Double Exponential Time Course Research Articles

The kinetics of exocytosis and endocytosis in the synaptic terminal of goldfish retinal bipolar cells.

1. The kinetics of exocytosis and endocytosis were studied in the giant synaptic terminal of depolarizing bipolar cells from the goldfish retina. Two techniques were applied: capacitance measurements of changes in membrane surface area, and fluorescence measurements of exocytosis using the membrane dye FM1-43. 2. Three phases of exocytosis occurred during maintained depolarization to 0 mV. The first component was complete within about 10 ms and involved a pool of 1200-1800 vesicles (with a total membrane area equivalent to about 1.6 % of the surface of the terminal). The second component of exocytosis involved the release of about 4400 vesicles over 1 s. The third component of exocytosis was stimulated continuously at a rate of about 1000 vesicles s-1. 3. After short depolarizations (< 200 ms), neither the FM1-43 signal nor the capacitance signal continued to rise, indicating that exocytosis stopped rapidly after closure of Ca2+ channels. The fall in capacitance could therefore be used to monitor endocytosis independently of exocytosis. The capacitance measured after brief stimuli began to fall immediately, recovering to the pre-stimulus baseline with a rate constant of 0.8 s-1. 4. The amount of exocytosis measured using the capacitance and FM1-43 techniques was similar during the first 200 ms of depolarization, suggesting that the most rapidly released vesicles could be detected by either method. 5. After a few seconds of continuous stimulation, the net increase in membrane surface area reached a plateau at about 5 %, even though continuous exocytosis occurred at a rate of 0.9 % s-1. Under these conditions of balanced exocytosis and endocytosis, the rate constant of endocytosis was about 0.2 s-1. The average rate of endocytosis during maintained depolarization was therefore considerably slower than the rate observed after a brief stimulus. 6. After longer depolarizations (> 500 ms), both the capacitance and FM1-43 signals continued to rise for periods of seconds after closure of Ca2+ channels. The continuation of exocytosis was correlated with a persistent increase in [Ca2+]i in the synaptic terminal, as indicated by the activation of a Ca2+-dependent conductance and measurements of [Ca2+]i using the fluorescent indicator furaptra. 7. The delayed fall in membrane capacitance after longer depolarizations occurred along a double exponential time course indicating the existence of two endocytic processes: fast endocytosis, with a rate constant of 0.8 s-1, and slow endocytosis, with a rate constant of 0.1 s-1. 8. Increasing the duration of depolarization caused an increase in the fraction of membrane recovered by slow endocytosis. After a 100 ms stimulus, all the membrane was recycled by fast endocytosis, but after a 5 s depolarization, about 50 % of the membrane was recycled by slow endocytosis. 9. These results demonstrate the existence of fast and slow endocytic mechanisms at a synapse and support the idea that prolonged stimulation leads to an increase in the amount of membrane retrieved by the slower route. The rise in cytoplasmic Ca2+ that occurred during longer depolarizations was correlated with stimulation of continuous exocytosis and inhibition of fast endocytosis. The results also confirm that transient and continuous components of exocytosis coexist in the synaptic terminal of depolarizing bipolar cells.

Abstracts of literature

Abstracts of literature

Characteristics of two slow inactivation mechanisms and their influence on the sodium channel activity of frog ventricular myocytes.

Inactivation of the fast Na+ current of heart muscle occurs in two kinetically distinct phases: a fast process operating on a millisecond time scale and a considerably slower process, the kinetic properties of which have not been explored fully. In this study, we analysed the slow inactivation process in isolated frog ventricular myocytes using the whole-cell variation of the patch-clamp method. Slow inactivation of the Na+ current followed a double-exponential time course, corresponding to slow and ultraslow components of Na+ channel inactivation. The individual time constants were 2-7 s (slow component) and 40-560 s (ultraslow component). Recovery from these slow inactivation processes also followed a double-exponential time course, but was characterized by significantly briefer time constants than those for the inactivation process. The relationship between transmembrane potential and steady-state slow or ultraslow inactivation was well described by the Boltzmann equation. The membrane potential at which half the Na+ channels are inactivated (V1/2) and the slope factor were estimated to be -48.1 and 13.6 mV, respectively, for the slow component alone. Under conditions in which the slow and ultraslow inactivation components were both present, these parameters were -53.1 and 8.7 mV respectively. When the fast and the two slow inactivation processes occurred concomitantly, the resultant steady-state inactivation curves were shifted to more negative potentials and the slope factor was decreased. Treatment with 1 mM Cd2+ externally did not affect the time course of slow inactivation, but produced a 3-7 mV depolarizing shift in its steady-state voltage dependency by virtue of cadmium's known effect on the cell surface potential. This study has thus identified two components of slow Na+ inactivation in heart muscle, operating on a time scale of seconds (slow inactivation) and minutes (ultraslow inactivation).

Reversal of cerebral vasospasm using an intrathecally administered nitric oxide donor.

Intrathecal bolus administration of (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)aminio]diazen++ +-1-ium-1,2-diolate (DETA/NO), a long half-life diazeniumdiolate-class nitric oxide (NO) donor, was evaluated for safety and efficacy in the treatment of delayed cerebral vasospasm in a canine model of subarachnoid hemorrhage (SAH). The baseline basilar artery (BA) diameter of 25 dogs was measured with the aid of angiography on Day 0. Vasospasm was then induced by intracisternal injection of autologous arterial blood on Days 0 and 2. Repeated arteriography on Day 7 revealed an average BA diameter of 58% of baseline. Each dog was then randomized to one of four groups: a pathology control group (SAH only, four animals); a treatment control group (SAH plus 2 micromol of the inactive drug carrier DETA, eight animals); a low-dose treatment group (SAH plus 0.2 micromol DETA/NO, six animals); or a high-dose treatment group (SAH plus 2 micromol DETA/NO, six animals). The drugs were administered in a 2-ml intrathecal bolus via the cisterna magna. Arterial caliber was monitored by angiography over the subsequent 4 hours. A 2-micromol dose of the drug was then given and serial arteriography continued for an additional hour to screen for tachyphylaxis. Intracranial pressure and respiratory and hemodynamic parameters were continuously monitored. Histopathological analyses of the animals' brains were performed after the dogs were killed on Day 8. The drug DETA/NO produced reversal of vasospasm in a dose-dependent fashion that roughly followed a double exponential time course. Doses of 2 micromol DETA/NO resulted in restoration of the angiographically monitored BA diameter to the prevasospasm size at 1.5 hours posttreatment, and this was sustained at 88% of baseline at 4 hours (p < 0.01, independent samples t-test). By contrast, the treatment control group remained on average at 54% of baseline diameter. The low-dose treatment group achieved only partial and more transitory relaxation. Histopathological analyses showed findings consistent with chronic SAH but did not demonstrate any toxicity associated with the NO donor. No adverse physiological changes were seen. This study indicates that long-acting NO donors are potentially useful as agents to restore circulation in patients suffering from cerebral vasospasm.

Electrophysiological characterization of voltage-gated Na+ current expressed in the highly metastatic Mat-LyLu cell line of rat prostate cancer.

Voltage-gated Na+ channels, classically associated with impulse conduction in excitable tissues, are also found in a variety of epithelial cell types where their possible functions are not known so well. We have previously reported expression of a voltage-gated Na+ channel specifically in the highly metastatic Mat-LyLu rat prostate cancer cell line; blockage of the current with tetrodotoxin (TTX) significantly reduced the invasiveness of the cells in vitro, suggesting that the channel may have a functional role in metastasis. The aim of the present study was to characterize this current using the whole-cell patch clamp recording technique, and compare it to Na+ currents found in various other tissues. The inward current of the Mat-LyLu cells was abolished completely, but reversibly, in Na+-free solution, confirming that Na+ was indeed the permeant ion. Activation occurred at -40 mV and currents reached a maximal amplitude at around 6 mV. Boltzmann fits to current activation and steady-state inactivation revealed that the currents were half activated at about -15 mV and half inactivated at -80 mV. Both current inactivation and recovery from inactivation followed a double-exponential time course with fast and slow components. The Na+ currents were highly sensitive to block by TTX (IC50 approximately 18 nM), whilst 1 microM mu-conotoxin GIIIA mostly had no effect. 100 microM Cd2+ also had no effect on the current, whilst 2.5 mM Cd2+, Mn2+, and Co2+ each caused a depolarizing shift in activation and a reduction in peak conductance of around 20%. In conclusion, the Na+ channel expressed in the highly metastatic Mat-LyLu cell line appeared to have electrophysiological and pharmacological properties of TTX-sensitive channels. Further work is needed, however, to elucidate the exact nature of the channel protein and the mechanism(s) of its involvement in cellular invasiveness.

Functional and pharmacological differences between recombinant N-methyl-D-aspartate receptors.

N-methyl-D-aspartic acid (NMDA) receptors transiently transfected into mammalian HEK-293 cells were characterized with subunit-specific antibodies and electrophysiological recordings. Deactivation time course recorded in response to fast-glutamate pulses were studied in isolated and lifted cells, as well as in outside-out membrane patches excised from cells expressing recombinant NR1 subunits in combination with the NR2A, NR2B, NR2C, or NR2D NMDA receptor subunits. Transfected cells were preidentified by the fluorescence emitted from the coexpressed Aequorea victoria jellyfish Green Lantern protein. Currents generated by NR1/NR2A channels displayed double exponential deactivation time course being faster than that in NR1/NR2B or NR1/NR2C channels. However, a large decay variability was observed within each cotransfection, suggesting that mechanisms additional to subunit composition may also regulate deactivation time course. NR1/NR2D channels displayed slowly deactivating currents. Channel deactivation was fast and comparable among receptors obtained by cotransfecting five distinct spliced variants of the NR1 subunit, each with the NR2A subunit. Additionally, recovery from desensitization was slower for NR1/NR2B than for NR1/NR2A channels. The average deactivation time course of responses to brief L-glutamate applications in cells where NR1/NR2A/NR2B cDNAs were cotransfected at variable ratio was intermediate between those of the NR1/NR2A and NR1/NR2B channels. Although immunocytochemical evidence indicates that the majority of cells are cotransfected by all plasmids in triple transfection, our experimental condition did not allow for a tight control of the expression of NMDA receptor subunits. This produced the result that many cells were characterized by deactivation time course and haloperidol sensitivities of separate NR1/NR2A and NR1/NR2B subunit heteromers. We also speculate on the possible formation of channels resulting from the coassembly in the same receptor of NR1/NR2A/NR2B subunits from a minority of cells that gave responses to brief application of L-glutamate characterized by slow deactivation time course and decreased haloperidol sensitivity.

Verapamil block of the delayed rectifier K current in chick embryo dorsal root ganglion neurons.

We have used the patch-clamp method in the whole-cell configuration to investigate the mechanism of block of the delayed rectifier K current (IDRK) by verapamil in embryonic chick dorsal root ganglion (DRG) neurons. Verapamil induced a dose-dependent decay of the current, without altering its activation kinetics. This observation, together with the good description of IDRK time course at various blocker concentrations with the computer simulation of a three-state chain model (closed left and right arrow open left and right arrow open-blocked), indicates that verapamil acts as a state-dependent, open-channel blocker. To account for the double-exponential time course of recovery from block, this minimal kinetics scheme was expanded to include a closed-blocked state resulting from channel closure (at hyperpolarized voltages) with verapamil still bound to it. The apparent block and unblock rate constants assessed from verapamil-induced current decay in the presence of external Na were 0.95 +/- 0.05 ms-1mM-1 and 0.0037 +/- 0.0016 ms-1, respectively. When external Na was replaced by K, only the unblock rate constant changed, to 0.02 +/- 0.009 ms-1. Under these ionic conditions it was also observed that the recovery from block was modified from the double-exponential time course in the presence of external Na (tau1 = 160 ms; tau2 = 1600 ms), to a faster single-exponential recovery (tau = 100 ms). We tested the voltage dependence of block by applying stimulation protocols aimed at eliminating bias easily introduced by the shift of the gating equilibrium and by the coupling of channel activation and block. Under these experimental conditions the resulting block rate constant was not measurably voltage dependent.

Voltage-induced slow activation and deactivation of mechanosensitive channels in Xenopus oocytes.

1. The relationship between stretch and voltage activation of mechanosensitive (MS) channels from Xenopus oocytes was studied in excised patches of membrane using the patch clamp technique. 2. As is characteristic of MS channels to oocytes, stretching the membrane by applying negative pressure to the patch pipette at -50 mV activated the MS channels rapidly. The channels then deactivated rapidly when the stretch was removed. The stretch-activated MS channels entered a main conductance level (45 pS) and one or more subconductance levels in the range of about 75-90% of the main conductance level. 3. In the absence of stretch, a depolarizing step from -50 to +50 mV activated apparent MS channels after long delays of typically 1-20 s (range, 100 ms to 6 min). Upon repolarization, the channels deactivated slowly with a single exponential (mean time constant of 4 s) or double exponential (mean time constants of 0.8 and 3 s) time course. 4. Delayed activation with depolarization and slow deactivation upon repolarization were also observed for apparent MS channels in on-cell patches. 5. The voltage-activated channels were cation selective and had the same selectivity and conductance levels as the stretch activated MS channels. Applying stretch during voltage-induced channel activity did not activate any additional channels, and the same maximal number of channels were typically activated by either stretch or by voltage. These observations suggest that voltage activates the same MS channels that are activated by stretch. 6. The opening of MS channels following steps to +50 mV occurred in an apparently co-operative manner in 70% of the excised patches containing multiple MS channels. 7. In the absence of stretch, the opening frequency and open probability of MS channels increased with depolarization in the examined voltage range of -60 to -20 mV. 8. Applying a brief stretch during the delay to activation at +50 mV activated the MS channels rapidly, which then remained active when the stretch was removed. In contrast, applying a brief stretch during the slow deactivation induced by stepping from +50 to -50 mV abruptly terminated the voltage induced channel activity upon release of the stretch and inhibited subsequent depolarization-induced activity. 9. Depolarizing steps from -50 to +50 mV inhibited any spontaneous channel activity that was present before the depolarizing step. If the potential was stepped back to -50 mV before the channels activated at +50 mV, a delayed activation could occur at -50 mV, followed by normal deactivation, indicating that the depolarizing step initiated activation processes that were initially masked by inhibition. 10. These observations suggest that voltage and stretch can induce different functional gating configurations of MS channels with associated structures, and that these different gating configurations can interconvert.

Alterations in Ito1, IKr and Ik1 density in the BIO TO-2 strain of syrian myopathic hamsters.

The BIO TO-2 strain of cardiomyopathic hamster provides a model of dilated low output heart failure. The goal of the study was to determine whether changes in potassium currents occur in this model of heart failure. The densities of Ito1, IKr and IK1 in 8-month-old myopathic hamsters were not significantly different from their age-matched controls. The half-maximum activation voltage (V1/2) of IKr and Ito1, as well as the voltage-dependence of Ito1 inactivation were also similar in both groups at 8 months. Ito1 inactivation exhibited a double exponential time-course; the slow component (tau2), but not the rapid component (tau1), was larger in the myopathic animals. The densities of Ito1, IKr and IK1 were not significantly different in the 8- and 10-month-old control animals. However, the densities of Ito1, IKr and IK1 were all significantly lower in the 10-month-old myopathic hamsters relative to the 10-month-old controls. The V1/2 for IKr and Ito1 activation was the same in myopathic and control animals. tau2, but not tau1, of Ito1 inactivation was again larger in the myopathic animals. The voltage-dependence of Ito1 inactivation was shifted slightly, but significantly, positive in the myopathic animals. Lastly, a sustained outwardly rectifying current that activated upon depolarization was found to be larger in the myopathic animals at both 8 and 10 months of age. In conclusion, many of the alterations in potassium current densities in the 10-month-old cardiomyopathic hamsters are qualitatively similar to the changes observed in the failing human heart.

Rapid Synaptic Transmission in the Avian Ciliary Ganglion Is Mediated by Two Distinct Classes of Nicotinic Receptors

We analyzed the kinetics and pharmacology of EPSCs in two kinds of neurons in the embryonic avian ciliary ganglion. Whole-cell voltage-clamp recordings revealed that the singly innervated ciliary neurons had large-amplitude (1.5-8.0 nA) EPSCs that could be classified according to the kinetics of their falling phases. Most of the neurons responded with an EPSC the falling phase of which followed a double exponential time course with time constants of approximately 1 and 10 msec. The EPSCs of the remaining ciliary neurons followed a single time constant ( approximately 8 msec). Multiple innervated choroid neurons had smaller-amplitude responses (0.2-1.5 nA when all inputs were activated) that appeared to contain only a slowly decaying component (tau = 12 msec). The fast and slow components of EPSC decay seen in most ciliary neurons could be pharmacologically isolated with two toxins against nicotinic acetylcholine receptors (AChRs). The fast component was blocked by 50 nM alpha-bungarotoxin (alpha-BuTx), which binds alpha7-subunit-containing AChRs. The slow component was selectively blocked by 50 nM alpha-conotoxin MII (alpha-CTx-MII), which blocks mammalian AChRs containing an alpha3/beta2 subunit interface. A combination of both alpha-BuTx and alpha-CTx-MII abolished nearly all evoked current. Similar pharmacological results were found for ciliary neurons with monoexponentially decaying EPSCs and for choroid neurons. These results suggest that nerve-evoked transmitter acts on at least two different populations of AChRs on autonomic motor neurons in the ciliary ganglion.

Neurosteroid Prolongs GABAAChannel Deactivation by Altering Kinetics of Desensitized States

Fast applications of GABA (1 mM) to nucleated and outside-out patches excised from granule neurons in cerebellar slices from developing rats evoked currents with a double exponential time course reminiscent of that of IPSCs. A neurosteroid 3alpha, 21dihydroxy-5alpha-pregnan-20-one (THDOC) remarkably increased the slow deactivation time constant and slowed down recovery from desensitization, as estimated by paired-pulse GABA applications. THDOC also reduced the amplitude of GABA currents, whereas it failed to affect the fast deactivation component and its relative contribution to peak amplitude. The effects of THDOC on slow deactivation were greater in rats younger than postnatal day 13 (P13) as compared with rats at P30-P35. THDOC failed to alter deactivation of short responses induced by a less-potent agonist taurine at saturating doses. These responses had deactivation kinetics described by a fast single exponential decay, little desensitization, and quick recovery. However, THDOC slowed deactivation if taurine responses were long enough to allow consistent desensitization, suggesting that desensitized states are required for the neurosteroid to modulate GABA responses. In outside-out patches, just as desensitized states prolonged GABA responses by producing reopening of channels activated by brief GABA pulses, THDOC increased the channel open probability by further increasing the number of late channel openings, resulting in a prolongation of the slow deactivation. Our data suggest that neurosteroid potentiates the inhibitory postsynaptic transmission via the prolongation of the slow deactivation and that the alteration of kinetics of entry and exit from desensitized states underlies the allosteric modification of GABAA receptors by neurosteroids.

P2X purinoceptors in cultured myenteric neurons of guinea-pig small intestine.

1. Fast excitatory postsynaptic currents (fEPSCs) and responses to exogenously applied purinoceptor agonists were studied in primary cultures of myenteric neurons from guinea-pig small intestine. Whole-cell and outside-out configurations of the patch clamp technique were used. Hexamethonium (100 microM) partly inhibited fEPSCs in 28% of neurons. Hexamethonium-resistant fEPSCs were inhibited by 97 +/- 2% by the P2X receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM). 2. ATP caused two types of inward currents. In 92% of neurons (n = 123), ATP caused a slowly desensitizing current that declined with a double exponential time course (tau 1 = 7.1 +/- 2.0 s; tau 2 = 57 +/- 7.4 s, n = 4). The rank order potency for purinoceptor agonists in these neurons was ATP > 2-methylthio-ATP (2-MeSATP) > > alpha, beta-methylene ATP (alpha, beta-me ATP) > beta, gamma-meATP > ADP. The EC50 values for ATP and 2-MeSATP were 40 and 65 microM, respectively. alpha, beta-MeATP acted as a partial agonist at these receptors. In 8% of neurons (n = 11), ATP-induced currents desensitized rapidly with a double exponential time course (tau 1 = 0.13 +/- 0.015 s; tau 2 = 2.2 +/- 1.3 s, n = 4); alpha, beta-meATP caused similar responses in these cells. Both types of ATP-induced current were associated with an increased conductance and an inwardly rectifying I-V relationship (Erev = 10 mV). Halving [Na+]o shifted the reversal potential of ATP currents by -22 +/- 6 mV. 3. ATP activated single channel currents in outside-out patches. The single channel I-V relationship was linear between -120 and 60 mV (Erev approximately 0 mV). Single channel conductance between -100 and -60 mV was 25 +/- 2 pS. Single channel open probability was voltage dependent and decreased from 0.05 +/- 0.01 at -100 mV to 0.007 +/- 0.002 at +40 mV. 4. These data show that P2X purinoceptors mediate some fEPSCs in cultured myenteric neurons. Myenteric neurons express the fast-desensitizing alpha, beta-me ATP-sensitive subtype of P2X receptor that has the properties of cloned P2X1 receptors and is similar to native receptors in smooth muscle cells. Myenteric neurons also express a P2X receptor that desensitized slowly and was alpha, beta-meATP-insensitive. This receptor has the properties of cloned P2X2 or P2X5 receptors and is similar to native receptors found in PC-12 cells and superior cervical ganglion neurons. The known distribution of P2X2 and P2X5 receptors suggests that myenteric neurons are likely to express predominantly P2X2 receptors.

Characterisation of the voltagemactivated calcium current in the marine ciliate Euplotes vannus

We have isolated the early Ca current (I ca) from the whole cell current that activates upon depolarisations in the marine ciliate Euplotes vannus. The peak of I ca activated within 4.2 ms at depolarisations to 5 mV with an amplitude of 2.5 ± 0.35 nA and was reduced to 1.0 ± 0.14 nA ( n = 5) when the extracellular Ca concentration was changed from 10 to 1 mM. The voltage-dependent activation curve was steeper and shifted to more negative values when external Ca 2+ was replaced by Ba 2+. The early inward current inactivated with a double-exponential time course including a fast and a slow component, and no inactivation was recorded with Ba 2+. The time constants for the recovery from inactivation varied between 44 and 153 ms according to the depolarisation-dependent Ca influx. At the common resting potential of -25 mV, I ca was not steady-state inactivated; I ca half-inactivated at -14.5 mV, and totally inactivated at -5 mV. I ca was inhibited by 10 mM extracellular Cd 2+. The peptides ω-conotoxin-GVIA (20 μM), ω-conotoxin-MVIIC (600 nM), ω-agatoxin-IVA (60 nM) and calciseptine (900 nM) did not block I ca. The benzothiazepine-derivative diltiazem (100 μM) and the dihydropiridine nifedipine (100 μM) inhibited 51% and 33% of I ca, respectively. The naphthalene sulfonamide W7 reduced I ca with an inhibition coefficient of 33 μM.

Amplitude and time course of evoked and spontaneous synaptic currents in rat submandibular ganglion cells.

1. Excitatory postsynaptic currents (EPSCs) were recorded in rat submandibular ganglion cells in vitro using the two-electrode voltage clamp technique. 2. The peak amplitude of EPSCs evoked by nerve impulses in single presynaptic fibres varied between 1.2 and 9.8 nA in different cells (mean = 4.6 +/- 2.6 nA; n = 23; -80 mV membrane potential; 22-25 degrees C). 3. Experiments were performed to re-investigate a previous hypothesis that different mechanisms underlie the generation of evoked versus spontaneous quantal EPSCs in submandibular cells. This hypothesis was based on the observation of different time courses of evoked and spontaneous EPSCs. 4. In agreement with previous studies, the time course of the decay phase of evoked EPSCs was described by the sum of two exponentials, with time constants tau 1 and tau 2 of 6.9 +/- 0.7 and 34.4 +/- 7.7 ms, respectively (n = 23; -80 mV membrane potential). 5. The double-exponential decay of evoked EPSCs persisted when transmitter release was reduced by bath addition of 100 microM cadmium chloride to the level of failures, one or several quanta. 6. Spontaneous EPSCs exhibited mean amplitudes of 81 +/- 24 pA (n = 5 cells; -80 mV membrane potential), and displayed an extremely wide range of peak amplitudes in the same cell (mean coefficient of variation (c.v.) = 0.37 +/- 0.09; n = 5 cells). In contrast to a previous report (see below), the decay phase of spontaneous EPSCs was found to exhibit a double-exponential time course with time constants similar to those of the evoked EPSC recorded in the same cell. 7. These results indicate that evoked and spontaneously released quanta of transmitter most probably act on the same population of postsynaptic receptors in submandibular ganglion cells. There is a large variability in the peak amplitudes of quantal EPSCs recorded in the same cell. This large variability is not due to electrotonic effects, since these cells lack dendrites.

Comparison of voltage-dependent potassium currents in rat pyramidal neurons acutely isolated from hippocampal regions CA1 and CA3

1. The properties of voltage-gated potassium currents were studied in acutely isolated rat hippocampal pyramidal cells from area CA1 and CA3 at postnatal ages of day 6-8, 9-14, and 26-29 (P6-8, P9-14, and P26-29) with the use of the whole cell version of the patch-clamp technique. 2. The outward current pattern of all cells under investigation could be separated in a fast transient A current (IA) and a delayed rectifier-like current (IK). 3. In both preparations, IA activated and inactivated rapidly. Vh describing steady-state inactivation was -84.5 mV in CA3 cells and -85.5 mV in CA1 cells. The activation behavior was characterized by Vh = -23.8 mV in CA3 cells and -27.2 mV in CA1 cells. The removal of inactivation was monoexponential both in CA1 and CA3 neurons with time constants of 32.1 and 28.5 ms, respectively. IA was insensitive to tetraethylammonium (TEA), dendrotoxin (300 nM), and mast cell degranulating peptide (200 nM), but could be blocked with 5 mM 4-aminopyridine (4-AP) by approximately 80%. In both preparations, A currents did not depend on Ca2+ influx. 4. Delayed rectifier currents (IK) in CA1 and CA3 pyramidal neurons decayed along a double exponential time course. Steady-state inactivation was described by Vh = -79.5 mV in CA3 cells and -76.0 mV in CA1 cells. The activation curves were characterized by midpoints of -3.8 mV in CA3 cells and of -1.4 mV in CA1 cells. The removal of inactivation was monoexponential in CA1 and CA3 neurons with time constants of 210.3 and 202.4 ms, respectively. All kinetic properties were identical for the differentially decaying components of IK. In CA1 cells IK was blocked by TEA at +30 mV with an IC50 of 0.98 mM. In CA3 cells the corresponding IC50 value was 1.05 mM. About 20% of IK were insensitive to TEA. IK was partially blocked by approximately 30% with 100 microM 4-AP. Mast cell degranulating peptide (100-200 nM) and dendrotoxin (50-300 nM) had no effect on IK. 6. Perfusion of charybdotoxin (30 nM), Cd2+ (300 microM), La3+ (10 microM), or Ca(2+)-free solutions resulted in the isolation of a small noninactivating outward current component. Around 10% of IK appeared to be Ca2+ dependent in CA1 neurons. In CA3 pyramidal cells Ca(2+)-dependent outward currents seemed to be somewhat larger with approximately 20%. 7. In CA1 as well as in CA3 cells, the kinetic and pharmacological properties of IA and IK remained stable during postnatal development. However, the contribution of IA and IK to the whole cell current varied with age. IA was more prominent in CA1 cells of age group P6-8 than in age-matched CA3 cells. CA3 cells had smaller A currents and larger delayed rectifier currents than CA1 pyramidal cells. Current densities of IA and IK were analyzed during development to assess changes in the expression of these currents. With increasing postnatal age, the expression of IA was downregulated in both preparations. This effect was more pronounced in CA3 than in CA1 cells. In contrast, IK was upregulated during the same developmental period. This increase in the expression of IK was with approximately 300% much larger in CA1 cells than in CA3 cells with only approximately 50%.

Dual activation of the cardiac Ca2+ channel alpha 1C-subunit and its modulation by the beta-subunit.

Ca2+ channels are heteromultimeric proteins in which the alpha 1-subunit forms the voltage-dependent Ca(2+)-selective ionic channel. We reported recently that coexpression of the beta-subunit with the cardiac alpha 1-subunit (alpha 1C) facilitates channel opening without affecting either the amplitude or the time course of the gating currents (13). Here we present evidence for the existence of two modes of channel opening. Xenopus oocytes expressing the alpha 1C-subunit alone display two modes of activation as indicated by the double-exponential time course of macroscopic ionic currents and the two open-time distributions of single channels. Coexpression of the beta-subunit potentiates Ca2+ currents by a relative increase of the fast-activating component, an acceleration of the slow component, and a larger proportion of long openings. We propose that multiple modes of gating are encoded in the alpha 1-subunit and that the beta-subunit increases Ca2+ channel opening by favoring a willing mode of gating in which the final transitions leading to channel opening are facilitated. In addition, we show that the carboxy terminus of alpha 1C also modulates the channel-gating behavior.

Potassium currents across the plasma membrane of protoplasts derived from rye roots: a patch-clamp study

Epidermal-cell protoplasts from rye (Secale cereale L.) roots were voltage-clamped in both the whole-cell and outside-out membrane-patch modes. Time-dependent inwardly-rectified (IR) and outwardly-rectified (OR) K+-currents were recorded, as well as a ubiquitous, timeindependent (instantaneous) K+-current. The IR current activated at voltages more negative than —100mV with two exponentially rising components. The time-constant of the shorter component was voltage-independent, whereas the time-constant of the longer component was voltage-dependent, increasing as the activating voltage became more negative. The IR current showed no inactivation. The IR current deactivated with a single exponential timecourse. The steady-state IR current could be fitted to a Boltzmann function with —135 mV as the voltage at which the current was half-maximal and a minimal gating charge of 1.93. These parameters were insensitive to changes in EK. One component of the IR current was K + , but other ions were also permeable. The IR current was inhibited by extracellular Ca2+ , Ba2+ , Cs+, and TEA+, but was insensitive to quinine. Single channels with unitary conductances of 56 pS and 110 pS (in c.100 mM K+) were recorded at negative voltages. Two OR currents were observed. One had sigmoidal activation kinetics and activated at low positive voltages. The other activated more rapidly, with apparently exponential kinetics, at voltages 50–100 mV more positive than the first. Neither current showed inactivation and deactivation of OR currents followed a double exponential time-course. Unitary-conductances of the channels mediating these OR currents were 24 pS and 57 pS (in c.100 mM K+), respectively. Only the first type of OR current was studied in detail. This current activated with a sigmoidal time-course, which could be described using a Hodgkin-Huxley function with the activation variable raised to the second power. Its voltage-dependence was modulated in response to changes in EK and analysis of single-channel recordings indicated that the channel was K+-selective. The current was inhibited by Ba2+ and TEA+, but not Ca2+, Cs+ or quinine. The instantaneous current was selective for monovalent cations and K+ , Na+ and Cs+ were all permeant. It was inhibited by extracellular quinine and the instantaneous inward K+-current was reduced by extracellular Ca2+, Ba2+ and TEA+, as well as by competing permeant monovalent cations. The kinetics and pharmacology of these currents are compared with K+-currents across the plasma membrane of protoplasts from other root-derived cells and with K+ channels in the plasma membrane of rye roots studied following incorporation into artificial, planar lipid bilayers.

Calcium concentration changes in the calyciform nerve terminal of the avian ciliary ganglion after tetanic stimulation

A study has been made of the changes in calcium concentration in the calyciform nerve terminal ([Ca] c) and in the neurone soma ([Ca] s) of avian ciliary ganglion cells following tetanic stimulation of the nerve terminal. Dissociated ciliary neurones were loaded with the calcium indicator Fura-2 and digital imaging techniques used to determine the spatial and temporal distribution of calcium in the cells during post-tetanic potentiation (PTP) and long-term potentiation (LTP). Stimulation of the calyciform terminal with an extracellular electrode at 10 Hz for 2 s increased both [Ca] c and [Ca] s over 3-fold, with the [Ca] increasing for each impulse in the facilitatory train. The increase in [Ca] s, could be prevented by allowing the terminal to degenerate in culture before stimulation. Stimulation of the calyciform terminal with a long tetanus of 30 Hz for 20 s gave an over 4-fold increase in both [Ca] c and [Ca] s by the end of the train. Analysis of the decline in [Ca] c after the train showed that it disappeared from the calyx along a double exponential time course with time constants of about 1 min and 50 min, respectively. These times are similar to those of PTP and LTP in the ganglia, and are almost independent of the extracellular calcium level. In order to determine whether the influx of calcium ions during a tetanus was through N-type calcium channels, these were blocked with adenosine (100 μM). Adenosine blocked the increase in both [Ca] s and [Ca] c that normally accompanies a tetanus. Thapsigargin (200 nM) did not affect [Ca] c or [Ca] s, but blocked transient increases in [Ca] caused by caffeine (10 mM) in both 3 mM and Ca 2+ free bath solutions. These results are discussed in relation to the role of intracellular calcium in initiating LTP after a tetanus to the nerve terminals.

Glutamate-activated currents in outside-out patches from spiny versus aspiny hilar neurons of rat hippocampal slices

The desensitization rate of non-NMDA glutamate receptors was investigated in outside-out membrane patches obtained from morphologically identified spiny "mossy cells" (SMCs) and aspiny hilar interneurons (AHIs) in young rat hippocampal slices. The fast application of a 1 mM step of L-glutamate for 50-100 msec in the presence of TTX and dizolcipine (MK-801) onto patches excised from these neurons produced large glutamate-activated currents (GACs) that decayed with a single or double exponential time course despite the continued presence of agonist. These desensitization rates of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-sensitive receptors differed markedly between patches obtained from the two cell types. The fast time constant of desensitization in AHIs (n = 34) averaged 3.3 +/- 0.93 msec (mean +/- SD), while that of SMCs (n = 57) averaged 6.8 +/- 2.0 msec. Current-voltage relationships of the GACs did not differ between SMCs and AHIs, with comparable reversal potentials and no evidence of inward rectification. We also failed to observe significant Ca2+ permeability in either cell type. However, brief (< 1 msec) pulses of 1 mM glutamate produced rapidly decaying GACs with distinct kinetics in the two neuronal classes. Furthermore, analysis of the single glutamate-activated channel currents in outside-out patches from hilar neurons revealed a larger predominant single-channel current in AHIs versus SMCs. Lastly, we observed a greater sensitivity to cyclothiazide in SMCs versus AHIs, with half-maximal removal of desensitization being 90 mM and 200 mM, respectively. Taken together, these differences in GACs between SMCs and AHIs might indicate a functional correlate to the substantial heterogeneity in the molecular structure of glutamate receptor subunits or might be related to posttranslational modifications of these subunits, perhaps provided by the unique microenvironment in the spines covering SMCs.

Analysis of anticholinesterase‐induced neuromuscular transmission failure

To define the underlying mechanism of neuromuscular transmission failure induced by anticholinesterases, we simultaneously performed surface recordings of compound muscle action potentials (CMAPs) and intracellular recordings of miniature end-plate potentials (MEPPs), miniature end-plate current (MEPCs), and end-plate potential (EPPs) in rat diaphragms exposed in vitro to 1 x 10(-4) to 2 x 10(-2) mmol/L neostigmine methylsulfate. At low concentrations of neostigmine, repetitive stimulation of the phrenic nerve resulted in decrement followed by complete recuperation of CMAP amplitudes. This bimodal pattern was associated with maximal end-plate depolarization at the beginning of the stimulation period, increased MEPP amplitudes, and prolonged time constants of MEPC decays. Higher concentrations of neostigmine resulted in a unimodal decline of amplitudes of CMAPs and EPPS, reduced MEPP amplitudes, and a double exponential time course of MEPC decays. These results indicate that low concentrations of anticholinesterases impaired neuromuscular transmission by producing transient depolarization of the end-plate region. Higher concentrations induced desensitization and direct blockade of the end-plate receptor channel, probably in its open conformation.