Voltage-dependent effect of tetraethylammonium on the nicotinic acetylcholine receptors of rat superior cervical ganglion neurons

The effect of tetraethylammonium (TEA) on the currents evoked in neurons of the rat superior cervical ganglion by iontophoretic application of acetylcholine (ACh) was studied using a whole-cell patch-clamp recording technique. Tetraethylammonium was used at a concentration of about 20 µM, providing no blocking effect on the ACh-induced membrane currents at a range of positive membrane potentials and reducing these currents recorded at a range of negative membrane potentials by about half. The blocking effect of TEA increased with hyperpolarization within the –50 to –90 mV membrane potential range, and did not depend on the membrane potential level within a range of 0 to –50 mV. The analysis of dose dependence showed that both the voltage-dependent and the voltage-independent blocking effects are due to TEA competitive action on the ganglionic nicotinic acetylcholine receptors (nAChR). The results suggest that the TEA-induced competitive blockade is voltage-dependent.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 63–66, January–February, 1995.     

Blocking of neuronal nicotinic acetylcholine receptors with d-sparteine derivatives

The effects of d-sparteine (d-SP) and its two derivatives, N-methylsparteine (IEM-1820) and N-phenylsparteine (IEM-1821), on nicotinic acetylcholine receptors (nAChR) of the rat superior cervical ganglion neurons were studied. Membrane currents evoked by iontophoretically applied acetylcholine were recorded using the patch-clamp recording technique in the whole-cell configuration. All three compounds were found to block nAChR competitively, the blocking activity being increased with an increase in the size of the blocking molecule. The EC₅₀ values for d-SP, IEM-1820, and IEM-1821 were equal to 2.06±0.38 µM (n=3), 1.64±0.41 µM (n=4), and 0.65±0.17 µM (n=3), respectively. It was assumed that the increase in efficiency of blocking is related to the decrease in the rate of dissociation of the blocker and receptor molecules.Neirofiziologiya/Neurophysiology, Vol. 26, No. 4, pp. 266–269, July–August, 1994.     

Mechanisms of tubocurarine action on nicotinic cholinoreceptors of rat sympathetic ganglia neurons

The effects of tubocurarine (TC) on current induced by acetylcholine (ACh) in neurons of rat upper cervical ganglia were investigated using techniques for voltage-clamping at the membrane. Reinforcement of TC-induced blockade was achieved by paired application of ACh following prior activation of nicotinic cholinoreceptors, indicating that TC blocked the channels opened by ACh. On average, the TC-open channel complex persisted for 9.8±0.5 sec (n=7) at –50 mV and 20–24°C. It was found that increases exponentially with hyperpolarization at the membrane (a shift in membrane potential of 61 mV corresponds to an e-fold change). Suppression of ACh-induced current (ACh current) was eliminated completely under the effects of 3–30 M with depolarization of up to 80–100 mV at the membrane. Suppression of ACh current produced by membrane potential at negative levels is intensified with increasing doses of ACh. Findings would indicate that blockade of ionic channels opened by ACh is the only mechanism of TC action on nicotinic cholinoreceptors in rat sympathetic ganglia.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 5, pp. 672–680, September–October, 1988.     

Effects of (±)-kavain on inactivation of voltage-operated Na+ channels

Effects of a kava-pyrone (±)-kavain on fast inactivation of Na⁺ channels were studied in experiments on isolated neurons from the rat hippocampus. (±)-Kavain was found to block Na⁺ channels, and its effect was voltage-dependent. At the holding potentials of –100 and –80 mV, IC₅₀ for (±)-kavain was 744.9 and 178.8 µM, respectively. The inactivation characteristic of Na⁺ channels was satisfactorily described with the Boltzmann's equation both in the control and under (±)-kavain application. (±)-Kavain at a 330 µM concentration shifted theV _1/2 toward more negative values by 14.4 mV and concurrently modified the slope factor: the latter was 5.7 mV in the control, while under the influence of 330 µM (±)-kavain it reached 6.7 mV. In agreement with Hille's hypothesis of a modulated receptor, inactivated Na⁺ channels demonstrated an increased sensitivity to kavain. (±)-Kavain effects resulted in an increase in the rate of depolarization-related fast inactivation, while the process of recovery from inactivation became slower when the membrane was hyperpolarized. Our data show that under the (±)-kavain effect the probability of the inactivated state of Na⁺ channels increases, and the state of fast inactivation is stabilized.Neirofiziologiya/Neurophysiology, Vol. 28, No. 4/5, pp. 218–224, July–October, 1996.     

Voltage dependence of the effects of (±)-kavain on the rate of inactivation of Na+ channels

The dependence of the efficacy of the influence of a kava-pyrone (±)-kavain (330 µM) on the frequency of activation of Na⁺ channels and voltage dependence of the effects of (±)-kavain on the rate of inactivation of these channels were studied in experiments on isolated neurons from the rat hippocampus. In all series of experiments, the holding potential equalled –100 mV. The efficacy of (±)-kavain-induced blockade of Na⁺ channels was independent of the frequency of stimulation within the range up to 10/sec. In the control experiments, the rate of inactivation increased with the rise of depolarization from –40 mV to +30 mV, and then the saturation effect was observed. At the membrane potential of –40 mV, the rate of (±)-kavain-evoked inactivation increased approximately by a factor of 2.5. At the more positive shifts of the membrane potential, the efficacy of the effects of (±)-kavain on the rate of inactivation became noticeably reduced, and at +30 mV (±)-kavain exerted no distinct influence on this parameter.Neirofiziologiya/Neurophysiology, Vol. 28, No. 6, pp. 312–315, November–December, 1996.     

Properties of the kainate channel in rat brain mRNA injected Xenopus oocytes: ionic selectivity and blockage

The properties of kainate receptor/channels were studied in Xenopus oocytes injected with mRNA that was isolated from adult rat striatum and cerebellum and partially purified by sucrose gradient fractionation. Kainate (3–1000 µ.M) induced a smooth inward current that was competitively inhibted by gamma-D-glutamyl-aminomethanesulfonate (GAMS, 300 µM). In striatal mRNA-injected oocytes, the kainate current displayed nearly linear voltage-dependence and mean reversal potential (E_r) of -6.1 ± 0.5 mV In cerebellar mRNA-injected oocytes; E_r was nearly identical (-5.1 ± 1.2 mV) but there was marked inward rectification of the kainate current. Ion replacement studies reveal that the kainate channel is selective for cations over anions, but relatively non-selective among small monovalent cations. Large monovalent cations such as tetrabutylammonium are impermeant and induce a non-competitive block of kainate current that is strongly voltage-dependent. Divalent cations are relatively impermeant in the kainate channel and Cd⁺⁺ and other polyvalent metals were shown to block kainate current by a mechanism that is only weakly voltage-dependent. A model of the kainate channel is proposed based upon these observations.     

Differentiated expression inXenopus oocytes of calcium channels from rat cerebellar and forebrain neurons

Calcium channels were expressed inXenopus laevis oocytes by means of matrix RNA (mRNA) extracted from the cerebellum (RNAc) and forebrain (RNAfb). In these oocytes, inward barium currents,I _Ba, evoked by 40 mM Ba²⁺ were investigated using a double microelectrode technique. Currents expressed after injection of both RNAc and RNAfb (further referred to as RNAc- and RNAfb-expressed currents) showed a voltage-dependent characteristic typical of high-threshold calcium channels of mammalian neurons. The threshold of activation was about –40 mV, the maximum amplitude was observed at +20 mV and reversal potential at +60 mV. In both groups of oocytes, no expression of low- or high-threshold calcium channels of other types was observed. Although in both cases the expression ofI _Ba had similar macrokinetics, characteristics of their stationary inactivation differed. The half-inactivation potential ranged between –32 and –16 mV, and the slope factor was 28 and 16.6 mV in RNAfb- and RNAc-injected oocytes, respectively. In both cases,I _Ba were insensitive to dihydropyridines; however their relation to other pharmacological agents was different. RNAfb-expressedI _Ba was completely blocked by Cd²⁺ (K _d=10 µM) and depressed up to 70% by -conotoxin (1 µM), being insensitive to either whole spider toxin fromAgelenopsis aperta venom or to its FTX fraction. On the contrary, RNAc-expressedI _Ba was more sensitive to Cd²⁺ (K _d=0.1 µM), stable to -conotoxin, and suppressed up to 75–90% by wholeA. aperta toxin in a dilution of 1:10000, and by FTX at a concentration of 0.5 µM. The findings allow us to suggest that the forebrain and cerebellum of mammals are the structures, whose mRNA differ and provide predominant expression of voltage-dependent calcium channels of N- and P-types, respectively.Neirofiziologiya/Neurophysiology, Vol. 26, No. 6, pp. 427–436, November–December, 1994.     

Argiopine,argiopinines, and pseudoargiopinines as glutamate receptor blockers in hippocampal neurons

A homologous set of low-molecular weight compounds selectively blocking ionic currents were purified from venom from the spiderArgiope lobata with a selective blocking action on ionic currents activated by applying glutamate and its agonist kainic acid (KA) to the membrane of neurons isolated from the rat hippocampus. Three groups of these compounds — argiopine, argiopinines, and pseudoargiopinines, produced voltage-dependent glutamate- and KA-activated ionic currents at concentrations of 10^–6-10^–4 M, interacting primarily with agonist-activated ionic channels without affecting K_d values of the agonist. The blocking action could be partially reversed by argiopine application but only slightly when argiopinines and pseudoargiopinines were used. Kinetics of toxin effects on Ka-activated ionic currents showed at least two exponential components with different time constants. Simple and reversed rate constants of interaction between toxins and ionic channels were estimated from the plot of the kinetics of ionic current blockade and recovery against toxin concentration. Argiopine, argiopinines, and pseudoargiopinines lend themselves to further research into glutamate receptors of the mammalian CNS employing electrophysiological and biochemical techniques.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev, M. M. Shemyakin Institute of Bioorganic Chemistry, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 748–756, November–December, 1989.     

Blockade of cardiac outwardly rectifying K+ channels by TEA and class III antiarrhythmics — evidence against a single drug-sensitive channel site

Elementary K⁺ currents through cardiac outwardly rectifying K⁺ channels were recorded in insideout patches excised from cultured neonatal rat cardiocytes at 19 °C and at 9 °C. By studying the inhibitory effects of tetraethylammonium (TEA), quinidine and verapamil, the properties of this novel type of K⁺ channel were further characterized. Internal TEA (50 mmol/1) evoked a reversible decline of i_unit to 62.7 + 2.7% of control (at –7 mV), without significant changes of open state kinetics, indicating a blockade of the open K⁺ pore with kinetics too fast to be resolvable at 1 kHz. This TEA blockade was e-fold voltage-dependent, with a decrease of the apparent K_{D( TEA)} from 102 mmol/1 at –37 mV to 65 mmol/1 at +33 mV and, furthermore, became accentuated on lowering the internal K⁺ concentration. Thus, TEA competes with the permeant K⁺ for a site located in some distance from the cytoplasmic margin, within the K⁺ pore. Quinidine (100 mol/l), like verapamil (40 mol/1) reversibly depressed i_unit to about 80% of the control value (at –7 mV), but drug-induced fast flicker blockade proved voltage-insensitive between –27 mV and +23 mV These drugs gain access to a portion of the pore distinct from the TEA binding site whose occupancy by drugs likewise blocks K⁺ permeation. Both drugs showed a greater potency to depress P_o which, with quinidine,decreased reversibly to38.6 ± 11.1% (at –7 mV) and, with verapamil to 24.9 ± 9.1%(at –7 mV), mainly by an increase of the prolonged closed state (C,). This alteration of the gating process also includes a sometimes dramatic shortening of the open state. Most probably, cardiac K_{(outw.-rect.)} ⁺K⁺ _outw.-rect. channels possess a second drug-sensitive site whose occupancy by quinidine or verapamil may directly or allosterically stabilize their non-conducting configuration. Correspondence to: M. Kohlhardt     

Mechanism Underlying the Effect of Mecamylamine on Nicotinic Acetylcholine Receptors of Rat Sympathetic Ganglion Neurons

On neurons of the superior cervical ganglion of 3-week-old rats, we studied the mechanism underlying the blocking effect of mecamylamine on transmembrane currents evoked by iontophoretic application of acetylcholine (ACh currents); these currents were recorded with the use of a patch-clamp technique in the whole-cell configuration. The IC₅₀ of the above agent equaled (2.7 ± 0.3) · 10^-10 M. The blocking effect of mecamylamine on ACh current did not depend on the membrane potential and decreased with rise in the concentration of the drug. Thus, a competitive blocking mechanism mostly underlies the above phenomenon.     

ATP-induced currents in submucous plexus neurons of the guinea pig small intestine

ATP-induced membrane durrents in the submucous neurons of the guinea pig small intestine were studied using the whole-cell patch-clamp recording technique. Being applied at –50 mV. ATP activated an inward non-selective cationic current in 68.3% of the investigated neurons. An increase in ATP concentration within the 1–1,000 µM range resulted in the s-like increase in the amplitude of ATP-induced current. The EC₅₀ was 150.0±18.5 µM, while the Hill number was 1.6. The current was selectively activated by ATP and was not blocked by P2 purinoreceptor antagonist suramin (50–300 µM).,-Methylene-ATP (100–200 µM) and,-methylene-ATP (100–200µM), which are P2-purinoreceptor agonists, as well as adenosine (100–300 µM), exerted no effects. Reactive blue 2, if applied up to 4 min, enhanced ATP-induced current, while its longer application partially suppressed this current. In most submucous neurons, acetylcholine (ACh) likewise activated an inward cationic current. The amplitude of ACh-induced current was lower if ACh was applied during a long-lasting application of ATP than if ACh only was applied. Hexamethonium (50 µM), d-tubocurarine (20–40 µM), and trimethaphan (30 µM) completely and reversibly blocked ACh-induced currents, regardless of the presence of ATP, and did not affect ATP-induced currents. The results suggest that ATP-induced currents in submucous neurons are due to activation of a unique type of P2 purinoreceptors, which function in connection with nicotinic ACh receptors.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 100–110, March–June, 1996.     

Expression of low voltage-activated calcium channels inXenopus oocytes

Calcium channels were expressed inXenopus oocytes by means of messenger RNA extracted from the rat thalamo-hypothalamic complex, mRNA(h). Inward barium currents,I _Ba, were recorded in Cl^–-free extracellular solution with 40 mM Ba²⁺ as a charge carrier, using two-microelectrode technique. Depolarizations from a very negative holding potential (V _h=–120 mV) began to activateI _Ba at about –80 mV; this current peaked at –30 to –20 mV and reversed at +50 mV, indicating that I _Ba may be transferred through the low voltage-activated (LVA) calcium channels. The time-dependent inactivation of the current during a prolonged depolarization to –20 mV was quite slow, followed a single exponential decay with a time constant of 1550 msec, and contained a residual component constituting 30% of the maximum amplitude. The current could not be completely inactivated at any holding potential. As expected for LVA current, a steady-state inactivation curve was shifted towards negative potentials. It could be described by the Boltzmann's equation with the half-inactivation potential of –78 mV, slope factor of 15 mV, and residual level of 0.3. ExpressedI _Ba could be blocked by flunarizine (K _d=0.42 µM), nifedipine (K _d=10 µM), and amiloride at a 500 µM concentration. Among the inorganic Ca²⁺ channel blockers, the most potent was La³⁺ (K _d=0.48 µM), while Cd²⁺ and Ni²⁺ were not very selective and almost thousand-fold less effective (K _d=0.52 mM andK _d=0.62 mM, respectively) than La³⁺. Our data show that mRNA(h) induces expression in the oocytes of almost exclusively LVA Ca²⁺ channels with voltage-dependent and pharmacological properties very similar to those observed for T-type Ca²⁺ current in native hypothalamic neurons, though kinetic properties of the expressed and natural currents are somewhat different.Neirofiziologiya/Neurophysiology, Vol. 27, No. 3, pp. 183–189, May–June, 1995.     

Single calcium channels in neurons of rat spinal ganglia

Using a refined patch clamp technique, a study was made of single calcium channels of spinal ganglia neurons on a cell-attached membrane site in newborn rats; these convey the basic (high threshold) component of calcium current. Findings show that currents carried by calcium ions at a concentration of 60 mM in the recording pipet changes from 0.58±0.05 to 0.43±0.05 pA with a change in potential of 20 mV. This corresponds to a channel conductance of 7±0.5 pS. The distribution of open time was monoexponential with a time constant of about 0.75 msec, independent of membrane potential. Distribution of closed time approached a biexponential time course. The fast component (0.8 msec) was voltage-dependent, while the slow component decreased from 22 to 4 msec when depolarization increased by 20 mV. Using experimentally obtained time parameters which describe single calcium channel function, and assuming a three-tier model of the channel, the numerical values of the constants of transition rates between individual states were determined.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 673–682, September–October, 1985.     

Modulation of the membrane potential of intact guinea pig aortic endothelium

The role of different ion conductances in regulation of the membrane potential (MP) of resting and agonist-stimulated intact endothelium from the guinea pig aorta was investigated. Under resting conditions, the MP measured by the patch-clamp technique varied within the range from –29 to –56 mV (the mean value of –40.8 ± 8.1 mV). Blockers of anomalous (inward) rectifier potassium channels cesium (100 µM) and barium (100 µM) exerted no effect on the MP of endothelium. Superfusion of preparations with calcium-free solution and application of 2 mM nickel depolarized the endothelium. ATP (10 µM) induced hyperpolarization of endothelium with the mean amplitude of 11.4 ± 0.6 mV. The initial phase of this hyperpolarization depended on the external potassium concentration and on the state of intracellular calcium stores, whereas the prolonged phase required the presence of external calcium. In the absence of external calcium, in 25% of recordings transient hyperpolarization was followed by depolarization, which was not observed after substitution of external NaCl for choline. It was concluded that basal activity of calcium-dependent potassium channels contributes to the regulation of the MP of resting endothelium. Stimulation with ATP led to activation of calcium-dependent potassium and nonselective cationic channels. Activation of the former channels produced the initial phase of hyperpolarization, whereas activation of the second channel type evoked the prolonged phase of hyperpolarization.Neirofiziologiya/Neurophysiology, Vol. 28, No. 6, pp. 260–266, November–December, 1996.     

Separation of sodium and calcium channels in the surface membrane of molluscan nerve cells

By intracellular dialysis of isolated neurons of the mollusksHelix pomatia andLimnaea stagnalis and by a voltage clamp technique the characteristics of transmembrane ionic currents were studied during controlled changes in the ionic composition of the extracellular and intracellular medium. By replacing the intracellular potassium ions by Tris ions, functional blocking of the outward potassium currents was achieved and the inward current distinguished in a pure form. Replacement of Ringer's solution in the extracellular medium with sodium-free or calcium-free solution enabled the inward current to be separated into two additive components, one carried by sodium ions, the other by calcium ions. Sodium and calcium inward currents were found to have different kinetics and different potential-dependence: _mNa=1±0.5 msec, _mCa=3±1 msec, _hNa=8±2 msec, _hCa=115±10 msec (V_m=0), G_Na=0.5 (V_m=–21±2 mV), G_Ca=0.5 (V_m=–8±2 mV). Both currents remained unchanged by tetrodotoxin, but the calcium current was specifically blocked by cadmium ions (2·10^–3 M), verapamil, and D=600, and also by fluorine ions if injected intracellularly. All these results are regarded as evidence that the soma membrane of the neurons tested possesses separate systems of sodium and calcium ion-conducting channels. Quantitative differences are observed in the relative importance of the systems of sodium and calcium channels in different species of mollusks.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 2, pp. 183–191, March–April, 1976.     

Ionic mechanisms for the antiarrhythmic action of cinnamophilin in rat heart

We investigated the electrophysiological effect and antiarrhythmic potential of cinnamophilin (Cinn), a thromboxane A₂ antagonist isolated fromCinnamomum philippinense, on rat cardiac tissues. Action potential and ionic currents in single rat ventricular cells were examined by current clamp or voltage clamp in a whole-cell configuration. In 9 episodes of ischemia-reperfusion arrhythmia, 10 µM Cinn converted 6 of them to normal sinus rhythm. Cinn suppressed the maximal rate of rise of the action potential upstroke (V_max) and prolonged the action potential duration at 50% repolarization (APD₅₀). Voltage clamp study showed that the suppression of V_max by Cinn was associated with an inhibition of sodium inward current (I_Na, IC₅₀=10.0 ± 0.4 µM). At 30 µM, V_1/2 for the steady-state inactivation curve of I_Na was shifted from –84.1 ± 0.2 to –93.0 ± 0.5 mV. Cinn also reduced calcium inward current (I_Ca) dose-dependently with an IC₅₀ value of 9.5 ± 0.3 µM. Cinn (10 µM) reduced the I_Ca with a negative shift of V_1/2 for the steady-state inactivation curve of I_Ca from –32.2 ± 0.3 to –50.7 ± 0.4 mV. The prolongation of APD₅₀ was associated with an inhibition of the integral of potassium outward current with IC₅₀ values between 4.8 and 7.1 µM. At 10 µM, Cinn reduced I_Na without a negative shift of its voltage-dependent steady-state inactivation curves. The inhibition of transient outward current (I_to) by Cinn (3–30 µM) was associated with an acceleration of its time constant of inactivation and negative shift of its potential-dependent steady-state inactivation curves. The equilibrium dissociation constant (K_d) of Cinn to inhibit open state I_to channels, as calculated from the time constant of developing block, was 18.3 µM. The time constant of recovery of I_to from inactivation state was unaffected by Cinn. The rate constant for the relief from the depolarization-dependent block of I_to was calculated to be 23.9 ms. As compared with its effect on I_to, Cinn exerted about half the potency to block I_Na and I_Ca. These results indicate that the inhibition of I_Na, I_Ca and I_to may contribute to the antiarrhythmic activity of Cinn against ischemia-reperfusion arrhythmia.     

Properties of the slow excitatory postsynaptic potential in mammalian sympathetic ganglion cells

Two types of slow excitatory postsynaptic potentials (EPSPs) with different properties were found in neurons of the rabbit superior cervical sympathetic ganglion. In our group of neurons slow EPSPs increased during artificial hyperpolarization and decreased during depolarization of the membrane. The input resistance of the cells fell or remained unchanged during the development of slow EPSPs. In the second group of cells slow EPSPs increased during depolarization and decreased during hyperpolarization. The reversal potential of these responses, determined by extrapolation, was –78.9±3.6 mV. Depolarization responses to activation of muscarinic cholinergic receptors by acetylcholine or carbachol developed in 53% of neurons with an increase in input resistance and had a reversal potential of –83.2±6.7 mV. It is suggested that in cells of the first group the ionic mechanism of the slow EPSPs is similar to that of the fast EPSPs, whereas in cells of the second group its main component is a decrease in the potassium conductance of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 4, pp. 371–379, July–August, 1981.     

The ionic basis of the resting potential of molluscan unstriated muscle

Summary The membrane potential (Vm) of unstriated, non-spiking fibres from the buccal retractor muscle of the opisthobranch molluscPhiline aperta is primarily determined by the distribution of the potassium ion across the membrane. In salines where potassium is varied and chloride remains constant or nearly so, the membrane potential varied with log external K⁺ with a slope of 50.6 (±2.3) mV per decade. In chloride-free salines the slope was 48.5 mV per decade. The experiments were conducted at temperatures of 18–20° C.A ten-fold reduction in external chloride concentration depolarised the fibres by around 10 mV, indicating that chloride permeability makes some contribution to Vm. In salines where [K]₀·[Cl]₀ is constant the Nernst slope was 55.8 mV per decade compared with the theoretical value of 58 mV.The experimental data suggest that the internal potassium concentration of the fibres is 247±31 mM and pNa/pK is 0.01, giving a predicted value of Vm in sea water of –72 mV. The membrane potential of 90 fibres measured in sea water was –74.2±1.3 mV. The membrane contains an electrogenic sodium pump which contributes 4–5 mV to the membrane potential.     

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.     

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.