The receptor potential of retinular cells of the blowflyCalliphora: The role of sodium,potassium and calcium ions

Summary Simultaneous membrane potential and membrane resistance measurements and current clamp experiments were performed on the visual sense cells ofCalliphora. The results suggest that the receptor potential is due to two different conductance variations: (i) a conductance increase for both Na⁺- and K⁺-ions upon illumination, and (ii) a light-independent conductance increase for K⁺-ions. Evidence is obtained that in the visual sense cells ofCalliphora the light-independent conductance increase is mediated by the influx of Ca²⁺-ions.The conclusion is that the increase of the intracellular Ca²⁺-ion concentration not only decreases the light-induced conductance, but also increases the light-independent conductance. So the potential of the sense cell is governed by two antagonistic conductances. The functional advantages of such a system are discussed.     

Na+K+-ATPase Activity and K+ Channels Differently Contribute to Vascular Relaxation in Male and Female Rats

Gender associated differences in vascular reactivity regulation might contribute to the low incidence of cardiovascular disease in women. Cardiovascular protection is suggested to depend on female sex hormones’ effects on endothelial function and vascular tone regulation. We tested the hypothesis that potassium (K⁺) channels and Na⁺K⁺-ATPase may be involved in the gender-based vascular reactivity differences. Aortic rings from female and male rats were used to examine the involvement of K⁺ channels and Na⁺K⁺-ATPase in vascular reactivity. Acetylcholine (ACh)-induced relaxation was analyzed in the presence of L-NAME (100 µM) and the following K⁺ channels blockers: tetraethylammonium (TEA, 2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 µM) and charybdotoxin (ChTX, 0.1 µM). The ACh-induced relaxation sensitivity was greater in the female group. After incubation with 4-AP the ACh-dependent relaxation was reduced in both groups. However, the dAUC was greater in males, suggesting that the voltage-dependent K⁺ channel (K_v) participates more in males. Inhibition of the three types of Ca²⁺-activated K⁺ channels induced a greater reduction in R_max in females than in males. The functional activity of the Na⁺K⁺-ATPase was evaluated by KCl-induced relaxation after L-NAME and OUAincubation. OUA reduced K⁺-induced relaxation in female and male groups, however, it was greater in males, suggesting a greater Na⁺K⁺-ATPase functional activity. L-NAME reduced K⁺-induced relaxation only in the female group, suggesting that nitric oxide (NO) participates more in their functional Na⁺K⁺-ATPase activity. These results suggest that the K⁺ channels involved in the gender-based vascular relaxation differences are the large conductance Ca²⁺-activated K⁺ channels (BK_Ca) in females and K_v in males and in the K⁺-induced relaxation and the Na⁺K⁺-ATPase vascular functional activity is greater in males.     

The respiration of brain mitochondria and its regulation by monovalent cation transport

The Na⁺ and K⁺ permeability properties of rat brain mitochondria were determined to explain the influences of these cations upon respiration. A new procedure for isolating exceptionally intact mitochondria with minimal contamination by synaptosomes was developed for this purpose.Respiration was uncoupled by Na⁺ and less so by K⁺. Uncoupling was maximal in the presence of EDTA plus P_i and was decreased by Mg²⁺. Maximal uncoupler-stimulated respiration rates were inhibited by Na⁺ but largely unaffected by K⁺. The inhibition by Na⁺ was relatively insensitive to Mg²⁺. Membrane Na⁺ and K⁺ conductances as well as neutral exchanges (Na⁺/H⁺ and K⁺/H⁺ antiport activities) were determined by swelling measurements and correlated with metabolic effects of the cations.Cation conductance, i.e. electrophoretic Na⁺ or K⁺ permeation, was increased by EDTA (Na⁺ > K⁺) and decreased by Mg²⁺. Magnesium preferentially suppressed Na⁺ conductance so as to reverse the cation selectivity (K⁺ > Na⁺). Neutral cation/H⁺ exchange rates (Na⁺ > K⁺) were not influenced by chelator or Mg²⁺.The extent of cation-dependent uncoupling of respiration correlated best with the inner membrane conductance of the ion according to an empirical relationship derived with the model K⁺ conductor valinomycin. The metabolic influences of Na⁺ and K⁺ can be explained in terms of coupled flow of these ions with protons and their effect upon the H⁺ electrochemical gradient although alternative possibilities are discussed. These in vitro studies are compared to previous observations in situ to assess their physiological significance.     

Voltage-dependent ionic conductances in the human malignant astrocytoma cell line U87-MG

Although the human malignant astrocytoma cell line U87-MG has been used in numerous studies, few findings are available on the properties of its membrane ion conductances. Characterization of the ion channels expressed in these cells will make it possible to study membrane ion conductance changes when a receptor is activated by its ligand. This will help to elucidate the functional properties of these receptors and their signal-transduction pathways in pathophysiological events. This work studied the voltage-dependent ionic conductances of U87-MG cells using the Whole-Cell Recording patch-clamp technique. Six types of voltage-dependent ionic currents were identified: (i) a TEA-, 4-AP- and CTX-sensitive Ca²⁺-dependent K⁺ current, (ii) a transient K⁺ current inhibited by 4-AP, (iii) an inwardly rectifying K⁺ current blocked by Ba²⁺ and 4-AP, (iv) a DIDS- and SITS-sensitive Cl^? current, (v) a TTX-sensitive Na⁺ conductance and (vi) a L-type Ca²⁺ conductance activated by BayK-8644 and inhibited by Ni and the L-type Ca²⁺ channel inhibitor, nifedipine. In addition, electrical depolarizations elicited inward currents due to voltage-independent, Ca²⁺-dependent K⁺ influx against the electrochemical gradient, probably via an ouabain-sensitive Na⁺-K⁺ pump.     

Equilibrium potential for the postsynaptic response in the squid giant synapse

The reversal potential for the EPSP in the squid giant synapse has been studied by means of an intracellular, double oil gap technique. This method allows the electrical isolation of a portion of the axon from the rest of the fiber and generates a quasi-isopotential segment. In order to make the input resistance of this nerve segment as constant as possible, the electroresponsive properties of the nerve membrane were blocked by intracellular injection of tetraethylammonium (TEA) and local extracellular application of tetrodotoxin (TTX). Thus, EPSP''s could be evoked in the isolated segment with a minimal amount of electroresponsive properties. The reversal potential for the EPSP (EEPSP) was measured by recording the synaptic potential or the synaptic current during voltage clamping. The results indicate that EEPSP may vary from +15 to +25 mV, which is more positive than would be expected for a 1:1 conductance change for Na⁺ and K⁺ (approximately -15 mV) and too negative for a pure Na⁺ conductance (⁺40 mV). This latter value (E _Na) was directly determined in the voltage clamp experiments. The results suggest that the synaptic potential is probably produced by a permeability change to Na⁺ to K⁺ in a 4:1 ratio. No change in time-course was observed in the synaptic current at clamp levels of -100 and +90 mV. The implications of a variable ratio for Na⁺-K⁺ permeability in subsynaptic-postsynaptic membranes are discussed.     

Taurine: a preventive agent of the acute ethanol depletive action on the isolated human amniotic membrane

Summary The preventive effect of taurine towards the acute ethanol reduction action was studied on the ionic transfer through the isolated human amniotic membrane. Taurine increased 3 components of the ionic transfer expressed by the conductance measurements (Na⁺ and K⁺ paracellular conductances through the intercellular spaces and coupling cell factor between 2 adjacent epithelial cells, expressed by a voltage ratio). These components were decreased by ethanol. Electrophysiological studies (conductance and voltage measurements) indicated that the addition of taurine (0.1–1 mM) before ethanol (0.4 g/l) hindered the decrease action of ethanol on the Na⁺ and K⁺ paracellular conductances and on the coupling cell factor. These data indicated a common target between taurine and ethanol: the membraneous phospholipids, particularly the distribution of the external fixed charges. The preventive action of taurineversus ethanol, on the human amniotic membrane, was exerted on the polar groups of phospholipids, hindering the incorporation of ethanol molecules.     

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.     

The Influence of H+ on the Membrane Potential and Ion Fluxes of Nitella

The resting membrane potential of the Nitella cell is relatively insensitive to [K]_o, but behaves like a hydrogen electrode. K⁺ and Cl^- effluxes from the cell were measured continuously, while the membrane potential was changed either by means of a negative feedback circuit or by external pH changes. The experiments indicate that P_K and P_Cl are independent of pH but are a function of membrane potential. Slope ion conductances, G_K, G_Cl, and G_Na were calculated from efflux measurements, and their sum was found to be negligible compared to membrane conductance. The possibility that a boundary potential change might be responsible for the membrane potential change was considered but was ruled out by the fact that the peak of the action potential remained at a constant level regardless of pH changes in the external solution. The conductance for H⁺ was estimated by measuring the membrane current change during an external pH change while the membrane potential was clamped at K⁺ equilibrium potential. In the range of external pH 5 to 6, H⁺ chord conductance was substantially equal to the membrane conductance. However, the [H]_i measured by various methods was not such as would be predicted from the [H]_o and the membrane potential using the Nernst equation. In artificial pond water containing DNP, the resting membrane potential decreased; this suggested that some energy-consuming mechanism maintains the membrane potential at the resting level. It is probable that there is a H⁺ extrusion mechanism in the Nitella cell, because the potential difference between the resting potential and the H⁺ equilibrium potential is always maintained notwithstanding a continuous H⁺ inward current which should result from the potential difference.     

The k/na selectivity of a cation channel in the plasma membrane of root cells does not differ in salt-tolerant and salt-sensitive wheat species

The characteristics of cation outward rectifier channels were studied in protoplasts from wheat root (Triticum aestivum L. and Triticum turgidum L.) cells using the patch clamp technique. The cation outward rectifier channels were voltage-dependent with a single channel conductance of 32 ± 1 picosiemens in 100 millimolar KCl. Whole-cell currents were dominated by the activity of the cation outward rectifiers. The time- and voltage-dependence of these currents was accounted for by the summed behavior of individual channels recorded from outside-out detached patches. The K⁺/Na⁺ permeability ratio of these channels was measured in a salt-sensitive and salt-tolerant genotype of wheat that differ in rates of Na⁺ accumulation, using a voltage ramp protocol on protoplasts in the whole-cell configuration. Permeability ratios were calculated from shifts in reversal potentials following ion substitutions. There were no significant differences in the K⁺/Na⁺ permeability ratios of these channels in root cells from either of the two genotypes tested. The permeability ratio for K⁺/Cl⁻ was greater than 50:1. The K⁺/Na⁺ permeability ratio averaged 30:1, which is two to four times more selective than the same type of channel in guard cells and suspension culture cells. Lowering the Ca²⁺ concentration in the bath solution to 0.1 millimolar in the presence of 100 millimolar Na⁺ had no significant effect on the K⁺/Na⁺ permeability ratios of the channel. It seems unlikely that the mechanism of salt tolerance in wheat is based on differences in the K⁺/Na⁺ selectivity of these channels.     

Voltage-activated ionic channels and conductances in embryonic chick osteoblast cultures

Summary Patch-clamp measurements were made on osteoblast-like cells isolated from embryonic chick calvaria. Cell-attachedpatch measurements revealed two types of high conductance (100–250 pS) channels, which rapidly activated upon 50–100 mV depolarization. One type showed sustained and the other transient activation over a 10-sec period of depolarization. The single-channel conductances of these channel types were about 100 or 250 pS, depending on whether the pipettes were filled with a low K⁺ (3mm) or high K⁺ (143mm) saline, respectively. The different reversal potentials under these conditions were consistent with at least K⁺ conduction. Whole-cell measurements revealed the existence of two types of outward rectifying conductances. The first type conducts K⁺ ions and activates within 20–200 msec (depending on the stimulus) upon depolarizing voltage steps from <–60 mV to >–30 mV. It inactivates almost completely with a time constant of 2–3 sec. Recovery from inactivation is biphasic with an initial rapid phase (1–2 sec) followed by a slow phase (>20 sec). The second whole-cell conductance activates at positive membrane potentials of >+50 mV. It also rapidly turns on upon depolarizing voltage steps. Activation may partly disappear at the higher voltages. Its single channels of 140 pS conductance were identified in the whole cell and did conduct K⁺ ions but were not highly Cl^– or Na⁺ selective. The results show that osteoblasts may express various types of voltage controlled ionic channels. We predict a role for such channels in mineral metabolism of bone tissue and its control by osteoblasts.     

Ion pathways in renal brush border membranes

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na⁺/H⁺ or Li⁺/H⁺ exchange was demonstrated by diluting Na₂SO₄ or Li₂SO₄ loaded vesicles into Na⁺- or Li⁺-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K⁺ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na⁺ conductance smaller than electroneutral Na⁺/H⁺ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na⁺ conductance greater than the H⁺ conductance was suggested. K⁺ gradients also induced changes of pH, at about 10% of the Na⁺ or Li⁺ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K⁺ was a low affinity substrate for the Na⁺/H⁺ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K⁺ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles had a low K⁺ conductance. Inward Cl^? gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl^? conductance greater than the H⁺ conductance and a Cl^?/OH^? exchange were present. The rate of Na⁺/H⁺ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent K_m for Na⁺ or Li⁺ of about 10 mM and an E_A of 14.3 kcal per mol. A 61-fold Na₂SO₄ gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower ΔpH even in the presence of gramicidin showing that the H⁺ and Cl^? pathways could alter the effects of the Na⁺/H⁺ exchange.     

Multiple modes of a conditional neural oscillator

We present a model for a conditional bursting neuron consisting of five conductances: Hodgkin-Huxley type time- and voltage-dependent Na⁺ and K⁺ conductances, a calcium activated voltage-dependent K⁺ conductance, a calcium-inhibited time- and voltage-dependent Ca⁺⁺ conductance, and a leakage Cl(_– conductance. With an initial set of parameters (versionS), the model shows a hyperpolarized steady-state membrane potential at which the neuron is silent. Increasingg _Na and decreasingg _Cl, whereg _i , is the maximal conductance for speciesi, produces bursts of action potentials (BursterN). Alternatively, an increase ing _Ca produces a different bursting state (BursterC). The two bursting states differ in the periods and amplitudes of their bursting pacemaker potentials. They show different steady-stateI–V curves under simulated voltage-clamp conditions; in simulations that mimic a steady-stateI–V curve taken under experimental conditions only BursterN shows a negative slope resistance region. ModelC continues to burst in the presence of TTX, while bursting in ModelN is suppressed in TTX. Hybrid models show a smooth transition between the two states.     

A possible mechanism for low affinity of silkworm Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase for K<Superscript>+</Superscript>

The affinity for K⁺ of silkworm nerve Na⁺/K⁺-ATPase is markedly lower than that of mammalian Na⁺/K⁺-ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K⁺ affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na⁺/K⁺-ATPase α and β subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and β subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na⁺/K⁺-ATPase α1 subunit. On the other hand, the amino acid identity of the β subunit with mammalian counterparts was as low as 30%. Cloned α and β cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na⁺/K⁺-ATPase. Na⁺/K⁺-ATPase expressed in the cultured cells showed a low affinity for K⁺ and a high affinity for Na⁺, characteristic of the silkworm nerve Na⁺/K⁺-ATPase. These results suggest that the β subunit is responsible for the affinity for K⁺ of Na⁺/K⁺-ATPase.     

Control of the Amiloride-Sensitive Na+ Current in Salivary Duct Cells by Extracellular Sodium

We have previously reported that intralobular salivary duct cells contain an amiloride-sensitive Na⁺ conductance (probably located in the apical membranes). Since the amiloride-sensitive Na⁺ conductances in other tight epithelia have been reported to be controlled by extracellular (luminal) Na⁺, we decided to use whole-cell patch clamp techniques to investigate whether the Na⁺ conductance in salivary duct cells is also regulated by extracellular Na⁺. Using Na⁺-free pipette solutions, we observed that the whole-cell Na⁺ conductance increased when the extracellular Na⁺ was increased, whereas the whole-cell Na⁺ permeability, as defined in the Goldman equation, decreased. The dependency of the whole-cell Na⁺ conductance on extracellular Na⁺ could be described by the Michaelis-Menten equation with a K _m of 47.3 mmol/1 and a maximum conductance (G _max) of 2.18 nS. To investigate whether this saturation of the Na⁺ conductance with increasing extracellular Na⁺ was due to a reduction in channel activity or to saturation of the single-channel current, we used fluctuation analysis of the noise generated during the onset of blockade of the Na⁺ current with 200 μmol/l 6-chloro-3,5-diaminopyrazine-2-carboxamide. Using this technique, we estimated the single channel conductance to be 4 pS when the channel was bathed symmetrically in 150 mmol/l Na⁺ solutions. We found that Na⁺ channel activity, defined as the open probability multiplied by the number of available channels, did not alter with increasing extracellular Na⁺. On the other hand, the single-channel current saturated with increasing extracellular Na⁺ and, consequently, whole-cell Na⁺ permeability declined. In other words, the decline in Na⁺ permeability in salivary duct cells with increasing extracellular Na⁺ concentration is due simply to saturation of the single-channel Na⁺ conductance rather than to inactivation of channel activity. Received: 27 July 1995/Revised: 7 December 1995     

Of cilia,titin, and neurosteroids

Missense mutations at arginine residues in the S4 voltage-sensor domains of Na_V1.4 are an established cause of hypokalemic periodic paralysis, an inherited disorder of skeletal muscle involving recurrent episodes of weakness in conjunction with low serum K⁺. Expression studies in oocytes have revealed anomalous, hyperpolarization-activated gating pore currents in mutant channels. This aberrant gating pore conductance creates a small inward current at the resting potential that is thought to contribute to susceptibility to depolarization in low K⁺ during attacks of weakness. A critical component of this hypothesis is the magnitude of the gating pore conductance relative to other conductances that are active at the resting potential in mammalian muscle: large enough to favor episodes of paradoxical depolarization in low K⁺, yet not so large as to permanently depolarize the fiber. To improve the estimate of the specific conductance for the gating pore in affected muscle, we sequentially measured Na⁺ current through the channel pore, gating pore current, and gating charge displacement in oocytes expressing R669H, R672G, or wild-type Na_V1.4 channels. The relative conductance of the gating pore to that of the pore domain pathway for Na⁺ was 0.03%, which implies a specific conductance in muscle from heterozygous patients of ∼10 µS/cm² or 1% of the total resting conductance.Unexpectedly, our data also revealed a substantial decoupling between gating charge displacement and peak Na⁺ current for both R669H and R672G mutant channels. This decoupling predicts a reduced Na⁺ current density in affected muscle, consistent with the observations that the maximal dV/dt and peak amplitude of the action potential are reduced in fibers from patients with R672G and in a knock-in mouse model of R669H. The defective coupling between gating charge displacement and channel activation identifies a previously unappreciated mechanism that contributes to the reduced excitability of affected fibers seen with these mutations and possibly with other R/X mutations of S4 of Na_V, Ca_V, and K_V channels associated with human disease.     

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.     

Effect of anisotonic media on volume,ion and amino-acid content and membrane potential of kidney cells (MDCK) in culture

Summary Effects of anisotonic media on a monolayer of confluent kidney cells in culture (MDCK) were studied by measuring: cell thickness and cross-section changes, ion and amino-acid content and membrane potential. The volume was also determined with cells in suspension. When cells in a monolayer were incubated in hypotonic media, the lateral and the apical membranes were rapidly stretched. Afterwards the lateral membranes returned to their initial state while the apical membranes remained stretched. This partial regulatory volume decrease (RVD) was verified with cells in suspension. RVD was accompanied by a loss of K⁺, Cl^– and amino acids, but there was no loss of inorganic phosphate. Also a transient hyperpolarization of the membrane potential was observed, suggesting an increase of the K⁺ conductance during RVD. Upon restoring the isotonic medium, a regulatory volume increase (RVI) was observed accompanied by a rapid Na⁺ and Cl^– increase and followed by a slow recovery of the initial K⁺ and Na⁺ content while amino acids remained at their reduced content. A transient depolarization of the membrane potential was measured during this RVI, suggesting that Na⁺ and Cl^– conductance could have increased. In hypertonic media, only a small and slow RVI was observed accompanied by an increase in K⁺ and Cl^– content but without any change of membrane potential. Quinine partly inhibited RVD in hypotonic media with cells in a monolayer while inhibiting RVD completely with cells in suspension. Incubation during four hours in a Ca²⁺ free medium had no effect on RVD. Furosemide and amiloride had no effect on RVD and RVI. Volume regulation, RVD or RVI, was not affected by replacing Cl^– by nitrate. When cells in a monolayer were incubated in a hypotonic K₂SO₄ medium, no RVD was observed. From these results, it seems that MDCK cells in a confluent monolayer regulate their volume by activating specific ion and amino-acid transport pathways. Selective K⁺ and Na⁺ conductances are activated during RVD and RVI, while the activated anion conductance has a low selectivity. The controlling mechanism might not be the free intracellular Ca²⁺ concentration.     

GABA and glutamate fluxes in developing nerve endings

Radiolabeled GABA and glutamate transport into 7 day, 14 day and adult cortical nerve ending preparations was examined. Transport was measured at several Na⁺ concentrations, 19, 27, 43 and 121 mM, and at two temperatures, 15 and 30°C. K_m and V_max values were calculated for all experimental conditions by means of Wilkinson (1961) analysis. A comparison of the day 14 and adult data shows higher K_m values at all Na⁺ concentrations on day 14 for both GABA and glutamate transport. In addition, the temperature dependence of transport was attenuated in the day 14 preparation. Finally, the specificity of GABA transport, as measured by the use of the transport inhibitors β-alanine and 2,4-diaminobutyric acid, was not different between the day 14 and adult preparations. Overall, it is concluded that both GABA and glutamate transport into day 14 nerve endings behave as if “adult” transporter molecules were existing in a more fluid lipid environment, which is the situation found in synaptic membranes prepared from day 14 nerve endings (Hitzemann and Johnson, 1983).Glutamate and GABA transport into 7 day nerve endings is complex and shows marked differences from the day 14 and adult data. Day 7 GABA transport was significantly more sensitive to β-alanine inhibition. Day 7 transport was more sensitive to Na⁺ manipulation and the temperature dependent kinetics show unique Na⁺ effects not seen in the day 14 or adult preparations. For example, at 19 mM Na⁺, 7 day glutamate transport was more temperature dependent than adult transport but as the Na⁺ concentration was increased the reverse was true. The opposite situation for temperature-Na⁺ effects was seen for GABA transport. Finally, no Ca⁺²-dependent component of GABA release could be found in 7 day nerve endings while a significant component was found at day 14. Overall, it is concluded that both glutamate and GABA fluxes in 7 day nerve endings differ both qualitatively from that seen in both day 14 and adult nerve endings.     

Ouabain switches Na+ transport to Na+/Na+ equivalent exchange in normal and apoptotic lymphoid cells

A theory of change of the ionic fluxes in the lymphoid cells in their transition from normal to apoptosis we have developed previously is applied to the analysis of Na⁺/Na⁺ exchange fluxes in human lymphoid cells U937 exposed to ouabain. We solve a system of equations describing changes in the intracellular concentrations of Na⁺, K⁺ and Cl^?, membrane potential and cell volume. It is shown that the Na⁺ influx (I _Na/Na) and output flux through the Na⁺/Na⁺ tract increased 4 times in 8 h after disconnecting Na⁺/K⁺-ATPase for normal cell U937. These fluxes increased 2.6 times for apoptotic cells. The value of I _Na/Na after 8 h off pump by ouabain is 97% of the total Na⁺ input for both cell types. It is concluded that ouabain not only inhibits the Na⁺/K⁺-ATPase, but also increases Na⁺ exchange fluxes through the Na⁺/Na⁺ tract, thereby switching sodium transport across the membrane of lymphoid cells to Na⁺/Na⁺ equivalent exchange.     

Ionic transporting systems of skeletal muscle in relation with innervation and their involvement in myotonic diseases

Excitation-contraction coupling describes the series of events that begins with propagated action potential on the muscle fiber surface membrane and leads to the twitch contraction of the fiber. The generation of an action potential during excitation requires rapid sequential changes in membrane conductances of Na⁺, Ca²⁺, and K⁺ ions that depend upon the opening and closing of the respective channels. Myotonic disorders are inherited diseases whose clinical manifestations include electrophysiological signs such as increased excitability and delayed relaxation of the muscles after voluntary contraction. All these disorders appears to be due to an abnormality of the muscle itself since they persist after section or blocking of the motor nerve after curarization. Most experimental and clinical data suggest that human myotonia arises from genetically-induced structural and functional alterations of the muscle membrane. The purpose of this article is to focus on the more recent developments in the molecular and pharmacological analysis of cation transporting systems such as ionic channels and (Na⁺, K⁺) ATPase in myotonic disorders.Special issue dedicated to Dr. Lawrence Austin.