Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.     

Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.     

Modification of cardiac Na+ channels by anthopleurin-A: effects on gating and kinetics

We used the whole cell patch clamp technique to investigate the characteristics of modification of cardiac Na⁺ channel gating by the sea anemone polypeptide toxin anthopleurin-A (AP-A). Guinea pig ventricular myocytes were isolated enzymatically using a retrograde perfusion apparatus. Holding potential was –140 mV and test potentials ranged from –100 to + 40 mV (pulse duration 100 or 1000 ms). AP-A (50–100 nM) markedly slowed the rate of decay of Na⁺ current (I _Na) and increased peak I _Na conductance (g _Na) by 38±5.5% (mean±SEM, P < 0.001, n = 12) with little change in slope factor (n = 12) or voltage midpoint of the g _Na/V relationship after correction for spontaneous shifts. The voltage dependence of steady-state I _Na availability (h ) demonstrated an increase in slope factor from 5.9±0.8 mV in control to 8.0±0.7 mV after modification by AP-A (P < 0.01, n = 14) whereas any shift in the voltage midpoint of this relationship could be accounted for by a spontaneous time-dependent shift. AP-A-modified I _Na showed a use-dependent decrease in peak current amplitude (interpulse interval 500 ms) when pulse duration was 100 ms (–15±2%, P < 0.01, n = 17) but showed no decline when pulse duration was 100 ms (–3±1%). This use-dependent effect was probably the result of a decrease in the rate of recovery from inactivation caused by AP-A which had a small effect on the fast time constant of recovery (from 4.1±0.3 ms in control to 6.0±1.1 ms after AP-A, P < 0.05) but increased the slow time constant from 66.2±6.5 ms in control to 188.9±36.4 ms (P< 0.002, n = 19) after exposure to AP-A. Increasing external divalent cation concentration (either Ca²⁺ or Mg²⁺) to 10 mM abolished the effects of AP-A on the rate of I _Na decay. These results demonstrate that modification of cardiac Na⁺ channels by AP-A markedly slowed I _Na inactivation and altered the voltage dependence of activation; these alterations in gating characteristics, in turn, caused an increase in g _Na presumably by increasing the number of channels open at peak I _Na. AP-A slows the rate of recovery of I _Na from inactivation which is probably the basis for a use-dependent decrease in peak amplitude. Finally, AP-A binding is sensitive to external divalent cation concentrations. Thus, increasing [Mg²⁺]_o or [Ca²⁺]_o displaces AP-A from binding, suggesting that they share related binding sites on the external surface of the Na⁺ channel.     

Antiarrhythmic effect ofRodiola rosea and its possible mechanism

Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 116, N ^o 8, pp. 175–176, August, 1993     

KCNQ-encoded channels regulate Na+ transport across H441 lung epithelial cells

H441 cells are a model of absorptive airway epithelia that are characterised by a pronounced apical Na⁺ flux through amiloride-sensitive Na⁺ channels. The flux of Na⁺ is intimately linked to Na⁺ handling by the cell as well as the membrane potential across the apical membrane. As KCNQ-encoded K⁺ channels influence chloride secretion in gastrointestinal epithelia, the goal of the present study was to ascertain the expression of KCNQ genes in H441 cells and determine the functional role of the expression products. Message for KCNQ3 and KCNQ5 was detected by RT-polymerase chain reaction and the translated proteins were observed by immunocytochemistry. Ussing experiments showed that the pan-KCNQ channel blocker XE991, but not KCNQ1 selective blockers, reduced the short circuit current and the amiloride-sensitive component. These data show for the first time that potassium channels encoded by KCNQ3 or KCNQ5 are crucial determinants of epithelial Na⁺ flux.     

Domain near TM1 influences agonist and antagonist responses of peptide-gated Na+ channels

A molecular biological approach was used to analyse the importance of different amino acids for ligand activation and for determining the action of amiloride on peptide- (Phe-Met-Arg-Phe-NH₂, FMRFamide)-gated Na⁺ channels, members of the degenerin/epithelial Na channel (DEG/ENaC) family. Amiloride is a low-affinity blocker of most DEG/ENa channels, but has an unusual enhancing effect on responses of some of them. Chimeras were expressed in Xenopus oocytes and studied electrophysiologically. Differences in properties of channels from Helix aspersa and Helisoma trivolvis highlighted a sequence of 50 residues of the extracellular domain, near the first transmembrane segment (TM1), that affected sensitivity to FMRFamide, and whether amiloride blocked or enhanced the response to FMRFamide. Comparisons of chimeras prepared from H. aspersa and the extracellular domains of two other species, Aplysia californica and Lymnaea stagnalis and the preparation of further constructs, showed that amino acids 128–134 in the H. aspersa sequence are important in determining the predominant effect of amiloride and influencing the EC₅₀ of FMRFamide. The results also showed that amino acids in this region are influenced by amino acids in other regions of the extracellular domain so as to affect not only the magnitude of responses, but also their time course and desensitisation.     

Age differences in hormonal regulation of Na+, K+-ATPase activity in the rat renal cortex

Laboratory of Physiological Genetics, Institute of Cytology and Genetics, Siberian Brach, Russian Academy of Sciences, Novosibirsk. (Presented by Academician of the Russian Academy of Medical SciencesV. P. Lozov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 114, No. 8, pp. 150–153, August, 1992.     

Modification of cloned brain Na+ channels by batrachotoxin

The effects of batrachotoxin (BTX) on cloned α-subunit Na⁺ channels were examined in CHO-K1 cells (a chinese hamster ovary cell line) transfected with rat brain NaIIA cDNA. Under whole-cell patch clamp conditions, BTX shifted the voltage dependence of the activation process by about 45 mV towards the hyperpolarizing direction and eliminated the inactivating phase of Na⁺ currents. Repetitive depolarizations greatly facilitated the binding of BTX with NaIIA channels while the membrane was held at −100 mV. In chloramine-T-pretreated cells, the association rate of BTX binding with the NaIIA channel was 6.5-fold faster than that in untreated cells. The estimated association rate constant for BTX binding with the open form of NaIIA channel was 1.11×10⁶ mol⁻¹·s⁻¹ at room temperature. BTX-modified NaIIA channels were blocked by tetrodotoxin (TTX) in a complicated manner. First, the TTX binding to the closed state of BTX-modified NaIIA channels was not voltage dependent. The K _D value of TTX was measured at 8.9 nM, which was similar to that of unmodified channels (K _D=14.2 nM). Second, the block of the open state of BTX-modified NaIIA channels by TTX was voltage dependent; depolarization reduced the potency of TTX block between −20 mV to +50 mV. Below −30 mV, the TTX affinity began to level off, probably because of the increased presence of the closed state. Unexpectedly, steady-state inactivation of BTX-modified NaIIA channels was minimal as measured by the two-pulse protocol, a phenomenon distinctly different from that found in GH₃ cells. Neutral local anesthetic benzocaine, however, drastically enhanced the steady-state inactivation of BTX-modified NaIIA channels, with its maximal effect around −60 mV. We conclude that BTX can bind and modify the NaIIA α-subunit. However, a specific subtype of α-subunits and/or an unidentified modulating process may be required for the optimal steady-state inactivation of BTX-modified Na⁺ channels.     

Anemonia sulcata toxins modify activation and inactivation of Na+ currents in a crayfish neurone

1. The effects of three toxins (ATX I, II, III) isolated from the sea anemoneAnemonia sulcata were studied in the soma membrane of a crustacean neurone under voltage-clamp conditions. 2. All three toxins affected the action potentials and the Na⁺ currents in a similar manner. The lowest concentrations tested (10 nM, 20 nM and 50 nM for AtX I, II and III, respectively) had pronounced selective effects on the Na⁺ current. No effect on K⁺ or Ca²⁺ currents was observed with concentrations up to 5 M. 3. In the presence of ATX the Na⁺ inactivation was incomplete even with pulses of 700 ms length or strong depolarizing prepulses. 4. Besides the effects on the inactivation process ATX affected also the activation of the Na⁺ current. 5. In cells treated with ATX the negative resistance branch of the peak Na⁺ current voltage relation was shifted by –5 mV to –20 mV. 6. The time to peak was increased for small depolarizations (up to –30 mV) and the rate of rise (I/t) was enlarged by ATX. A slow activating current component was also observed after depolarizing prepulses or if the Na⁺ current was outward. 7. The decay of the Na⁺ tail currents was considerably prolonged after the application of ATX if the membrane was repolarized to potentials more positive than about –60 mV. 8. Repetitive stimulation led to a shortening of the action potential in ATX II treated neurones. A simultaneous and parallel decrement of the peak and plateau current was observed with depolarizing voltage steps.     

Kinetic studies on the effects of ouabain on Na+ fluxes in frog skin

Among 48 pieces of paired frog skins ofRana pipiens in Ringer's solution, 10 pieces showed a strictly monotone decrease in the short circuit current (SCC) following ouabain treatment (10^–4 M). In 9 cases a transient attenuation, and in 27 cases a distinct wave in the ebb of the SCC, was seen. In 2 instances, two waves were seen. Associated with the not-monotone events was a transient rise in electrical skin conductance. The reasons for these mixed skin responses are unknown. One possible reason is considered here: Early during the ouabain action, some of the Na⁺ entering from the mucosal side is trapped in the skin by electroneutral processes, in keeping with the already known fact that ultimately cellular KCl is partly replaced by NaCl. Computer assisted model studies show how monotone, and not-monotone transepithelial net Na⁺ flux curves can be generated. Essential conditions for the generation of notmonotone Na⁺ flux curves are: 1. Presence of two distinct cellular, active Na⁺ pools in the model. 2. Presence of a loop pathway in which a principal transepithelial Na⁺ transport compartment, and a constitutent Na⁺/K⁺ maintenance compartment, are connected to each other and to the extracellular compartment. The model, then, predicts under which kinetic conditions monotone and not-monotone transepithelial Na⁺ flux curves will be seen.     

Influence of Mitragyna Ciliata (MYTA) on the Microsomal Activity of ATPase Na+/K+ Dependent Extract on a Rabbit Heart

Mitragyna ciliata (MYTA) (Rubiaceae) inhibits plasmodia activity. MYTA induces a cardiotonicity of the digitalic type on rat''s isolated heart. In this work we studied the effect of MYTA on microsomal Na⁺/K⁺ dependant ATPase (Na⁺, K⁺ ATPase) extracted from the heart of a rabbit since digitalics inhibit Na⁺, K⁺ ATPase. Our results revealed that the Na⁺/K⁺ ATPase has an optimum pH of 7.4 and temperature of 37°C respectively. There is a linear relationship between the organic phosphate formed and the incubation time over 25 mins incubation period. The ATP hydrolysis rate in the presence of MYTA was 0.775 µM/min. LINEWEAVER and BURK plots showed that MYTA did not alter K_M (1.31 mM) but decreased V_MAX. This study shows that MYTA exerts a non-competitive inhibition on the microsomal Na⁺/K⁺ ATPase extracted from rabbit heart with a Ci₅₀ of 48 µg / ml. We conclude that the mechanism of action of MYTA is linked to the inhibition of the Na⁺/K⁺ ATPase like cardiotonics of the digitalic type.     

Erythrocyte cations and Na+,K+-ATPase pump activity in athletes and sedentary subjects

Summary The chronic effect of training on intraerythrocyte cationic concentrations and on red cell Na⁺,K⁺-ATPase pump activity was studied by comparing well-trained athletes with sedentary subjects at rest. Also the acute effect of a 50-min cross-country run on these erythrocyte measurements was studied in the athletes. At rest the intraerythrocyte potassium concentration was increased (P<0.01) in the athletes compared to that of the control subjects. The intraerythrocyte concentrations of sodium and magnesium and red cell Na⁺, K⁺-ATPase pump activity were, however, similar in the trained and the untrained subjects.As compared with the resting condition, the intraerythrocyte potassium concentration was decreased (P<0.05) after exercise in the athletes, and this was accompanied by a minor increase in the intraerythrocyte sodium concentration. Red cell Na⁺,K⁺-ATPase pump activity was slightly increased (P<0.05) after exercise.     

Inactivation modifiers of Na+ currents and the gating of rat brain Na+ channels in planar lipid membranes

Rat brain Na⁺ channels whose inactivation process had been removed either by batrachotoxin (BTX) or veratridine (VT) were reconstituted into planar lipid membranes. The voltage dependence of the open probability (P _o) of the channel, of the opening and closing rate constants, and the conductance and relative permeability for Na⁺ and K⁺ were studied in voltage-clamp conditions in the presence of agents known to modify the inactivation of Na⁺ currents. In relation to alkaloids (BTX, VT, and aconitine), it was found that once a Na⁺ channel was modified by BTX or VT, the addition of another alkaloid did not change further the gating and permeation properties of the channel over a period of about 1 h. Once the inactivation process of the channels is removed by BTX, the addition of a proteolytic enzyme (trypsin) or an halogenated compound (chloramine-T, CT) induced profound and specific modifications on the opening and closing events of Na⁺ channels: (1) the voltage dependence of the channel P _o shifted to more hyperpolarized potentials; (2) this voltage shift can be explained by equal hyperpolarizing voltage shifts of the opening and closing rate constants of the channel; (3) although the gating properties of the channel were modified by these compounds, the permeation properties of the channel, as evaluated by the conductance and the selectivity to Na⁺ and K⁺ ions, were unaltered; (4) trypsin and CT were active only in the intracellular side of the channel and were irreversible within the time course of the experiments, suggesting covalent modifications of the channel. Inactivation modifiers also affected the gating of toxin-activated single Na⁺ channels. This alteration is compatible with a simple increase in the intracellular potential as seen by the voltage sensor of the channel.     

Platelet Na+/H+ antiport activity in patients with insulin-dependent diabetes mellitus with and without diabetic nephropathy

Summary The incidence of diabetic nephropathy in patients with insulin-dependent diabetes mellitus (IDDM) may depend on factors other than the quality of diabetes control. Hypertension is an additional factor associated with a high degree of renal involvement in IDDM. One abnormality consistantly observed in various tissues of patients with essential hypertension is enhanced activity of the Na⁺/H⁺ antiport. In the present study we have therefore studied platelet antiport activity in 41 healthy subjects (control), in 22 patients with untreated essential hypertension (EH), and in 35 normotensive IDDM patients (type 1). Of these patients 17 exhibited signs of diabetic nephropathy (group 1) while 18 had no evidence for renal involvement of IDDM in spite of a duration of IDDM of at least 10 years (group 2). The two IDDM patient groups were undistinguishable with respect to age, body mass index, and arterial blood pressure (group 1, 117.9±2.4/78.4±1.5 mmHg; group 2, 113.9±3.6/76.1±1.8 mmHg). Antiporter activity was determined from the rate of cell volume changes induced by propionic acid. Platelet Na⁺/H⁺ exchange activity averaged 23.43±0.43 10^–3·s^–1 in control subjects and was markedly elevated in EH (28.38±0.62 10^–3·s^–1 P<0.01). Antiport activity in group 2 patients without nephropathy averaged 24.54±0.57 10^–3·s^–1 and was undistinguishable from the control group. However, platelet Na⁺/H⁺ antiport activity was significantly stimulated in group 1 patients with nephropathy as compared to group 2(26.95±0.73 10^–3. s^–1 ; P<0.025). Our results show that renal involvement in IDDM is associated with enhanced activity of the platelet Na⁺/H⁺ antiport.Abbreviations EH essential hypertension - HbA_1c glycosyla-ted hemoglobin - IDDM insulin-dependent diabetes mellitus Dedicated to Prof. Dr. N. Zöllner on the occasion of his 70th birthday     

Regulation of the cytosolic pH set point for activation of the Na+/H+ antiport in human platelets: the roles of the Na+/Ca2+ exchange,the Na+-K+-2Cl− cotransport and cellular volume

To explore further the mechanisms that regulate the Na⁺/H⁺ antiport in human platelets, we examined the effect of Na⁺ pump inhibition by ouabain and K⁺ removal from the extracellular medium on parameters of this transport system. Treatment with ouabain resulted in increased cytosolic free Ca²⁺ and Na⁺, coupled with an alkaline shift in the cytosolic pH set point for the Na⁺/ H⁺ antiport. Inhibition of the Na⁺ pump by the removal of K⁺ from the medium increased the cytosolic Na⁺ but not the cytosolic Ca²⁺; yet this treatment also produced a substantial alkaline shift in the cytosolic pH set point for the Na⁺/H⁺ antiport. This effect appeared to relate to a decline in cellular volume and it was attenuated by the Na⁺-K⁺-2Cl^– cotransport inhibitor, bumetanide. These findings indicate: (a) a link between the Na⁺ pump and the Na⁺/H⁺ antiport, mediated by the Na⁺/Ca²⁺ exchange and the cytosolic free Ca²⁺, and (b) a link between the Na⁺/H⁺ antiport and the Na⁺-K⁺-2Cl^– cotransport through cellular volume.This work was supported by grants from the National Heart, Lung, and Blood Institute (HL34807, HL42856) and the American Diabetes Association. M. Kimura is a postdoctoral research fellow of the American Heart Association, New Jersey Affiliate     

Aldosterone actions on basolateral Na+/H+ exchange in Madin-Darby canine kidney cells

In recent studies, there has been a re-evaluation of the polarity of Na⁺/H⁺ exchange in Madin-Darby canine kidney (MDCK) cells. This study was designed to examine aldosterone actions on basolaterally located Na⁺/H⁺ exchange of MDCK cell monolayers grown on permeant filter supports; pH_i was analysed in the absence of bicarbonate by using the pH-sensitive fluorescent probe 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein. Pre-exposure of MDCK cells to aldosterone led within 10–20 min to an alkalization of pH_i ( 0.3 pH unit); this effect is prevented by an addition of dimethylamiloride to the basolateral superfusate. Addition of aldosterone led to stimulation of the basolaterally located Na⁺/H⁺ exchange activity (Na⁺-dependent recovery from an acid load); this effect required preincubation (more then 3 min) and was observed at 0.1 nM aldosterone. Preexposure (15 min) of MDCK monolayers to phorbol 12-myristate 13-acetate also led to an activation of Na⁺/H⁺ exchange; pre-exposure to 8-bromo-cAMP led to inhibition of Na⁺/H⁺ exchange activity. An inhibitory effect of aldosterone was observed if Na⁺/H⁺ exchange activity was analysed in the presence of aldosterone; the highest inhibitory effects (20%–30%) occurred at concentrations of 5 nM and higher. Aldosterone-dependent inhibition does not require preincubation and is fully reversible; it was only observed at low (20 mM) but not at high Na⁺ concentrations (130 mM). The data suggest that aldosterone has an instantaneous inhibitory effect on basolaterally located Na⁺/H⁺ exchange activity under conditions of low Na⁺, but stimulates the rate of transport activity upon preincubation under conditions of physiological Na⁺ concentrations.     

Copper toxicity in cultured human skeletal muscle cells: the involvement of Na+/K+-ATPase and the Na+/Ca2+-exchanger

Copper (Cu²⁺) intoxication has been shown to induce pathological changes in various tissues. The mechanism underlying Cu²⁺ toxicity is still unclear. It has been suggested that the Na⁺/K⁺-ATPase and/or a change of the membrane permeability may be involved. In this study we examined the effects of Cu²⁺ on the Na⁺ and Ca²⁺ homeostasis of cultured human skeletal muscle cells using the ion-selective fluorescent probes Na⁺-binding benzofuran isophtalate (SBFI) and Fura-2, respectively. In addition, we measured the effect of Cu²⁺ on the Na⁺/K⁺-ATPase activity. Cu²⁺ and ouabain increase the cytoplasmic free Na⁺ concentration ([Na⁺]_i). Subsequent addition of Cu²⁺ after ouabain does not affect the rate of [Na⁺]_i increase. Cu²⁺ inhibits the Na⁺/K⁺-ATPase activity with an IC₅₀ of 51 M. The cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) remains unaffected for more than 10 min after the administration of Cu²⁺. Thereafter, [Ca²⁺]_i increases as a result of the Na⁺/Ca²⁺-exchanger operating in the reversed mode. The effects of Cu²⁺ on the Na⁺ homeostasis are reversed by the reducing and chelating agent dithiothreitol and the heavy metal chelator N,N,N,N,-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). In conclusion, SBFI is a good tool to examine Na⁺ homeostasis in cultured human skeletal muscle cells. Under the experimental conditions used, Cu²⁺ does not modify the general membrane permeability, but inhibits the Na⁺/K⁺-pump leading to an increase of [Na⁺]_i. As a consequence the operation mode of the Na⁺/Ca²⁺-exchanger reverses and [Ca²⁺]_i rises.The authors thank staff and coworkers of the Department of Neurology of the University Hospital Nijmegen, Nijmegen for their kind cooperation in obtaining muscle biopsies. Mr. Arie Oosterhof is gratefully acknowledged for culturing of the human muscle cells. The Prinses Beatrix Fonds and the Dutch-Chinese scientific exchange program contributed financial support for this study.     

Influence of the external K+-concentration on the electrical activity and the Na+-, K+-content of the retinal tissue

Summary The effect of the external K⁺-concentration in a range from 0 to 10 mMol/l on the exposure potential (ERG) and the Na⁺-, K⁺-distribution in the retinal tissue of Rana esculenta was investigated. After 45 min of perfusion with the test solution moist combustion of the tissue and analysis by flame photometry was carried out.At concentrations of less than 1 mMol/l an extensive loss of retinal potassium could be observed which corresponded to a slow and steady decrease of the exposure potential. Simultaneously the mean intracellular Na⁺-content increased. Above 2 mMol/l a rapid decline of the potential _b to a constant level was recorded. At the same time there was a slight increase in retinal potassium and a decrease in sodium.The quick transition to steady values of the ERG at high external K⁺-concentrations is attributed to the limitation of the potassium uptake of retinal cells which brings about a constant distribution of electrolytes in a short time. The slow drop in potential at a low potassium content of the bathing solution is ascribed to the diffusion process of K⁺ through cellular membranes of the retina.Partly supported by the Deutsche Forschungsgemeinschaft.     

Effect of Cs+, Li+ and Na+ on the potassium conductance and gating kinetics in the frog node of Ranvier

The effects of monovalent internal cations Cs⁺, Li⁺ and Na⁺ on potassium channel conductance in the frog node of Ranvier were studied by means of the voltage clamp. As previously reported, when 10–80% of the internal K⁺ was replaced by one of the above cations, the steady-state current-voltage relationship was significantly modified. The main effect was a voltage-dependent attenuation of the currents. We demonstrate that the current attenuation is associated with a change in the channel gating kinetics. For small depolarizations the kinetics can be described by the usual potassium conductance activation time constant, τ _n . However, under certain experimental conditions (e.g. substitution of the intracellular K⁺ with 10% Cs⁺), during larger depolarizations, stepping the membrane potential to values above 40–60 mV, the conductance develops with two time constants: τ _n and a new, slower time constant that, in contrast to τ _n , grows with membrane potential. These results can be explained by assuming that the catins may occupy two different sites in the channel; when the first site is occupied the channel is blocked, while occupation of the second site results in slowing of the gating kinetics in the affected channels.