Shift In Reversal Potential Research Articles

Substrate influences rat osteoclast morphology and expression of potassium conductances.

1. We studied the electrophysiological properties of freshly isolated rat osteoclasts using the whole-cell configuration of the patch-clamp technique. Membrane currents were recorded from cells plated on three substates: dentine, type I collagen and glass. 2. Based on their morphology, we defined two categories of osteoclasts. 'Rounded' osteoclasts were dome-shaped and lacked lamellipodia. 'Spread' osteoclasts were flattened and had lamellipodia. The proportion of 'rounded' osteoclasts was significantly greater when cells were plated on dentine or type I collagen than when cells were plated on glass. 3. 'Spread' osteoclasts expressed an inwardly rectifying K+ conductance regardless of the substrate on which they were plated. 4. 'Rounded' osteoclasts, on all substrates, expressed a transient, outwardly rectifying conductance that was selective for K+ based on: reversal of deactivation tail currents at -74 mV; a 60 mV shift in tail current reversal potential for 10-fold change in [K+]o; and blockade of outward current by extracellular 4-aminopyridine, charybdotoxin, and intracellular Cs+. The outward K+ current had an activation threshold of approximately -50 mV, with half-activation at -29 mV. The current also exhibited voltage-dependent inactivation, with half-inactivation at approximately -40 mV. 5. Outward K+ current in 'rounded' osteoclasts was reduced when extracellular Ca2+ was removed and upon addition of Ni2+, but was unaffected by Cd2+ or nifedipine. 6. 'Rounded' osteoclasts had large whole-cell capacitance for their apparent surface area. Capacitance was positively correlated with K+ conductance. The additional surface membrane we detected through capacitance measurements may be the 'ruffled border' of actively resorbing osteoclasts. 7. We conclude that substrate influences the expression of osteoclast phenotype, as defined by morphology and K+ conductances. 'Rounded' osteoclasts express an outwardly rectifying K+ conductance, with no apparent inwardly rectifying K+ conductance. In contrast, 'spread' osteoclasts exhibit an inwardly rectifying K+ conductance with no outwardly rectifying K+ conductance. The 'spread' phenotype may represent a motile phase, while the 'rounded' phenotype may represent a resorptive phase of osteoclastic activity.

Resting membrane potential and potassium currents in cultured parasympathetic neurones from rat intracardiac ganglia.

1. Whole-cell K+ currents contributing to the resting membrane potential and repolarization of the action potential were studied in voltage-clamped parasympathetic neurones dissociated from neonatal rat intracardiac ganglia and maintained in tissue culture. 2. Rat intracardiac neurones had a mean resting membrane potential of -52 mV and mean input resistance of 850 M omega. The current-voltage relationship recorded during slow voltage ramps indicated the presence of both leakage and voltage-dependent currents. The contribution of Na+, K+ and Cl- to the resting membrane potential was examined and relative ionic permeabilities PNa/PK = 0.12 and PCl/PK < 0.001 were calculated using the Goldman-Hodgkin-Katz voltage equation. Bath application of the potassium channel blockers, tetraethylammonium ions (TEA; 1 mM) or Ba2+ (1 mM) depolarized the neurone by approximately 10 mV. Inhibition of the Na(+)-K+ pump by exposure to K(+)-free medium or by the addition of 0.1 mM ouabain to the bath solution depolarized the neurone by 3-5 mV. 3. In most neurones, depolarizing current pulses (0.5-1 s duration) elicited a single action potential of 85-100 mV, followed by an after-hyperpolarization of 200-500 ms. In 10-15% of the neurones, sustained current injection produced repetitive firing at maximal frequency of 5-8 Hz. 4. Tetrodotoxin (TTX; 300 nM) reduced, but failed to abolish, the action potential. The magnitude and duration of the TTX-insensitive action potential increased with the extracellular Ca2+ concentration, and was inhibited by bath application of 0.1 mM Cd2+. The repolarization rate of the TTX-insensitive action potential was reduced, and after-hyperpolarization was replaced by after-depolarization upon substitution of internal K+ by Cs+. The after-hyperpolarization of the action potential was reduced by bath application of Cd2+ (0.1 mM) and abolished by the addition of Cd2+ and TEA (10 mM). 5. Depolarization-activated outward K+ currents were isolated by adding 300 nM TTX and 0.1 mM Cd2+ to the external solution. The outward currents evoked by step depolarizations increased to a steady-state plateau which was maintained for > 5 s. The instantaneous current-voltage relationship, examined under varying external K+ concentrations, was linear, and the reversal (zero current) potential shifted in accordance with that predicted by the Nernst equation for a K(+)-selective electrode. The shift in reversal potential of the tail currents as a function of the extracellular K+ concentration gave a relative permeability, PNa/PK = 0.02 for the delayed outward K+ channel(s).(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiological studies of anion secretion in cultured human epididymal cells.

1. Primary monolayer cultures from adult human epididymis were grown on Petri dishes and previous supports. The epithelia so formed were used for whole-cell patch clamp recording and short-circuit current (ISC) measurement. 2. After 50 days of culture, the cells formed a tight epithelium with transepithelial potential of 5.5 +/- 1.3 mV (mean +/- S.E.M.., n = 16), apical side negative, and a basal ISC of 6.9 +/- 0.9 microA cm-2 (mean +/- S.E.M., n = 16). 3. Adrenaline, when added to the basolateral side, at a concentration of 0.23 mumol l-1 increased the ISC by 3.0 +/- 1.2 microA cm-2 (mean +/- S.E.M., n = 4). This increase was blockable by diphenylamine-2-carboxylate (DPC, 1 mmol l-1). Forskolin (10 mumol l-1) also evoked a similar response to adrenaline. 4. In whole-cell patch clamp experiment, the resting membrane potential of the cells after dialysis with pipette solution containing 135 mmol l-1 KCl was found to be -30 +/- 14 mV (mean +/- S.E.M., n = 15). 5. About 90% of the cells successfully forming patches responded to 1 mumol l-1 adrenaline by an increase in inward current at -70 mV holding potential (delta I = -1600 +/- 900 pA, mean +/- S.E.M., n = 15). This increase in current was accompanied by a shift in reversal potential to -2 +/- 1 mV (mean +/- S.E.M., n = 15). 6. The adrenaline-induced inward current was found to be blockable by the Cl- channel blocker, DPC (0.25 mmol l-1). Ion substitution experiments showed that the adrenaline-evoked current was carried mainly by Cl-. 7. The effect of adrenaline on the whole-cell current was found to be mimicked by forskolin and could be abolished by including GDP beta S or a protein kinase A inhibitor in the pipette solution. Propranolol, but not phentolamine, completely abolished the effect of adrenaline. 8. Inclusion of 20 mmol l-1 EGTA or 2 mmol l-1 BAPTA + 100 mumol l-1 TMB-8 (to inhibit intracellular Ca2+ release) in the pipette did not seem to have any marked effect on adrenaline-evoked whole-cell current. Lowering the pipette Ca2+ concentration to 1 nmol l-1 or raising it to 10 mumol l-1 had no effect on the whole-cell current response to adrenaline. 9. This study shows that adrenaline stimulates Cl- secretion in cultured human epididymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations.

We measured the permeability ratios (PX/PNa) of 3 wild-type, 1 hybrid, 2 subunit-deficient, and 22 mutant nicotinic receptors expressed in Xenopus oocytes for alkali metal and organic cations using shifts in the bi-ionic reversal potential of the macroscopic current. Mutations at three positions (2', 6', 10') in M2 affected ion selectivity. Mutations at position 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) near the intracellular end of M2 changed the organic cation permeability ratios as much as twofold and reduced PCs/PNa and PK/PNa by 16-18%. Mutations at positions 6' and 10' increased the glycine ethyl ester/Na+ and glycine methyl ester/Na+ permeability ratios. Two subunit alterations also affected selectivity: omission of the delta subunit reduced PCs/PNa by 16%, and substitution of Xenopus delta for mouse delta increased Pguanidinium/PNa more than twofold and reduced PCs/PNa by 34% and PLi/PNa by 20%. The wild-type mouse receptor displayed a surprising interaction with the primary ammonium cations; relative permeability peaked at a chain length equal to four carbons. Analysis of the organic permeability ratios for the wild-type mouse receptor shows that (a) the diameter of the narrowest part of the pore is 8.4 A; (b) the mouse receptor departs significantly from size selectivity for monovalent organic cations; and (c) lowering the temperature reduces Pguanidinium/PNa by 38% and Pbutylammonium/PNa more than twofold. The results reinforce present views that positions -1' and 2' are the narrowest part of the pore and suggest that positions 6' and 10' align some permeant organic cations in the pore in an interaction similar to that with channel blocker, QX-222.

Tris+/Na+ permeability ratios of nicotinic acetylcholine receptors are reduced by mutations near the intracellular end of the M2 region.

Tris+/Na+ permeability ratios were measured from shifts in the biionic reversal potentials of the macroscopic ACh-induced currents for 3 wild-type (WT), 1 hybrid, 2 subunit-deficient, and 25 mutant nicotinic receptors expressed in Xenopus oocytes. At two positions near the putative intracellular end of M2, 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) and -1', point mutations reduced the relative Tris+ permeability of the mouse receptor as much as threefold. Comparable mutations at several other positions had no effects on relative Tris+ permeability. Mutations in delta had a greater effect on relative Tris+ permeability than did comparable mutations in gamma; omission of the mouse delta subunit (delta 0 receptor) or replacement of mouse delta with Xenopus delta dramatically reduced relative Tris+ permeability. The WT mouse muscle receptor (alpha beta gamma delta) had a higher relative permeability to Tris+ than the wild-type Torpedo receptor. Analysis of the data show that (a) changes in the Tris+/Na+ permeability ratio produced by mutations correlate better with the hydrophobicity of the amino acid residues in M2 than with their volume; and (b) the mole-fraction dependence of the reversal potential in mixed Na+/Tris+ solutions is approximately consistent with the Goldman-Hodgkin-Katz voltage equation. The results suggest that the main ion selectivity filter for large monovalent cations in the ACh receptor channel is the region delimited by positions -1' and 2' near the intracellular end of the M2 helix.

Modulatory effects of FMRF-NH2 on outward currents and oscillatory activity in heart interneurons of the medicinal leech

Using single-electrode voltage clamp, heart interneurons of the medicinal leech were shown to possess both a rapidly inactivating outward current, IA, and a more slowly inactivating outward current, IK. IA and IK could be separated by their voltage sensitivity and kinetic properties. FMRF-NH2 (Phe-Met-Arg-Phe-NH2) modulates IK by shifting both steady state activation and inactivation to more hyperpolarized potentials, but it does not affect the time constants. IA and IK appear to use K+ as a charge carrier; a change in the external [K+] produced a shift in the apparent reversal potential in the direction predicted with potassium as the charge carrier. Both IA and IK are sensitive to tetraethylammonium (TEA) and 4-aminopyridine (4-AP), and TEA and 4-AP both interfere with the effects of FMRF-NH2 on IK. The biophysical properties of IA and of IK in the presence and absence of FMRF-NH2 were incorporated into a Hodgkin-Huxley model of these currents that could reproduce voltage-clamp data. FMRF-NH2 produces two apparently dissimilar effects on the heartbeat rhythm--acceleration and disruption. We suggest that both effects could result from the hyperpolarizing shifts in steady state activation and inactivation of IK.

CAMP-activated chloride conductance in the colonic cell line, Caco-2.

In this study we investigated the properties of adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl- efflux in Caco-2 monolayers by measuring 125I efflux rates from preloaded cells and using patch-clamp electrophysiology. The addition of a cocktail containing 100 microM dibutyryl cAMP (DBcAMP), 10 microM forskolin, and 1 mM 3-isobutyl-1-methylxanthine caused a significant (P less than 0.05) increase in the rate of 125I efflux. Dissipation of cell potential by adding valinomycin (4.5 microM) with 135 mM extracellular KCl reduced the cAMP-evoked 125I efflux. These results suggest that cAMP-stimulated anion efflux occurs through a conductive pore or channel. Whole cell currents evoked with DBcAMP or forskolin were anion selective, PCl greater than PI greater than Pgluconate, and exhibited a linear current-voltage (I-V) relationship. Currents evoked with depolarizing or hyperpolarizing voltage steps showed no evidence of time-dependent activation or inactivation. Single Cl- channels were stimulated in cell-attached patches after treatment with cAMP. Onset of channel activity occurred after 20-30s of cAMP treatment, and the response was long lasting. The I-V relationship for the channel activated in cell-attached patches by cAMP was best fit using two linear regressions. The slope conductance of the channel was 3.2 +/- 0.6 and 7.4 +/- 0.3 pS at hyperpolarizing and depolarizing potentials, respectively. Substitution of 140 mM NaCl with 70 mM NaCl in the patch pipette resulted in a positive shift in reversal potential, indicating that the channel is anion selective.(ABSTRACT TRUNCATED AT 250 WORDS)

Slow depolarizing afterpotentials in neocortical neurons are sodium and calcium dependent.

Depolarizing afterpotentials (DAPs) were studied in intracellular recordings from neocortical slices bathed in tetrodotoxin (TTX) (1 microM) and tetraethylammonium chloride (TEA) (24 mM), to block voltage-dependent Na+ currents and most K+ currents. The DAP was Ca(2+)-dependent, in that its magnitude varied as a function of the duration of the preceding Ca2+ plateau. It had an apparent reversal potential of between -40 and -5 mV. The DAP was blocked when choline replaced all extracellular Na+; there was a hyperpolarizing shift in apparent reversal potential when extracellular Na+ was lowered. The DAP was blocked by amiloride (1 mM), which also decreased the preceding Ca2+ plateau. The data are consistent with the hypothesis that the DAP is due to electrogenic Na+/Ca2+ exchange.

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 Ca(2+) 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.

An outward-rectifying potassium channel in primary cultures of sweat glands from cystic fibrosis subjects

We have previously described a high conductance calcium-activated ‘maxi K’ channel in primary cultures of human eccrine sweat gland cells both from normal subjects and those with cystic fibrosis. In further studies we have now identified a potassium-selective channel of much lower conductance which shows outward-reactification and which is present in sweat glands isolated from cystic fibrosis subjects. In experiments with inside-out patches using symmetrical pipette and bath solutions containing 140 mM K + the channel showed an outward slope conductance (at +50 mV) of ≈ 26 pS and an inward conductance (at −50 mV) of ≈ 11 pS. When K + in the bath was replaced by Na + the reversal potential shifts to reveal a permeability ratio P K/ P Na ≈ 40 Unlike the maxi-K + channel, the outward-rectifying channel does not show sensitivity to Ca 2+. Channels were found in cells cultured from the glands of four out of five cystic fibrosis subjects. In cells cultured from 30 subjects who did not have cystic fibrosis, an outward-rectifying potassium channel was seen in only one out of ≈ 3000 patches.

Calcium permeability of neuronal nicotinic acetylcholine receptor channels in PC12 cells

The conductance properties of neuronal nicotinic acetylcholine receptors (neuronal nAChR channels) in PC12 cells were studied using single channel and whole cell gigaohm seal voltage clamp techniques. Acetylcholine receptor agonists were applied using external pipettes. Neuronal nAChR channels were selective for monovalent cations, and were impermeable to anions. Based on measurements of shifts in reversal potential with changes in external Na and K ion concentrations and evaluation of the GHK constant field relation, P K/P Na was 1.45 (ignoring Ca permeability). P Ca/P K was 1.75 based on the shift in reversal potential seen when raising the external Ca concentration from 2 to 50 mM (holding the external Na ion concentration constant), and determining the best fit to the extended GHK constant field relation using ionic activities. This value is larger than that determined for the muscle-type nAChR channel. Evaluation of the extended GHK current equations using these permeability coefficients indicated that at subthreshold voltages approximately 5% of total current through neuronal nAChR channels will be carried by Ca ions. Thus, these channels may be important mediators of activity-dependent Ca influx in the nervous system.

Concentration-related effects of extracellular application of ATP on the action potential and membrane currents of the guinea-pig vas deferens

The ionic mechanisms underlying concentration-related alterations in the action potential configuration caused by ATP were studied using preparations of the guinea-pig vas defercns voltage-clamped by a double sucrose gap method. Under current-clamp conditions, ATP at concentration of 1.6 μM enhanced the rates of rise and of repolarisation of the action potential whereas at concentration of 1.6 mM it reduced both rates. Under voltage-clamp conditions, lower concentrations increased the maximum inward Ca current without altering kinetics or reversal potential. Higher concentrations reduced the maximum inward Ca current with slowing of rates of activations and inactivation, but also caused a negative shift in reversal potential without affecting conductance. These results suggest that a low ATP concentration activates the voltage-dependent Ca current channels and that the action of a high ATP concentration is related to the internal Ca ion concentration.

Properties of single fast chloride channels from rat cerebral cortex neurons.

1. Properties of Cl- channels from surface membranes of acutely dissociated rat cerebral cortical neurons were studied with the patch clamp technique. These channels were present in the majority of excised inside-out membrane patches. 2. Cl- channels were rarely observed in cell-attached membrane patches, and usually several minutes elapsed following excision of the patch before Cl- channels became active. 3. Under asymmetric ionic conditions (1000 mM-KCli, 140 mM-KClo), neuronal Cl- channels are fairly selective for Cl- over K+ and Na+, with permeability ratios, determined by reversal potential shifts of 4.8 for both PCl/PK and PCl/PNa. 4. Neuronal Cl- channel kinetic activity remained stable over periods of time long enough to collect up to 500,000 open and closed intervals. Occasionally, the channels entered altered modes of activity. In the 'buzz mode' the open and closed interval durations became much shorter than normal for several hundreds of intervals. In the 'subconductance mode' the channel opened to a current level about two-thirds of the normal level. 5. Using the method of maximum likelihood, sums of exponentials were fitted to the distributions of open and closed interval durations. Open interval distributions required at least two exponential components with time constants of less than 1 ms. At least six or seven exponential components were required to fit the closed interval distributions with time constants ranging from 30 microseconds to several hundreds of milliseconds. This suggests that neuronal Cl- channels enter at least two open and six or seven closed kinetic states during normal activity. 6. Cl- channels often entered long-duration closed states of several minutes which could not be accounted for by the sums of exponentials fitted to the distribution of closed interval durations. 7. Neuronal Cl- channels exhibit a marked voltage dependence with the percentage of time the channels are open increasing with depolarization. Most of the observed voltage dependence can be accounted for by a decrease in the mean closed interval duration with depolarization. The mean open interval was relatively independent of voltage. 8. These results suggest a high degree of similarity in kinetic behaviour and conductance properties between the fast Cl- channels of tissue-cultured rat skeletal muscle and fast Cl- channels in acutely dissociated rat cerebral cortical neurons.

Acetylcholine‐evoked currents in cultured neurones dissociated from rat parasympathetic cardiac ganglia.

1. The properties of acetylcholine (ACh)-activated ion channels of parasympathetic neurones from neonatal rat cardiac ganglia grown in tissue culture were examined using patch clamp recording techniques. Membrane currents evoked by ACh were mimicked by nicotine, attenuated by neuronal bungarotoxin, and unaffected by atropine, suggesting that the ACh-induced currents are mediated by nicotinic receptor activation. 2. The current-voltage (I-V) relationship for whole-cell ACh-evoked currents exhibited strong inward rectification and a reversal (zero current) potential of -3 mV (NaCl outside, CsCl inside). The rectification was not alleviated by changing the main permeant cation or by removal of divalent cations from the intracellular or extracellular solutions. Unitary ACh-activated currents exhibited a linear I-V relationship with slope conductances of 32 pS in cell-attached membrane patches and 38 pS in excised membrane patches with symmetrical CsCl solutions. 3. Acetylcholine-induced currents were reversibly inhibited in a dose-dependent manner by the ganglionic antagonists, mecamylamine (Kd = 37 nM) and hexamethonium (IC50 approximately 1 microM), as well as by the neuromuscular relaxant, d-tubocurarine (Kd = 3 microM). Inhibition of ACh-evoked currents by hexamethonium could not be described by a simple blocking model for drug-receptor interaction. 4. The amplitude of the ionic current through the open channel was dependent on the extracellular Na+ concentration. The direction of the shift in reversal potential upon replacement of NaCl by mannitol indicates that the neuronal nicotinic receptor channel is cation selective and the magnitude suggests a high cation to anion permeability ratio. The cation permeability (PX/PNa) followed the ionic selectivity sequence Cs+ (1.06) greater than Na+ (1.0) greater than Ca2+ (0.93). Anion substitution experiments showed a relative anion permeability, PCl/PNa less than or equal to 0.05. 5. The nicotinic ACh-activated channels described mediate the responses of postganglionic parasympathetic neurones of the mammalian heart to vagal stimulation.

Adenosine triphosphate‐evoked currents in cultured neurones dissociated from rat parasympathetic cardiac ganglia.

1. The excitatory response of cultured neurones of rat parasympathetic cardiac ganglia to extracellular adenosine 5'-triphosphate (ATP) was examined using the whole-cell isolated membrane patch recording configurations of the patch clamp technique. The short latency between ATP application and activation of the membrane current (less than 20 ms) suggests a direct coupling between purinergic receptor and ion channel. The response was maintained during exposure to ATP suggesting that receptor desensitization is not a factor in current decay. 2. The current-voltage (I-V) relationship for macroscopic ATP-evoked currents showed strong inward rectification in the presence and absence of external divalent cations and a reversal potential of +10 mV (NaCl outside, CsCl inside). Unitary ATP-activated currents in cell-attached membrane patches exhibited a linear (ohmic) I-V relationship with a slope conductance of approximately 60 pS. 3. The order of agonist potency for the purinergic receptor-mediated response was 2-methylthioATP = ATP greater than ADP greater than AMP greater than adenosine = alpha,beta-methylene ATP greater than beta,gamma-methylene ATP, a sequence consistent with a P2y receptor subtype. ATP-evoked currents were attenuated by alpha,beta-methylene ATP (IC50 approximately 10 microM) and reversibly inhibited in a dose-dependent manner by Reactive Blue 2 (Kd = 1 microM). 4. The amplitude of the ATP-evoked current was dependent on the extracellular Na+ concentration. The direction of the shift in reversal potential when NaCl was replaced with mannitol indicated that the purinergic receptor channel is cation selective. The cation permeability relative to Na+ followed the ionic selectivity sequence Ca2+ (1.48) greater than Na+ (1.0) greater than Cs+ (0.67). Anions were not measurably permeant. 5. ATP and ACh-evoked responses in rat intracardiac neurones are mediated by distinct receptor channels. The ATP-activated channels in cardiac neurones may contribute to non-cholinergic, non-adrenergic neurotransmission and mediate, in part, the vagal innervation of the mammalian heart.

Pharmacological properties and H+ sensitivity of excitatory amino acid receptor channels in rat cerebellar granule neurones.

1. N-Methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate receptor channels have been examined in rat cerebellar granule neurones with whole-cell and single-channel patch-clamp methods. The whole-cell peak and steady-state aspartate and NMDA currents were reversibly inhibited by extracellular protons; the IC50 (concentration producing half-maximal inhibition) for the full H+ inhibition curve for NMDA receptors corresponded to pH 7.3, near to physiological pH. (S)-AMPA and kainate whole-cell currents were inhibited by protons with IC50 values that corresponded to pH 6.3 and 5.7, respectively; these receptors were, however, insensitive to H+ concentrations that inhibited NMDA receptor responses. 2. Proton inhibition of the NMDA, AMPA and kainate receptor-mediated responses was voltage insensitive, and did not involve a shift in reversal potential. 3. The EC50 (concentration producing half-maximal effect) for aspartate calculated from the whole-cell dose-response curve was similar at pH 6.8 and 7.6 (mean 11.2 microM). Although the EC50 for glycine potentiation of the aspartate response was marginally increased from 273 nM at pH 7.6 to 373 nM at pH 6.8, H+ inhibition was not overcome by up to 1 mM-external glycine. Inhibiting concentrations of H+ appropriate for AMPA and kainate receptors did not markedly alter the EC50 values determined for (S)-AMPA (3.4 microM) and kainate (114 microM) at pH 7.2. 4. Treatment of neurones with N-ethylmaleimide, iodoacetic acid, dithiothretiol or diethyl pyrocarbonate did not influence proton inhibition of NMDA receptor responses. However, treatment with diethyl pyrocarbonate, which potentiated aspartate responses, appeared to reduce the effectiveness of Zn2+ inhibition of NMDA receptors. 5. Desensitization of whole-cell NMDA and (S)-AMPA currents was studied with ionophoretic application of agonist to the cell soma. Whole-cell aspartate currents desensitized rapidly, irrespective of the glycine concentration. Increased H+ concentrations did not detectably alter the ratio of peak/steady-state current, or the time constants describing the onset of, or recovery from, desensitization. The time constant describing desensitization of (S)-AMPA-induced whole-cell currents also appeared unchanged by inhibiting pH (6.2). 6. The amplitudes of aspartate- or NMDA-activated single-channel multiple conductance levels were unchanged by decreasing the pH to 6.8.(ABSTRACT TRUNCATED AT 400 WORDS)

Epileptiform activity in the hippocampus produced by tetraethylammonium

1. The epileptiform discharges in the CA3 region of the rat hippocampal slice produced by bath application of the potassium channel blocker tetraethylammonium (TEA) were investigated. The effects of a convulsant (5 mM) and subconvulsant (0.5 mM) concentration of TEA on the mossy fiber-evoked synaptic currents were studied by the use of voltage-clamp techniques to determine whether TEA, like 4-aminopyridine (4-AP), another potassium channel blocker and convulsant, increased both inhibitory and excitatory components of the synaptic response. 2. At extracellular potassium concentrations of 2.5 mM, TEA (5 mM) was found to produce spontaneously occurring epileptiform discharges that could be recorded extracellularly. The intracellular correlate of the epileptiform discharge, the paroxysmal depolarizing shift (PDS), could be reversed in polarity by depolarizing the membrane and was associated with a large increase in membrane conductance. These results suggest that a synaptically mediated potential underlies the generation of the epileptiform discharge. 3. The reversal potential for the PDS was dependent on the time, relative to the extracellularly recorded field discharge, at which the measurement was made. In current clamp the mean reversal potential of the PDS measured at the midpoint of the extracellular discharge was -3.3 +/- 2.9 (SE) mV (n = 9). The reversal potential of the PDS was considerably more negative when measured either before or after the midpoint of the extracellular discharge, suggesting the presence of an inhibitory synaptic component. In voltage clamp similar results were obtained and a large conductance change was found to be associated with the PDS. These results suggest that the synaptic conductance associated with the PDS has both inhibitory and excitatory components. 4. TEA increased significantly the mossy fiber-evoked, early-inhibitory conductance. A convulsant concentration (5 mM) increased the conductance measured 15 ms after the stimulus from 39.7 +/- 8.7 to 87.2 +/- 8.0 nS (n = 6). The reversal potential associated with the conductance depolarized from -68.3 +/- 3.4 to -58.3 +/- 4.0 mV after 5 mM TEA. A subconvulsant concentration of TEA (0.5 mM) also increased the conductance of the mossy fiber-evoked response at 15 ms after the stimulus from 49.5 +/- 3.1 to 63.1 +/- 6.1 nS (n = 4) without an associated shift in reversal potential. 5. The late-inhibitory component of the mossy fiber-evoked response, when present, was increased by 5 mM TEA and unchanged by 0.5 mM TEA. 6. The excitatory mossy fiber-evoked synaptic current was studied in the presence of picrotoxin and was found to be increased and prolonged by 5 mM TEA.(ABSTRACT TRUNCATED AT 400 WORDS)

Whole-cell patch study of GABAergic inhibition in CA1 neurons of immature rat hippocampal slices

Inhibitory processes mediated by γ-aminobutyric acid (GABA) were studied in immature rat hippocampal slices using the whole-cell patch clamp technique. Orthodromically evoked hyperpolarizing inhibitory postsynaptic potentials (IPSPs) were observed in CA1 neurons of postnatal 2–5 (P2–5) and 7–13 (P7–13) day old rats under conditions of low internal [Cl −]. In the whole-cell voltage-clamp mode, applications of GABA evoked outward currents which reversed at −55 mV and −62 mV in P2–5 and P7–13 CA1 neurons, respectively, with comparable reversal potentials for the IPSPs for each age. An increase of internal [Cl −] caused a depolarizing shift of the GABA reversal potential which followed the Nernst equation. In both groups of neurons, the IPSPs and GABA currents were blocked with the bath applications of bicuculline (10 μM) and picrotoxin (100 μM). We conclude that the GABA A-mediated inhibitory synaptic process exists in P2–5 CA1 neurons and hypothesize that the absence of such IPSPs noted in previous studies of immature CA1 neurons was likely due to higher internal [Cl −] in the more immature neurons.

Permeation of both cations and anions through a single class of ATP-activated ion channels in developing chick skeletal muscle.

Micromolar concentrations of extracellular adenosine 5'-triphosphate (ATP) elicit a rapid excitatory response in developing chick skeletal muscle. Excitation is the result of a simultaneous increase in membrane permeability to sodium, potassium, and chloride ions. In the present study we quantify the selectivity of the ATP response, and provide evidence that a single class of ATP-activated ion channels conducts both cations and anions. Experiments were performed on myoballs using the whole-cell patch-clamp technique. We estimated permeability ratios by measuring the shift in reversal potential when one ion was substituted for another. We found that monovalent cations, divalent cations, and monovalent anions all permeate the membrane during the ATP response, and that there was only moderate selectivity between many of these ions. Calcium was the most permeant ion tested. To determine if ATP activates a single class of channels that conducts both cations and anions, or if ATP activates separate classes of cation and anion channels, we analyzed the fluctuations about the mean current induced by ATP. Ionic conditions were arranged so that the reversal potential for cations was +50 mV and the reversal potential for anions was -50 mV. Under these conditions, if ATP activates a single class of channels, ATP should not evoke an increase in noise at the reversal potential of the ATP current. However, if ATP activates separate classes of cation and anion channels, ATP should evoke a significant increase in noise at the reversal potential of the ATP current. At both +40 and -50 mV ATP elicited a clear increase in noise, but at the reversal potential of the ATP current (-5 mV), no increase in noise above background was seen. These results indicate that there is only a single class of excitatory ATP-activated channels, which do not select by charge. Based on analysis of the noise spectrum, the conductance of individual channels is estimated to be 0.2-0.4 pS.

Properties of GABA-activated whole-cell currents in bipolar cells of the rat retina

This paper describes experiments on GABA-activated whole-cell membrane currents in bipolar cells freshly isolated from the adult rat retina. The main goal was to determine whether bipolar cell responses to GABA could be resolved in terms of mediation by the GABAA receptor, the GABAB receptor, or both. Bipolar cells were isolated by gentle enzymatic dissociation and identified by their distinct morphology. GABA agonists and antagonists were applied focally by pressure and the resultant currents were recorded under whole-cell voltage clamp. In all bipolar cells tested, GABA (0.1-100 microM) induced a monophasic response associated with a conductance increase (IGABA). The shift in reversal potential for IGABA as a function of pipet [Cl-] paralleled that predicted based on the Nernst equation for Cl-. IGABA was mimicked by muscimol (5-20 microM) and antagonized by bicuculline (20-100 microM). Baclofen (0.1-1.0 mM) produced no apparent conductance change. "Hot spots" of sensitivity to GABA which might be associated with regions of synaptic contact were not found; both the soma and processes of all bipolar cells were responsive to focally applied GABA. Furthermore, all bipolar cells tested responded to glycine. In conclusion, we have established the presence of GABAA receptors on rat retinal bipolar cells. Our data suggest further that these cells lack GABAB receptors. Finally, our observation that bipolar cells in the rat retina are relatively homogeneous in terms of their sensitivity to GABA and glycine lead us to postulate that the functional significance of the presence of receptors and their distribution on a neuron may be dictated more by the topography of the presynaptic inputs than by its inherent chemosensitivity.