Pre- and post-synaptic actions of the cerebral ventral giant cell on buccal muscles ofPhiline

Summary The cerebral ventral giant cell ofPhiline exterts a selective presynaptic inhibitory modulatory action on the terminals of a buccal excitatory motoneuron in two buccal muscles. Other excitatory inputs to the muscles are not affected. The ventral giant cell also makes direct synaptic contacts on the fibres of the same muscles. In the retractor muscle M4, 5 the junction potentials are usually depolarising when measured in sea water, but in the fibres of M6 they may have either polarity. The mean membrane potential of the fibres of M4, 5 and M6 was –74.7±0.65 mV and –64±0.95 mV respectively. Depolarization of the muscles fibres by around 15 mV by immersion in 20 mM K saline abolished the junction potential in M4, 5 and converted the depolarizing potential in M6 to a hyperpolarizing response.It is concluded that the VGC junction potential results from an increase in membrane conductance to an ion with a reversal potential between –60 and –70 mV.     

Microelectrode studies of the effect of lanthanum on the electrical potential and resistance of outer and inner cell membranes of isolated frog skin

Summary Microelectrodes were used to investigate the effect of 0.5mm mucosal lanthanum (La³⁺) on the intracellular potential and the resistance of outer and inner isolated frog skin (Rana esculenta) cell membranes. Under short-circuit conditions, the transapical membrane potentialV _o ^sc (mean value=–65.4±3.2 mV, inside negative) hyperpolarized to –108.7±2.3 mV in control skins, after addition of the sodium blocker amiloride. Current-voltage curves for the outer and inner membranes were constructed from the amiloride-inhibitable current versus the outer membrane potentialV _o or the inner membrane potentialV _t . The outer, and to a lesser degree the inner, membrane showed a characteristic nonlinearity with two slope resistances. Addition of La³⁺ to the outer medium increased the short-circuit current to 190% of the control value.V _o ^sc concomitantly changed to –28±3.5 mV and outer and inner membrane resistances fell, considerably attenuating the nonlinearity seen in control skins. La³⁺ is suggested to raise the conductance by its effect on the surface potential. A secondary long-term inhibitory effect of La³⁺ on short-circuit current has been observed. It is ascribed to the penetration of La³⁺ into the sodium channels.     

Electrical effects of potassium and bicarbonate on proximal tubule cells ofNecturus

Summary The effects of stepwise concentration changes of K⁺ and HCO ₃ ^– in the basolateral solution on the basolateral membrane potential (V _bl) of proximal tubule cells of the doubly-perfusedNecturus kidney were examined using conventional microelectrodes. Apparent transference numbers were calculated from changes inV _bl after alterations in external K⁺ concentration from 1.0 to 2.5mm (t _{K, 1.0–2.5}), 2.5 to 10, and in external HCO ₃ ^– concentration (at constant pH) from 5 to 10mm (t _{HCO3, 5–10}), 10 to 20, or 10 to 50.t _{K, 2.5–10} was 0.38±0.02 under control conditions but was sharply reduced to 0.08±0.03 (P>0.001) by 4mm Ba⁺⁺. This concentration of Ba⁺⁺ reducedV _bl by 9±1 mV (at 2.5 external K⁺). Perfusion with SITS (5×10^–4 m) for 1 hr hyperpolarizedV _bl by 10±3 mV and increasedt _{K, 2.5–10} significantly to 0.52±0.01 (P<0.001). Ba⁺⁺ application in the presence of SITS depolarizedV _bl by 22±3 mV. In control conditionst _{HCO3, 10–50} was 0.63±0.05 and was increased to 0.89±0.07 (P<0.01) by Ba⁺⁺ but was decreased to 0.14±0.02 (P<0.001) by SITS. In the absence of apical and basolateral chloride, the response ofV _bl to bicarbonate was diminished but still present (t _{HCO3, 10–20} was 0.35±0.03). Intracellular pH, measured with liquid ion-exchange microelectrodes, increased from 7.42±0.19 to 7.57±0.17 (P<0.02) when basolateral bicarbonate was increased from 10 to 20mm at constant pH. These data show that the effects of bicarbonate onV _bl are largely independent of effects on the K⁺ conductance and that there is a significant current-carrying bicarbonate pathway in the basolateral membrane. Hence, both K⁺ and HCO ₃ ^– gradients are important in the generation ofV _bl, and their relative effects vary reciprocally.     

Fused cells of frog proximal tubule: II. Voltage-dependent intracellular pH

Summary Experiments were performed in intact proximal tubules of the doubly perfused kidney and in fused proximal tubule cells ofRaha esculenta to evaluate the dependence of intracellular pH (pH_i) on cell membrane potential applying pH-sensitive and conventional microelectrodes. In proximal tubules an increase of the K^– concentration in the peritubular perfusate from 3 to 15 mmol/liter decreased the peritubular cell membrane potential from –55±2 to –38±1 mV paralleled by an increase of pH _i , from 7.54±0.02 to 7.66±0.02. The stilbene derivative DIDS hyperpolarized the cell membrane potential from –57 ± 2 to –71 ±4 mV and led to a significant increase of the K^–-induced cell membrane depolarization, but prevented the K^–-induced intracellular alkalinization. Fused proximal tubule cells were impaled by three microelectrodes simultaneously and cell voltage was clamped stepwise while pH _i changes were monitored. Cell membrane hyperpolarization acidified the cell cytoplasm in a linear relationship. This voltage-induced intracellular acidification was reduced to about one-third when HCO₃ ions were omitted from the extracellular medium. We conclude that in proximal tubule cells pH _i depends on cell voltage due to the rheogenicity of the HCO ₃ ^– transport system.     

Thiocyanate transport across fish intestine (Pleuronectes platessa)

Summary When bathed on both sides with identical chloride-containing salines thein vitro preparation of the plaice intestine maintains a negative (serosa to mucosa) short-circuit current of 107±11 A/cm², a transepithelial potential difference of 5.5±0.6 mV (serosa negative), and a mean mucosal membrane potential of –45.4±0.6 mV. Under these conditions the intracellular chloride activity is 32mm.If chloride in the bathing media is partially, or completely substituted by thiocyanate the measured electrical parameters do not change but transepithelial flux determinations show a reduction in chloride fluxes and the presence of a significant thiocyanate flux. The addition of piretanide (10^–4 m) reduced the short-circuit current and the mucosa-to-serosa fluxes of chloride and thiocyanate; this inhibition is similar to the effect of piretanide on chloride transport in this tissue.The results indicate that thiocyanate is transported in this tissue via the piretanide-sensitive chloride pathway and are compared with the effects of thiocyanate on other tissues reported in the literature.     

Conditioning prepulses and kinetics of potassium conductance in the frog node

Summary The kinetics of potassium conductance were analyzed in response to voltage-clamp steps with holding potential (–75 mV) as initial condition and after a positive prepulse to-wards +45 mV of 10-msec duration. As the potassium reversal potentialE _K altered during potassium current flow, a method to obtain the conductance independent ofE _K was used. Conductance kinetics at 15°C were analyzed according to the Hodgkin-Huxley (HH) model. The time constant of potassium activation, with holding potential as initial condition, is a monotonous decreasing function of membrane potential. Its value ofca. 9 msec at –50 mV decreases to 1 msec at +30 mV. Changes inE _K did not affect the voltage dependency of this time constant. The time constant of potassium deactivation, i.e. the off-response following a 10-msec prepulse towards +45 mV, shows a completely different voltage dependency. At a membrane potential of –90 mV it is approximately 2 msec and gradually increases for more positive voltages towards a maximum value of about 6 msec, that is reached between –5 and 0 mV. At still larger values of membrane voltage this time constant starts to fall again. It is concluded that a HH-model, as applied for a single population of potassium channels, has to be rejected. Computer simulations indicate that an extension to two populations of independent potassium channels, each with HH-kinetics, is also inconsistent with the observed results.     

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.     

Comparison of the pacemaker properties of chick embryonic atrial and ventricular heart cells

Summary We have investigated the pacemaker properties of aggregates of cells dissociated from the atria and ventricles of 10 to 14-day-old chick embryonic hearts using a two-microelectrode current and voltage-clamp technique. These preparations usually beat spontaneously and rhythmically in tissue culture medium containing 1.3mm potassium with a beat rate typically in the range of 15–60 beats per minute. The beat rate results show considerable variability, which precludes any statistically significant comparison between the spontaneous activity of atrial and ventricular cell preparations at 10–14 days of development. However, the shapes of pacemaker voltage changes do exhibit differences characteristic of cell type. Spontaneous atrial preparations rapidly depolarize from maximum diastolic potential (–90 mV) to a plateau range of pacemaker potentials (–80 to –75 mV). The membrane subsequently depolarizes more gradually until threshold (–65 mV) is reached. In contrast, spontaneously beating ventricular cell preparations slowly hyperpolarize after maximum diastolic potential to the –100 to –95 mV range before gradually depolarizing toward threshold. Voltage-clamp analysis reveals a virtual lack of any time-dependent pacemaker current in atrial preparations. These preparations are characterized by an approximately linear background current (I _bg) having a slope resistance of 100 K cm². Ventricular preparations have a potassium ion pacemaker current with slow kinetics (I _{K 2}), and a second time-dependent component (I _x) which is activated at potentials positive to –65 mV. The background current of these preparations displays inward rectification. Computer simulations of pacemaking reveal that the initial rapid phase of pacemaker depolarization in atrial cells is determined by the membrane time constant, which is the product of membrane capacitance and the slope resistance ofI _bg. The hyperpolarization after maximum diastolic potential of ventricular cells is caused byI _{K 2}. The final slow phase of depolarization in both cell types is caused in part by the steady-state amplitude of the fast inward sodium current (I _Na). This component has negative slope conductance which effectively increases the slope resistance in the vicinity of threshold compared to TTX-treated preparations. This mechanism is sufficient to produce interbeat intervals several seconds in duration, even in the absence of time-dependent pacemaker current, provided that the background current is at the appropriate level.     

Electrophysiologic changes associated with potassium depletion of frog skin

Summary Skins from the frogRana pipiens pipiens were studied under short-circuited conditions during the course of removing and replacing potassium in the inner bathing media in 14 experiments. The intracellular potential (V _SC), fractional resistance (FR), short-circuit current (I _SC) and total tissue conductance (g _T) were constantly monitored during impalements of the epithelial cells. The mean value (±se) forV _SC was –79 (±3) mV under baseline conditions. Removal of potassium from the inner bathing solution transiently stimulated the short-circuit current and hyperpolarized the basolateral membrane; with sufficiently long incubations, the basolateral membrane was eventually depolarized. Restoration of potassium to the inner solution within 43 min after initiating the perfusion with K⁺-free solution depolarized the basolateral membrane. However, restoration of potassium after at least 11/2 hr of incubation hyperpolarized the membrane. Ouabain consistently depolarized the basolateral membrane, even after extended periods of potassium depletion as long as 320 min. In the presence of ouabain, restoration of potassium depolarized the basolateral membrane. The data provide further evidence for the concept that the Na–K exchange pump of frog skin is rheogenic. Furthermore, the results suggest that the pump continues to be active even during prolonged periods of potassium depletion, reaccumulating potassium which has leaked out of the epithelial cells.     

Intracellular potential and K+ activity in rat kidney proximal tubular cells in acidosis and K+ depletion

Summary Techniques were developed for the measurement of intracellular potentials and potassium activities in rat proximal tubule cells using double barreled K⁺ liquid-ion-exchanger microelectrodes. After obtaining measurements of stable and reliable control values, the effects of K⁺ depletion and metabolic and respiratory acidosis on the intracellular potential and K⁺ activity in rat kidney proximal tubular cells were determined. At a peritubular membrane potential of –66.3±1.3 mV (mean±se), intracellular K⁺ activity was 65.9±2.0 mEq/liter in the control rats. In metabolic acidosis [70 mg NH₄ Cl/100 g body wt) the peritubular membrane potential was significantly reduced to –47.5±1.9 mV, and cellular K⁺ activity to 53.5±2.0 mEq/liter. In contrast, in respiratory acidosis (15% CO₂) the peritubular membrane potential was significantly lowered to –46.1±1.39 mV, but the cellular K⁺ activity was maintained at an almost unchanged level of 63.7±1.9 mEq/liter. In K⁺ depleted animals (6 weeks on low K⁺ diet), the peritubular membrane potential was significantly higher than in control animals, –74.8±2.1 mV, and cellular K⁺ activity was moderately but significantly reduced to 58.1±2.7 mEq/liter. Under all conditions studied, cellular K⁺ was above electrochemical equilibrium. Consequently, an active mechanism for cellular K⁺ accumulation must exist at one or both cell membranes. Furthermore, peritubular HCO₃ ^– appears to be an important factor in maintaining normal K⁺ distribution across the basolateral cell membrane.     

Intracellular chloride activity of leech neurones and glial cells in physiological,low chloride saline

Leech blood apparently contains considerably less chloride than generally used in physiological experi ments. Instead of 85–130 mM Cl^– used in experimental salines, leech blood contains around 40 mM Cl^– and up to 45 mM organic anions, in particular malate. We have reinvestigated the distribution of Cl^– across the cell membrane of identified glial cells and neurones in the central nervous system of the leech Hirudo medicinalis L., using double-barrelled Cl^–- and pH-selective micro electrodes, in a conventional leech saline, and in a saline with a low Cl^– concentration (40 mM), containing 40 mM malate. The interference of anions other than Cl^–to the response of the ion-selective microelectrodes was estimated in Cl^–-free salines (Cl^– replaced by malate and/or gluconate). The results show that the absolute intracellu lar Cl^– activities (aCl_i) in glial cells and neurones, but not the electrochemical gradients of Cl^– across the glial and the neuronal cell membranes, are altered in the low Cl^–, malate-based saline. In Retzius neurones, aCl_i is lower than expected from electrochemical equilibrium, while in pressure neurones and in neuropil glial cells, aCl_i is distributed close to its equilibrium in both salines, re spectively. The steady-state intracellular pH values in the glial cells and Retzius neurones are little affected (0.1 pH units) in the low Cl^–, malate-based saline.     

Glass microelectrode measurements of sieve tube membrane potentials in internode discs and petiole strips of tomato (Solanum lycopersicum L.)

Summary In tissue slices of tomato (Solanum lycopersicum L.) sieve tube membrane potentials (E_m) were measured by use of glass microelectrodes. In internode discs, the potential differences (pd) of phloem cells near the cut surface fell into two distinct categories with average values of –66 and –109 mV. More distant from the cut surface the values decreased to averages of –71 and –140 mV. These pds were associated with phloem parenchyma cells and sieve tube/companion cell complexes, respectively. In petiole strips, pds were recorded from cells which were identified by iontophoretic injection of fluorescent dye. Averages in two different bathing media, were –140/–146mV, –149/–152mV, and –70/–68mV for sieve tubes, companion cells, and phloem parenchyma cells, respectively. The membrane potentials recorded from sieve tubes were transiently reduced upon sucrose addition. Reduction by CCCP and KCN was more permanent. Sieve tube E_ms recovered more slowly from potassium than from sucrose-induced depolarizations. Light/ dark (L/D) responses were minute (±3 mV). The limitations of the present experimentation are evaluated with special reference to the question as whether the recorded E_ms represent sieve tube membrane potentials occurring in the intact plant.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - D dark(ness) - E_m membrane potential - L light - LYCH Lucifer yellow CH - pd potential difference - SE standard error     

Effects of the anion transport inhibitor,SITS, on the proximal straight tubule of the rabbit perfusedin vitro

Summary Conventional microelectrodes were used to study the effects of SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonate) on the basolateral membrane potentialVbl of the superficial proximal straight tubule (PST) of the rabbit kidney perfusedin vitro. Addition of 0.1mm SITS to the bathing solution resulted in a slow and irreversible hyperpolarization ofVbl from –42.5±1.17 (37) mV to –77.3±0.83 (52) mV. The new steady-state potential was reached in 10 to 15 min and was accompanied by visible cell swelling. Associated with thisVbl hyperpolarization was: 1) an increased steady-state depolarization (from 6.2±0.77 (17) mV to 25.7±0.83 (29) mV) in response to increasing bath potassium concentration from 5 to 16.7mm (HK); 2) a decreased transient depolarization (from 19.8±1.88 (8) mV to 0.43±0.37 (8) mV) in response to decreasing bath bicarbonate concentration from 22 to 6.6mm at constant bath pH (L-HCO₃); and 3) inhibition of a depolarizing overshoot and a decreased steady-state depolarization (from 35.9±1.84 (12) mV to 4.7±1.37 (13) mV) in response to reducing bath sodium concentration from 144 to zero (0-Na). Sodium, chloride and NMDG (N-methyl-d-glucamine) were used as the substituting ions, respectively. These results are consistent with the presence of a coupled sodium-bicarbonate carrier in the basolateral membrane which is electrogenic and SITS inhibitable. Comparison of the time course of SITS effects on these ion-substitution responses suggests that the inhibition of the bicarbonate exit pathway(s) is the primary event and that the changes inVbl and in the steady-stateVbl responses to HK and 0-Na are secondary events which may be related to changes in intracellular composition and/or basolateral membrane properties.     

Membrane properties of identified embryonic nerve and glial cells of the leech central nervous system

Summary The membrane potential of identified nerve (Retzius) cells and neuropil glial cells from 11 (±1) day-old embryos of the leechHirudo medicinalis was recorded using conventional intracellular microelectrodes. At this stage all ganglia of the segmental nervous system are formed. The membrane potential of Retzius cells was –68±4 mV (±SD,n=8), and showed a slope of 42 mV between 10 mM and 100 mM external K concentration. Retzius cells were able to fire action potentials which had a fast Na-dependent component, and, under appropriate conditions, also generated slow Ca (Ba) action potentials. The mean membrane potential of the neuropil glial cell at physiological K concentration (4 mM) was –83±5 mV (±SD,n=10), and showed a dependence of 56 mV for a tenfold change in the external K concentration (> 4mM). Neuropil glial cells showed no signs of voltage-activated excitability, but they repeatedly depolarized in the presence of 0.1 mM 5-HT.     

Electrical characteristics of stomatal guard cells: The contribution of ATP-dependent, “Electrogenic” transport revealed by current-voltage and difference-current-voltage analysis

Summary The steady-state, current-voltage (I–V) characteristics of stomatal guard cells fromVicia faba L. were explored by voltage clamp using conventional electrophysiological techniques, but with double-barrelled microelectrodes containing 50mm K⁺-acetate. Attention was focused, primarily, on guard cell response to metabolic blockade. Exposures to 0.3–1.0mm NaCN and 0.4mm salicylhydroxamic acid (SHAM) lead consistently to depolarizing (positive-going) shifts in guard cell potentials (V _m ), as large as +103 mV, which were generally complete within 60–90 sec (mean response half-time, 10.3±1.7 sec); values forV _m in NaCN plus SHAM were close or positive to –100 mV and well removed from the K⁺ equilibrium potential. Guard cell ATP content, which was followed in parallel experiments, showed a mean half-time for decay of 10.8±1.9 ([ATP] _t=0, 1.32±0.28mm; [ATP] _{t=60–180sec}, 0.29±0.40mm). In respiring cells, theI–V relations were commonly sigmoid aboutV _m or gently concave to the voltage axis positive toV _m . Inward- and outward-rectifying currents were also observed, especially near the voltage extremes (nominally –350 and +50 mV). Short-circuit currents (atV=0 mV) were typically about 200–500 mA m^–2. The principal effect of cyanide early on was to linearize theI–V characteristic while shifting it to the right along the voltage axis, to decrease the membrane conductance, and to reduce the short-circuit current by approx. 50–75%. The resulting difference-current-voltage (dI–V) curves (±cyanide) showed a marked sensitivity to voltages negative from –100 mV and, when clamp scans had been extended sufficiently, they revealed a distinct minimum near –300 mV before rising at still more negative potentials. The difference currents, along with changes in guard cell potential, conductance and ATP content are interpreted in context of a primary, ATP-consuming ion pump. FittingdI–V curves to reaction kinetic model for the pump [Hansen, U.-P., et al. (1981)J. Membrane Biol. 63:165; Blatt, M.R. (1986)J. Membrane Biol. 92:91] implicates a stoichiometry of one (+) charge transported outward for each ATP hydrolyzed, with pump currents as high as 200 mA m^–2 at the free-running potential. The analysis indicates that the pump can comprise more than half of the total membrane conductance and argues against modulations of pump activity alone, as an effective means to controlling K⁺ transport for stomatal movements.     

Studies on the perfused plasmalemma ofChara corallina: I. Current-voltage curves: ATP and potassium dependence

Summary The electrical properties of theChara cell membrane have been studied using a perfusion method based on that of Williamson, R.E. 1975.J. Cell Sci. 17655. The vacuole, tonoplast, and inner cytoplasm are removed by a brief rapid perfusion. Electrical properties of the plasmalemma indicate that it remains intact after this perfusion.The membrane potential difference after perfusion and with no ATP was close to the potassium equilibrium potential; the current-voltage characteristic had a slope that was time- and voltage-dependent, indicating that the steady-state potassium conductance increased with depolarization. At –125 mV the membrane conductance of the plasmalemma depended on [K⁺]₀. This dependence was inhibited by perfusing with 2.0mm ATP or by clamping at a more negative membrane potential. The addition of ATP to the perfusion medium of unclamped cells caused a hyperpolarization ofca. 50 mV, presumably by activating the proton pump. In clamped cells, perfusion with ATP caused currents ofca. 20 mA m^–2, whose magnitude depended on pH₀. ATP induced membrane conductance changes which were variable. 2.0mm ADP inhibited the proton pump. The intersection points of current-voltage characteristics can set limits on the stalling potential; the resulting stoichiometry of the proton pump appears to be 1.5–2.0 H⁺ per ATP.     

Effect of HgCl2 on acetylcholine,carbachol, and glutamate currents ofAplysia neurons

Summary 1. Using conventional two-microelectrode voltage-clamp techniques we studied the effects of inorganic mercury (HgCl₂) on acetylcholine-, carbachol-, and glutamate-activated currents onAplysia neurons. Hg²⁺ was applied with microperfusion.2. Acetylcholine and carbachol activated an inward, sodium-dependent current in the anterior neurons of the pleural ganglion. The medial neurons gave a biphasic current to acetylcholine and carbachol, which was outward at resting membrane potential. The faster component was Cl^– dependent and reversed at about –60 mV, while the slower component was K⁺ dependent and reversed at greater than –80 mV.3. Hg²⁺ (0.1–10 µM) caused a dramatic increase in the acetylcholine- and carbachol-induced inward current in anterior neurons and the fast Cl^– current in medial neurons. With only a 1-min preapplication of Hg²⁺, the acetylcholine- or carbachol-activated sodium or chloride currents were increased to 300% and the effect was only partly reversible. The threshold concentration was 0.1 µM Hg²⁺.4. Contrary to the effects on sodium and chloride currents, concentrations of 0.1–10 µM Hg²⁺ caused a complete and irreversible blockade of K⁺-dependent acetylcholine and carbachol currents. The block of the potassium current was relatively fast and increased with time. The concentration of HgCl₂ that gave a half-maximal blockade of the carbachol-activated potassium current was 0.89 µM. The chloride-dependent current elicited by glutamate on medial neurons was increased by HgCl₂ as well.5. These results suggest that actions at agonist-activated channels must be considered as contributing to mercury neurotoxicity. It is possible that the toxic actions of Hg²⁺ on synaptic transmission at both pre- and postsynaptic sites are important factors in the mechanism of Hg²⁺ toxicity.     

Intracellular potassium activity and the role of potassium in transepithelial salt transport in the human reabsorptive sweat duct

Summary We have measured the intracellular potassium activity, [K⁺]_i and the mechanisms of transcellular K⁺ transport in reabsorptive sweat duct (RSD) using intracellular ion-sensitive microelectrodes (ISMEs). The mean value of [K⁺]_i in RSD is 79.8±4.1mm (n=39). Under conditions of microperfusion, the [K⁺]_i is above equilibrium across both the basolateral membrane, BLM (5.5 times) and the apical membrane, APM (7.8 times). The Na⁺/K⁺ pump inhibitor ouabain reduced [K⁺]_i towards passive distribution across the BLM. However, the [K⁺]_i is insensitive to the Na⁺/K⁺/2 Cl^– cotransport inhibitor bumetanide in the bath. Cl^– substitution in the lumen had no effect on [K⁺]_i. In contrast, Cl^– substitution in the bath (basolateral side) depolarized BLM from –26.0±2.6 mV to –4.7^*±2.4 mV (n=3;^* indicates significant difference) and decreased [K⁺]_i from 76.0±15.2mm to 57.7^* ±12.7mm (n=3). Removal of K⁺ in the bath decreased [K⁺]_i from 76.3±15.0mm to 32.3^*±7.6mm (n=4) while depolarizing the BLM from –32.5±4.1 mV to –28.3^*±3.0 mV (n=4). Raising the [K⁺] in the bath by 10-fold increased [K⁺]_i from 81.7±9.0mm to 95.0^*±13.5mm and depolarized the BLM from –25.7±2.4 mV to –21.3^*±2.9 mV (n=4). The K⁺ conductance inhibitor, Ba²⁺, in the bath also increased [K⁺]_i from 85.8±6.7mm to 107.0^*±11.5mm (n=4) and depolarized BLM from –25.8±2.2 mV to –17.0^*±3.1 mV (n=4). Amiloride at 10^–6 m increased [K⁺]_i from 77.5±18.8mm to 98.8^*±21.6mm (n=4) and hyperpolarized both the BLM (from –35.5±2.6 mV to –47.8^*±4.3 mV) and the APM (from –27.5±1.4 mV to –46.0^* ±3.5 mV,n=4). However, amiloride at 10^–4 m decreased [K⁺]_i from 64.5±0.9mm to 36.0^*±9.9mm and hyperpolarized both the BLM (from –24.7±1.4 mV to –43.5^*±4.2 mV) and APM (from –18.3±0.9 mV to –43.5^*±4.2 mV,n=6). In contrast to the observations at the BLM, substitution of K⁺ or application of Ba²⁺ in the lumen had no effect on the [K⁺]_i or the electrical properties of RSD, indicating the absence of a K⁺ conductance in the APM. Our results indicate that (i) [K⁺]_i is above equilibrium due to the Na⁺/K⁺ pump; (ii) only the BLM has a K⁺ conductance; (iii) [K⁺]_i is subject to modulation by transport status; (iv) K⁺ is probably not involved in carrier-mediated ion transport across the cell membranes; and (v) the RSD does not secrete K⁺ into the lumen.     

Electrophysiological properties ofDictyostelium derived from membrane potential measurements with microelectrodes

Summary Electrical membrane properties of the cellular slime moldDictyostelium discoideum were investigated with the use of intracellular microelectrodes. The rapid potential transients (1 msec) upon microelectrode penetration of normal cells had a negative-going peak-shaped time course. This indicates that penetration of a cell with a microelectrode causes a rapid depolarization, which can just be recorded by the microelectrode itself. Therefore, the initial (negative) peak potential transient valueE _p (–19 mV) should be used as an indicator of the resting membrane potentialE _m ofD. discoideum before impalement, rather than the subsequent semistationary depolarized valueE _n (–5 mV). Using enlarged cells such as giant mutant cells (E _p=–39 mV) and electrofused normal cells (E _p=–30 mV) improved the reliability ofE _p as an indicator ofE _m. From the data we concluded thatE _m ofD. discoideum cells bathed in (mm) 40 NaCl, 5 KCl and 1 CaCl₂ is at least –50 mV. This potential was shown to be dependent on extracellular potassium. The average input resistanceR _i of the impaled cells was 56 M for normalD. discoideum. However, our analysis indicates that the membrane resistance of these cells before impalement is >1 G. Specific membrane capacitance was 1–3 pF/cm². Long-term recording of the membrane potential showed the existence of a transient hyperpolarization following the rapid impalement transient. This hyperpolarization was associated with an increase inR _i of the impaled cell. It was followed by a depolarization, which was associated with a decrease inR _i. The depolarization time was dependent on the filling of the microelectrode. The present characterization of the electrical membrane properties ofDictyostelium cells is a first step in a membrane electrophysiological analysis of signal transduction in cellular slime molds.     

Control of membrane potential by external H concentration in Bacillus subtilis as determined by an ion-selective electrode

The membrane potential of intact bacteria was monitored by measuring the tetraphenylphosphonium ion distribution across the membrane using poly-(vinyl chloride) matrix-type electrode selective to tetraphenylphosphonium ion. It was found that the tetraphenylphosphonium ion was not countertransported against H⁺ movement. The membrane potential of Bacillus subtilis was estimated to be 80–120 mV inside-negative at external pH 7. The effect of the external pH on the membrane potential was studied. It varied from 30 to 40 mV/decade change in the external [H⁺] in the pH region of greater than 6.5, increasing pH making it more inside-negative. The addition of carbonyl cyanide m-chlorophenylhydrazone depolarized the membrane, and the membrane potential approached the H⁺ equilibrium potential. The addition of N,N′-dicyclohexylcarbodiimide did not abolish the pH dependence of the membrane potential. Increasing the external [K⁺] did not affect the pH dependence. CN⁻ partially depolarized the membrane. A parallel conductance model for membrane potential could explain the results qualitatively.