Effects of K+, Ba2+ and Ca2+ on the excitable membrane in Paramecium tetraurelia

• 1.1. The effects of Ba²⁺ and K⁺ ions on the membrane currents of Paramecium tetraurelia under a voltage clamp were investigated.
• 2.2. External Ba²⁺ suppresses the inward-going K-current and the Ca-induced K-outward current and changes the activation and inactivation kinetics of transient inward current through the Ca-channel.
• 3.3. K⁺ increases the Ca-induced K-conductances but little affects the leakage conductance.
• 4.4. The resting potentials by changing those ionic concentrations shift the voltage sensitivities of all voltage sensitive channels, simultaneously.
• 5.5. The competition between ions to the channel responses was discussed.

     

Ba2+ ions block K+-induced contractures by antagonizing K+-induced membrane depolarization in frog skeletal muscle fibres

The effects of Ba2+ ions on twitches, K+-induced contractures, and on intracellularly recorded membrane potentials (Em) and depolarizations of frog skeletal muscle fibres were investigated. Exposure of toe muscles to choline--Ringer's solution with 10(-3) M Ba2+ with Ca2+ (1.08 mM) eliminated or very greatly reduced contractures produced by 60 mM K+. In contrast, not only did the same concentration of Ba2+ ions fail to depress the twitch tension of isolated semitendinosus fibres when added to Ringer's with Ca2+, but it even restored twitches that had been eliminated in a zero Ca2+ Ringer's solution. The resting Em of sartorius muscle fibres in choline--Ringer's solution was reduced about 20 mV by 10(-3) M Ba2+. This Ba2+ ion concentration also antagonized the K+-induced depolarization. Thus in the presence of 1 mM Ba2+, 20 mM K+ hyperpolarized rather than depolarized the fibres and 60 or 123 mM K+ produced only very slowly developing, small depolarizations. These results suggest that the loss of the K+-induced contracture in choline-Ringer's caused by Ba2+ ions is due to an inhibition of the K+-induced depolarization. The latter result is consistent with previous findings of other workers that Ba2+ ions block membrane K+ channels.     

Ba2+ uptake and the inhibition by Ba2+ of K+ flux into rat liver mitochondria

Summary Rapid uptake of Ba²⁺ by respiring rat liver mitochondria is accompanied by a transient stimulation of respiration. Following accumulation of Ba²⁺, e.g. at a concentration of 120 nmol per mg protein, the mitochondria exhibit reduced rates of state 3 and uncoupler-stimulated respiration. ADP-stimulated respiration is inhibited at a lower concentration of Ba²⁺ than is required to affect uncoupler-stimulated respiration, suggesting a distinct effect of Ba²⁺ on mechanisms involved in synthesis of ATP. Ba²⁺, which has an ionic radius similar to that of K⁺, inhibits unidirectional K⁺ flux into respiring rat liver mitochondria. This effect on K⁺ influx is observable at concentrations of Ba²⁺, e.g. 23 to 37 nmol per mg protein, which cause no significant change in state 4 or uncoupler-stimulated respiration. The accumulated Ba²⁺ decreases the measuredV _max of K⁺ influx, while having little effect on the apparentK _m for K⁺. The inhibition of K⁺ influx by Ba²⁺ is seen in the presence and absence of mersalyl, an activator of K⁺ influx. In contrast, under the conditions studied, Ba²⁺ has no apparent effet on the rate of unidirectional K⁺ efflux. These data are consistent with the idea that K⁺ may enter and leave mitochondria via spearate mechanisms.     

Role of the slow inward current during the repolarisation process in frog atrial trabeculae

Voltage clamp experiments were performed to analyse the influence of the slow inward current (isi) on the repolarisation process in frog atrium. MnCl2 was used as isi blocker. The action potential was prolonged by depolarising pulses applied during the plateau. This was an expected result considering that, at positive potentials the time constant for isi inactivation increases. This effect was abolished by Mn ions which in turn block isi. The results suggest that inactivation of isi is of primary importance in determining the repolarisation rate. The action of Mn was not selective, since it reduced the background K current. This effect seems to be related to the decrease in Ca influx.     

Ionic interactions of Ba2+ blockades in the MthK K+ channel

The movement and interaction of multiple ions passing through in single file underlie various fundamental K⁺ channel properties, from the effective conduction of K⁺ ions to channel blockade by Ba²⁺ ions. In this study, we used single-channel electrophysiology and x-ray crystallography to probe the interactions of Ba²⁺ with permeant ions within the ion conduction pathway of the MthK K⁺ channel. We found that, as typical of K⁺ channels, the MthK channel was blocked by Ba²⁺ at the internal side, and the Ba²⁺-blocking effect was enhanced by external K⁺. We also obtained crystal structures of the MthK K⁺ channel pore in both Ba²⁺–Na⁺ and Ba²⁺–K⁺ environments. In the Ba²⁺–Na⁺ environment, we found that a single Ba²⁺ ion remained bound in the selectivity filter, preferably at site 2, whereas in the Ba²⁺–K⁺ environment, Ba²⁺ ions were predominantly distributed between sites 3 and 4. These ionic configurations are remarkably consistent with the functional studies and identify a molecular basis for Ba²⁺ blockade of K⁺ channels.     

Stable membrane potentials and mechanical K+ responses activated by internal Ca2+ in HeLa cells

Stable membrane potentials have been measured with microelectrodes in HeLa cells and their average value (-38 mV) compares well with the Cl- electrochemical potential (-34 mV). A hyperpolarizing response is observed upon microelectrode penetrations and following a mechanical or electrical shock; this response is due to a large increase in the K+ permeability, which can also be triggered by Ca2+ injection or blocked by EGTA injection through the microelectrode. Stable hyperpolarizations are obtained with the Ca2+ ionophore A 23187.     

Residues and mechanisms for slow activation and Ba2+ block of the cardiac muscarinic K+ channel, Kir3.1/Kir3.4

Mechanisms and residues responsible for slow activation and Ba(2+) block of the cardiac muscarinic K(+) channel, Kir3.1/Kir3.4, were investigated using site-directed mutagenesis. Mutagenesis of negatively charged residues located throughout the pore of the channel (in H5, M2, and proximal C terminus) reduced or abolished slow activation. The strongest effects resulted from mutagenesis of residues in H5 close to the selectivity filter; mutagenesis of residues in M2 and proximal C terminus equivalent to those identified as important determinants of the activation kinetics of Kir2.1 was less effective. In giant patches, slow activation was present in cell-attached patches, lost on excision of the patch, and restored on perfusion with polyamine. Mutagenesis of residues in H5 and M2 close to the selectivity filter also decreased Ba(2+) block of the channel. A critical residue for Ba(2+) block was identified in Kir3.4. Mutagenesis of the equivalent residue in Kir3.1 failed to have as pronounced an effect on Ba(2+) block, suggesting an asymmetry of the channel pore. It is concluded that slow activation is principally the result of unbinding of polyamines from negatively charged residues close to the selectivity filter of the channel and not an intrinsic gating mechanism. Ba(2+) block involves an interaction with the same residues.     

Effect of Ba2+ on the K+ conductance pathways in the frog cornea

Two types of transepithelial potential difference (PD) responses have been observed in the bullfrog, Rana catesbeiana, when the K+ concentration is changed in the aqueous solution. (1) A normal response, that is, a decrease in the positivity of the aqueous solution when the K+ is increased in this solution. (2) An anomalous response, that is, an increase in PD when K+ is increased from 0 to 4 mM in the aqueous solution. In present experiments 2 mM Ba2+ results in a significant decrease in transepithelial PD and an increase in resistance (R), consistent with the well-known effect of Ba2+ on the K+ conductance in other biological membranes. In the presence of Ba2+ compared to its absence the normal PD responses were decreased when K+ was increased from 4 to 20 or to 79 mM in the aqueous solution. Barium enhanced, but not significantly, the anomalous PD response (PD increase) when K+ was increased from 0 to 4 mM. An anomalous PD response (PD decrease) was obtained with Ba2+ when K+ was changed from 4 to 0 mM while in its absence the response was normal (PD increase) or did not change. These findings support the concept that anomalous PD responses as a result of the electrogenic (Na+ + K+)-ATPase may be obtained when the resistance of the simple K+ pathway is increased.     

Effect of external K+, Ca2+, and Ba2+ on membrane potential and ionic conductance in rat astrocytes

Summary 1. The purpose of this study was (a) to identify if astrocytes show a similar non-Nernstian depolarization in low K⁺ or low Ca²⁺ solutions as previously found in human glial and glioma cells, and (b) to analyze the influence of the K⁺ conductance on the membrane potential of astrocytes.2. The membrane potential (E_m) and the ionic conductance were studied with whole-cell patch-clamp technique in neonatal rat astrocytes (5–9 days in culture) and in human glioma cells (U-251MG).3. In 3.0 mM K⁺, E_m was –75 ± 1.0 mV (mean ± SEM,n=39) in rat astrocytes and –79 ± 0.7 mV (n=5) in U-251MG cells. In both cell types E_m changed linearly to the logarithm of [K⁺]₀ between 3.0 and 160 mM K⁺. K⁺ free medium caused astrocytes to hyperpolarize to –93 ± 2.7 mV (n=21) and U-251MG cells to depolarize to –27 ± 2.1 mV (n=3).4. The I-E curve did not show inward rectification in astrocytes at this developmental stage. The slope conductance (g) exhibited only a small decrease (–19%) in K⁺ free solution and no significant change in 160 mM K⁺.5. Ba²⁺ (1.0 mM) depolarized astrocytes to –45 ± 2.9 mV (n=11), decreasing the slope conductance (g) by 42.4 ± 8.3% (n=11). Ca²⁺ free solution depolarized astrocytes to –53 ± 3.4 mV (n=12) and resulted in a positive shift of the I-E curve, increasing g by 15.3 ± 8.2% (n=8).6. Calculations indicated that a block of K⁺ channels explains the depolarizing effect of Ba²⁺. The effects of K⁺ free or Ca²⁺ free solutions on E_m can be explained by a transformation of K⁺ channels to non-specific leakage channels. That astrocytes show a different reaction to low K⁺ than glioma cells can be related to the lack of inwardly rectifying K⁺ channels in astrocytes at this developmental stage.     

Ca2+-binding of S-100 protein in the presence of K+ and Mg2+

Ca2+-binding of S-100 protein was studied using a Ca2+ electrode at pH 6.80. In the presence of 0.1 M KCl and 10 mM MgCl2 (ionic strength 0.13), Ca2+-binding to S-100 protein occurred in three steps with positive cooperativity. The numbers of bound Ca2+ ions in the three steps were 2, 2, and 4. The Ca2+-binding constants were 6.9 x 10(3) M-1, 2.9 x 10(3) M-1, and 3.7 x 10(2) M-1, respectively. The Ca2+-binding constants of the first and second steps obtained in the presence of 33.3 mM MgCl2 or 0.1 M KCl (ionic strength 0.10) were 1.4 times larger than those described above. This suggests that Mg2+ does not inhibit Ca2+-binding of S-100 protein. The increase of KCl concentration from 0.1 to 0.2 M caused a decrease of the Ca2+-binding constants to ca. 50%.     

Ba2+-Induced conductance fluctuations of spontaneously fluctuating K+ channels in the apical membrane of frog skin (Rana temporaria)

Summary We studied the influence of mucosal Ba²⁺ ions on the recently described (Zeiske & Van Driessche, 1979a, J. Membrane Biol. 47:77) transepithelial, mucosa towards serosa directed K⁺ transport in the skin ofRana temporaria. The transport parametersG (conductance), PD (potential difference),I _sc (short-circuit current, K⁺ current), as well as the noise ofI _sc were recorded. Addition of millimolar concentrations of Ba²⁺ to the mucosal K⁺-containing solution resulted in a sudden but quickly reversible drop inI _sc.G andI _sc decreased continuously with increasing Ba²⁺ concentration, (Ba²⁺) _o . The apparent Michaelis constant of the inhibition by Ba²⁺ lies within the range 40–80 m. The apical membrane seems to remain permselective for K⁺ up to 500 m (Ba²⁺) _o . Higher (Ba²⁺) _o , however, appears to induce a shunt (PD falls,G increases). This finding made an accurate determination of the nature of the inhibition difficult but our results tend to suggest a K⁺-channel block by K⁺–Ba²⁺ competition. In the presence of Ba²⁺, the power spectrum of the K⁺ current shows a second Lorentzian component in the low-frequency range, in addition to the high-frequency Lorentzian caused by spontaneous K⁺-channel fluctuations (Van Driessche & Zeiske, 1980). Both Lorentzian components are only present with mucosal K⁺ and can be depressed by addition of Cs⁺ ions, thus indicating that Ba²⁺ ions induce K⁺-channel fluctuations. The dependence of the parameters of the induced Lorentzian on (Ba²⁺) _o , shows a rise in the plateau values to a maximum around 60 m (Ba²⁺) _o , followed by a sharp and progressive decrease to very low values. The corner frequency which reflects the rate of the Ba²⁺-induced fluctuations, however, increases quasi-linearly up to 1mm (Ba²⁺) _o with a tendency to saturate at higher (Ba²⁺) _o . Based on a three-state model for the K⁺ channel (having one open state, one closed by the spontaneous fluctuation and one blocked by Ba²⁺) computer calculations compared favorably with our results. The effect of Ba²⁺ could be explained by assuming reversible binding at the outer side of the apical K⁺ channel, thereby blocking the open channel in competition with K⁺. The association-dissociation of Ba²⁺ at its receptor site is thought to cause a chopping of the K⁺ current, resulting in modulated current fluctuations.     

Effect of Li+ and Ba2+ on the electrocyte membrane-bound (Na+ + K+)-ATPase

• 1.1. Membrane-bound (Na⁺ + K⁺)-ATPase activity from the non-innervated and innervated faces of Electrophorus electricus (L.) electric organ, obtained by differential centrifugation, was measured using AChE as an enzyme marker for membranes derived from the post-synaptic area (fraction P₃) of the electrolyte.
• 2.2. The effect of Li⁺ and Ba²⁺ on (Na⁺ + K⁺)-ATPase activity of the two membrane fractions (P₂ and P₃) was analysed with respect to K⁺ and Mg²⁺ ions, after the I₅₀ estimation.
• 3.3. The kinetics of the reactions with these cations were investigated showing that Li⁺ inhibits P₂ uncompetitively and for P₃ presented a mixed type inhibition.
• 4.4. Ba²⁺ behaved as an hyperbolic mixed type inhibitor for P₂ and a linear mixed type inhibitor for P₃ fraction.

     

Ba2+ unmasks K+ modulation of the Na+-K+ pump in the frog retinal pigment epithelium

This paper presents electrophysiological evidence that small changes in [K+]o modulate the activity of the Na+-K+ pump on the apical membrane of the frog retinal pigment epithelium (RPE). This membrane also has a large relative K+ conductance so that lowering [K+]o hyperpolarizes it and therefore increases the transepithelial potential (TEP). Ba2+, a K+ channel blocker, eliminated these normal K+-evoked responses; in their place, lowering [K+]o evoked an apical depolarization and TEP decrease that were blocked by apical ouabain or strophanthidin. These data indicate that Ba2+ blocked the major K+ conductance(s) of the RPE apical membrane and unmasked a slowing of the normally hyperpolarizing electrogenic Na+-K+ pump caused by lowering [K+]o. Evidence is also presented that [K+]o modulates the pump in the isolated RPE under physiological conditions (i.e., without Ba2+). In the intact retina, light decreases subretinal [K+]o and produces the vitreal-positive c-wave of the electroretinogram (ERG) that originates primarily in the RPE from a hyperpolarization of the apical membrane and TEP increase. When Ba2+ was present in the retinal perfusate, the apical membrane depolarized in response to light and the TEP decreased so that the ERG c-wave inverted. The retinal component of the c-wave, slow PIII, was abolished by Ba2+. The effects of Ba2+ were completely reversible. We conclude that Ba2+ unmasks a slowing of the RPE Na+-K+ pump by the light-evoked decrease in [K+]o. Such a response would reduce the amplitude of the normal ERG c-wave.     

Rabbit distal colon epithelium: III. Ca2+-activated K+ channels in basolateral plasma membrane vesicles of surface and crypt cells

Summary In the mammalian distal colon, the surface epithelium is responsible for electrolyte absorption, while the crypts are the site of secretion. This study examines the properties of electrical potential-driven⁸⁶Rb⁺ fluxes through K⁺ channels in basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon epithelium. We show that Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels are present in both surface and crypt cell derived vesicles with half-maximal activation at 5×10^–7 m free Ca²⁺. This suggests an important role of cytoplasmic Ca²⁺ in the regulation of the bidirectional ion fluxes in the colon epithelium.The properties of K⁺ channels in the surface cell membrane fraction differ from those of the channels in the crypt cell derived membranes. The peptide toxin apamin inhibits Ca²⁺-activated K⁺ channels exclusively in surface cell vesicles, while charybdotoxin inhibits predominantely in the crypt cell membrane fraction. Titrations with H⁺ and tetraethylammonium show that both high-and low-sensitive⁸⁶Rb⁺ flux components are present in surface cell vesicles, while the high-sensitive component is absent in the crypt cell membrane fraction. The Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels can be solubilized in CHAPS and reconstituted into phospholipid vesicles. This is an essential step for further characterization of channel properties and for identification of the channel proteins in purification procedures.     

Properties of the K+ inward rectifier in the plasma membrane of xylem parenchyma cells from barley roots: Effects of TEA+, Ca2+, Ba2+ and La3+

Xylem parenchyma cells are situated around the (apoplastic) xylem vessels and are involved in the control of the composition of the xylem sap by exporting and resorbing solutes. We investigated properties of the K⁺ inward rectifier in the plasma membrane of these cells by performing patch clamp experiments on protoplasts in the whole-cell configuration. Inward currents were sensitive to the K⁺ channel blocker TEA⁺ at a high concentration (20 mm). Barium, another classical K⁺ channel blocker, inhibited K⁺ currents with a K _i of about 1.3 mm. In contrast to guard cells, the cytosolic Ca²⁺ level proved to be ineffective in regulating the K⁺ conductance at hyperpolarization. External Ca²⁺ blocked currents weakly in a voltage-dependent manner. From instantaneous current-voltage curves, we identified a binding site in the channel pore with an electrical distance of about 0.2 to 0.5. Lanthanum ions reduced the inward current in a voltage-dependent manner and simultaneously displaced the voltage at which half of the channels are in the open state to more positive values. This finding was interpreted as resulting from a sum of two molecular effects, an interaction with the mouth of the channel that causes a reduction of current, and a binding to the voltage sensor, leading to a shielding of surface charges and, subsequently, a modulation of channel gating.A comparison between the K⁺ inward rectifier in xylem parenchyma cells, guard cells and KAT1 from Arabidopsis leads to the conclusion that these rectifiers form subtypes within one class of ion channels. The ineffectiveness of Ca²⁺ to control K⁺ influx in xylem parenchyma cells is interpreted in physiological terms.