (pH 6.4), 1^?10^?5 M Mn²⁺ and 1 · 10^?4 M ouabain depressed the capacity of the low-affinity sites to a range of 154–291 nmol/mg. The dissociation constants of the high- and low-affinity sites and the capacity of the high-affinity sites were not affected by these agents.     

pH gradient across the lysosomal membrane generated by selective cation permeability and donnan equilibrium

The pH within isolated Triton WR 1339-filled rat liver lysosomes was determined by measuring the distribution of [¹⁴C]methylamine between the intra- and extralysosomal space. The intralysosomal pH was found to be approximately one pH unit lower than that of the surrounding medium. Increasing the extralysosomal cation concentration lowered the pH gradient by a cation exchange indicating the presence of a Donnan equilibrium. The lysosomal membrane was found to be significantly more permeable to protons than to other cations. The relative mobility of cations through the lysosomal membrane is H⁺ ? Cs⁺ > Rb⁺ > K⁺ Na⁺ > Li⁺ ? Mg²⁺, Ca²⁺. The presented data suggest that the acidity within isolated Triton WR 1339-filled lysosomes is maintained by: (1) a Donnan equilibrium resulting from the intralysosomal accumulation of nondifussible anions and (2) a selective permeability of the lysosomal membrane to cations.     

ADP binding to (Na + K)-activated ATPase

[¹⁴C]ADP binding to EDTA-washed ox brain cell membranes was increased by Na⁺, but decreased by K⁺, Mg²⁺ and Ca²⁺. Na⁺ abolished the effect of K⁺ on ADP binding by a competitive mechanism, but could not reverse the inhibitory action of Mg²⁺ and Ca²⁺. It is concluded that the cation-induced changes in ADP binding reflect properties of (Na⁺ + K⁺)-activated ATPase.     

Ion permeation properties of a cloned human 5-HT3 receptor transiently expressed in HEK 293 cells

Summary Human 5-HT₃ receptors expressed in HEK 293 cells were studied using patch-clamp techniques. The permeability ratios of cations to Na⁺ were Li⁺, 1.16; K⁺, 1.04; Rb⁺, 1.11; Cs⁺ 1.11; NMDG⁺, 0.04; Ca²⁺, 0.49, and Mg²⁺, 0.37. The permeability sequence of the alkali metal cations was Lⁱ⁺ > Rb⁺ = Cs⁺ > K⁺ > Na⁺. Increased external concentrations of Ca²⁺ or Mg²⁺ decreased 5-HT-induced currents at all potentials tested in a voltage-independent manner. The single-channel conductance of human 5-HT₃ receptors measured by fluctuation analysis of whole-cell currents was 790 ± 100fS. Differences in the basic properties of 5-HT₃ receptors between species may explain interspecies differences in pharmacological properties.     

Lithium induced changes in intracellular free magnesium concentration in isolated rat ventricular myocytes

We have examined the effect of exposing isolated rat ventricular myocytes to lithium while measuring cytosolic free magnesium ([Mg²⁺]_i) and calcium ([Ca²⁺]_i) levels with the fluorescent, ion sensitive probes mag-fura-2 and fura-2. There was a significant rise in [Mg²⁺]_i after a 5 min exposure to a solution in which 50% of the sodium had been replaced by Li⁺, but not when the sodium had been replaced by bis-dimethylammonium (BDA). However, there were significant increases in [Ca²⁺]_i when either Na⁺ substitute was used. The possibility that Li⁺, which enters the cells, interferes with the signal from mag-fura-2 was eliminated as Li⁺ concentrations up to 10 mM had no effect on the dye's fluorescence signal. A possible explanation for these findings is that Li⁺ displaces Mg²⁺ from intracellular binding sites. Having considered the binding constants for Mg²⁺ and Li⁺ to ATP, we conclude that Li⁺ can displace Mg²⁺ from Mg-ATP, thus causing a rise in [Mg²⁺]_i. This work has implications for other studies where Li⁺ is used as a Na⁺ substitute.     

Kinetics of Ca+ adsorption and cationic selectivity with a synaptic membrane protein

A study was conducted on the adsorption of ⁴⁵Ca²⁺ to a surface film of a hydrophobic protein derived from synaptic membranes isolated from bovine cerebellum. A kinetic analysis of Ca²⁺ displacement from the protein by various metal and organic cations could be described by a rate law based on diffusion and displacement. The relative rate constants for the displacement of bound Ca²⁺ were in the order Li⁺ <Na⁺, Rb⁺ <Cs⁺ <K⁺, NH₄⁺. Among the alkaline earth series the sequence was Mg²⁺, Sr²⁺ <Ba²⁺. Ca²⁺ adsorption could be described by a theoretical formulation which takes into account an interfacial energy and potential barrier as well as the diffusional process. An attempt was made to consider the effect of energy of hydration of the cations, surface charge, and the chemical environment at the interface on catonic selectivity. The behavior of the cations in this system significantly resemble their behavior in natural membranes, particularly excitatory ones. The structural and physicochemical environment of the protein at the interface is discussed in relation to Ca²⁺ binding and cationic selectivity.     

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.     

3H-mepyramine binding to histamine H1-receptors in guinea pig lung: Characteristics and regulation by ions

The antogonist [³H]-mepyramine is used to label histamine H₁-receptors in guinea pig lung. Scatchard analysis reveals two classes of binding sites. Monovalent cations decrease steady-state binding (Na⁺ > Li⁺ > K⁺), while divalent cations (Mg⁺⁺, Ca⁺⁺, Mn⁺⁺, Ba⁺⁺) exhibit a biphasic curve, increasing binding at low concentrations and decreasing it at higher levels. Na⁺ decreases both affinity and number of binding sites. Dissociation curve shows two components, and Na⁺ accelerates the rate of dissociation of the slower component. GTP does not affect the binding of the antagonist ³H-Mepyramine.     

Ca-Mg-ATPase activity in brain coated microvesicles purified on immunosorbents

Coated microvesicle fractions isolated from ox forebrain cortex by the ultracentrifugation procedure of Pearse (1) and by the modified, less time consuming method of Keen et al (2) had comparable Ca²⁺+Mg²⁺ dependent ATPase activities (about 9 μmol/h per mg protein). The Na⁺+K⁺+Mg²⁺ dependent ATPase activity was 3.2 μmol/h per mg (±1.0, S.D., n=3) when microvesicles were prepared according to (1) and 1.5 μmol/h per mg (±1.0, S.D., n=3) when prepared according to (2).Oligomycin, ruthenium red, and trifluoperazine, inhibitors of Ca²⁺ transport in mitochondria and erythrocyte membranes had no effect on Ca²⁺+Mg²⁺ dependent ATPase from any of the preparations.As demonstrated both by ATPase assays and electron microscopy, coated microvesicles could be bound to immunosorbents prepared with poly-specific antibodies against a coated microvesicle fraction obtained by the method of Pearse (1). The binding could be inhibited by dissolved coat protein using partially purified clathrin. The fraction of coated vesicles eluted from the immunosorbent was purified relative to the starting material as judged by electron microscopy.The Ca²⁺+Mg²⁺ ATPase activity and calmodulin content was copurified with the coated microvesicles and the specific activity of Na⁺+K⁺+Mg²⁺ ATPase was decreased.Na⁺+K⁺+Mg²⁺ dependent ATPase activity in the coated microvesicle fraction could be ascribed to membranes with the appearance of microsomes. These membranes were also bound to the immunosorbents, but the binding was not influenced by clathrin. The capacity of the immunosorbents for these membranes was less than for the coated microvesicles, resulting in a decrease of Na⁺+K⁺+Mg²⁺ dependent ATPase activity in the eluted coated microvescile fraction.It was concluded that Ca²⁺+Mg²⁺ ATPase activity is not a contamination from plasma membrane vesicles or mitochondrial membranes but seems to be an integral part of the coated vesicle membrane.     

Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels

Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K⁺, Na⁺, Cs⁺ and Li⁺; organic cations: TMA and TEA, and divalents: Ba²⁺, Ca²⁺, Mg²⁺ and Mn²⁺. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs⁺, Na⁺ and K⁺ had chord conductances in the range of 35–55 pS with the exception of Li⁺, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K⁺, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba²⁺ and 15 pS for Ca²⁺ at -80 mV and 10 pS for Mg²⁺ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K⁺ currents much like the divalents. As expected, the channel K⁺ conductance increased with K⁺ concentration saturating at ~ 45 pS and the K_D of K⁺ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.     

Anomalous DNA binding by E2 regulatory protein driven by spacer sequence TATA

We have investigated the anomalously weak binding of human papillomavirus (HPV) regulatory protein E2 to a DNA target containing the spacer sequence TATA. Experiments in magnesium (Mg²⁺) and calcium (Ca²⁺) ion buffers revealed a marked reduction in cutting by DNase I at the CpG sequence in the protein-binding site 3′ to the TATA spacer sequence, Studies of the cation dependence of DNA-E2 affinities showed that upon E2 binding the TATA sequence releases approximately twice as many Mg²⁺ ions as the average of the other spacer sequences. Binding experiments for TATA spacer relative to ATAT showed that in potassium ion (K⁺) the E2 affinity of the two sequences is nearly equal, but the relative dissociation constant (K_d) for TATA increases in the order K⁺ < Na⁺ < Ca²⁺ < Mg²⁺. Except for Mg²⁺, K_d for TATA relative to ATAT is independent of ion concentration, whereas for Mg²⁺ the affinity for TATA drops sharply as ion concentration increases. Thus, ions of increasing positive charge density increasingly distort the E2 binding site, weakening the affinity for protein. In the case of Mg²⁺, additional ions are bound to TATA that require displacement for protein binding. We suggest that the TATA sequence may bias the DNA structure towards a conformation that binds the protein relatively weakly.     

Terbium binding to axonal membrane vesicles from lobster (Homarus Americanus) peripheral nerve. A probe of calcium binding sites

Tb³⁺, a fluorescent trivalent cation with physicochemical properties similar to Ca²⁺, binds to peripheral nerve membrane vesicles prepared from the walking leg nerve bundle of the lobster (Homarus americanus). Saturable binding is measured for at least two classes of binding site. Bound Tb³⁺ can be displaced by other cations in the order: Ca²⁺ > Mg²⁺ = Zn²⁺ > NH₄⁺. The binding of Tb³⁺ to the lower affinity site (K_D(app) = 6.0 μM) is inhibitable by Na⁺, Mg²⁺ and Ca²⁺, whereas the higher affinity site (K_D(app) = 2.2 μM) is only sensitive to Ca²⁺. Using this spectral probe the role of Ca²⁺ in peripheral nerve membrane function can be investigated.     

Gating and Conduction Properties of a Sodium-activated Cation Channel from Lobster Olfactory Receptor Neurons

The gating and conduction properties of a channel activated by intracellular Na⁺ were studied by recording unitary currents in inside-out patches excised from lobster olfactory receptor neurons. Channel openings to a single conductance level of 104 pS occurred in bursts. The open probability of the channel increased with increasing concentrations of Na⁺. At 210 mm Na⁺, membrane depolarization increased the open probability e-fold per 36.6 mV. The distribution of channel open times could be fit by a single exponential with a time constant of 4.09 msec at −60 mV and 90 mm Na⁺. The open time constant was not affected by the concentration of Na⁺, but was increased by membrane depolarization. At 180 mm Na⁺ and −60 mV, the distribution of channel closed times could be fit by the sum of four exponentials with time constants of 0.20, 1.46, 8.92 and 69.9 msec, respectively. The three longer time constants decreased, while the shortest time constant did not vary with the concentration of Na⁺. Membrane depolarization decreased all four closed time constants. Burst duration was unaffected by the concentration of Na⁺, but was increased by membrane depolarization. Permeability for monovalent cations relative to that of Na⁺ (P _X /P _Na ), calculated from the reversal potential, was: Li⁺ (1.11) > Na⁺ (1.0) > K⁺ (0.54) > Rb⁺ (0.36) > Cs⁺ (0.20). Extracellular divalent cations (10 mm) blocked the inward Na⁺ current at −60 mV according to the following sequence: Mn²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺ > Ba²⁺. Relative permeabilities for divalent cations (P _Y /P _Na ) were Ca²⁺ (39.0) > Mg²⁺ (34.1) > Mn²⁺ (15.5) > Ba²⁺ (13.8) > Na⁺ (1.0). Both the reversal potential and the conductance determined in divalent cation-free mixtures of Na⁺ and Cs⁺ or Li⁺ were monotonic functions of the mole fraction, suggesting that the channel is a single-ion pore that behaves as a multi-ion pore when the current is carried exclusively by divalent cations. The properties of the channel are consistent with the channel playing a role in odor activation of these primary receptor neurons. Received: 17 September 1996/Revised: 15 November 1996     

The cardiac ryanodine receptor: Looking for anomalies in permeation properties

Anomalies in the permeation properties of the cardiac RyR channel reconstituted into bilayer lipid membranes were investigated systematically. We tested the presence of the anomalous mole fraction effect (AMFE) for the ion conductance and the reversal potential with varying mole fractions of two permeant ions, while the total ion concentration was lower, as in previous studies, to avoid the masking effect of the channel pore saturation with ions. Mixtures of Ba²⁺ with other divalents (Ca²⁺, Sr²⁺), of Ca²⁺ with monovalents (Li⁺, Cs⁺), and of Na⁺ with other monovalents (Cs⁺, Li⁺) were used. We revealed a clear anomaly only for the ion conductance measured in the Na⁺-Cs⁺ and Ca²⁺-Li⁺ mixtures as computed by a Poisson-Nernst-Planck/density functional theory (PNP/DFT) model. Furthermore, we found a significant minimum in the concentration dependence of the reversal potential determined under Li⁺/Ca²⁺ bi-ionic conditions. Our study led to new observations that may have important implications for understanding the mechanisms involved in ion handling in the RyR channel pore; furthermore our results could be useful for further validation of ion permeation models developed for the RyR channel.     

Short communication. Selectivity of the fast activating vacuolar cation channel

The FV channel dominates the ion conductance of the vacuolar membrane at physiological Ca²⁺ concentrations. Patch-clamp measurements on whole barley (Hordeum vulgare) mesophyll vacuoles and on excised tonoplast patches showed small differences in a selectivity sequence NH4⁺ > K⁺ Rb⁺ Cs⁺ >Na⁺ >Li⁺. Less permeant cations decreased the open probability. The FV channel allows the uptake of small monovalent cations especially NH4⁺ into the vacuole.     

K+-Dependent Selectivity and External Ca2+ Block of Shab K+ Channels

Potassium channels allow the selective flux of K⁺ excluding the smaller, and more abundant in the extracellular solution, Na⁺ ions. Here we show that Shab is a typical K⁺ channel that excludes Na⁺ under bi-ionic, Na_o/K_i or Na_o/Rb_i, conditions. However, when internal K⁺ is replaced by Cs⁺ (Na_o/Cs_i), stable inward Na⁺ and outward Cs⁺ currents are observed. These currents show that Shab selectivity is not accounted for by protein structural elements alone, as implicit in the snug-fit model of selectivity. Additionally, here we report the block of Shab channels by external Ca²⁺ ions, and compare the effect that internal K⁺ replacement exerts on both Ca²⁺ and TEA block. Our observations indicate that Ca²⁺ blocks the channels at a site located near the external TEA binding site, and that this pore region changes conformation under conditions that allow Na⁺ permeation. In contrast, the latter ion conditions do not significantly affect the binding of quinidine to the pore central cavity. Based on our observations and the structural information derived from the NaK bacterial channel, we hypothesize that Ca²⁺ is probably coordinated by main chain carbonyls of the pore´s first K⁺-binding site.