Noninactivating Ca2 Research Articles

Realistic synaptic inputs applied to coupled oscillator model of the dopamine neuron

Midbrain dopamine neurons are involved in motivation and the control of movement, and have been implicated in various pathologies such as Parkinson's disease, schizophrenia and drug abuse. Dopamine neurons in the presence of their afferent inputs in vivo can exhibit one of several firing modes: silence, regular single-spike firing, irregular single-spike firing, and bursting. Bursts in dopamine neurons are thought to convey the reward prediction and salience signals. Dopamine neurons in a slice preparation fire spontaneously in a regular, pacemaker-like manner at a low frequency (~2–3 Hz). This regular firing appears to be driven by a subthreshold oscillation caused by interaction of a low voltage threshold, noninactivating Ca2+ current and a Ca2+-activated small conductance K+ current. Somatic injection of depolarizing bias current can increase the frequency of sustained firing only up to 10 Hz before the neurons go into depolarization block. Bursts observed in vivo have higher instantaneous frequencies. Two theories have been advanced for how higher frequencies are achieved in vivo. One is that during pacemaking, the natural frequencies of the soma and proximal dendrites drive the subthreshold oscillation, whereas during bursting the NMDA input to the dendrites amplifies the current associated with the distal dendritic oscillation, and drives the soma, resulting in high-frequency spiking. The other hypothesis is that the rapidly varying synaptic input (particularly the AMPA component) is not equivalent to a constant depolarizing pulse but this rapid variation can drive faster spiking that can be observed in response to a constant pulse. We have constructed a realistic multicompartmental model to test the contribution of intrinsic and synaptic currents in the dendrites to the firing pattern. The oscillatory dendrites regularize the firing pattern, decrease the frequency compared to a model in which the subthreshold oscillation is confined to the soma, and contribute to grouping of spikes into bursts.

The intrinsic rhythmicity of spike-burst generation in pancreatic beta-cells and intercellular interaction within an islet.

The pancreatic beta-cell has four types of Ca2+ channel (L-type, T-type, low-threshold slowly inactivating, and low-threshold non-inactivating Ca2+), although the low-threshold non-inactivating Ca2+ channel has not yet been confirmed experimentally. Beside these, there are at least three types of K+ channels (K(ATP), K(Ca,V), and K(V)), and transporters (GLUT-2, Na+/Ca(2+)-countertransporter, and Na+/K(+)-pump) as schematically shown in Fig.4. Opinions on the mechanism of spike-burst are converging to the following view: At intermediate glucose concentrations, the intracellular ATP/ADP ratio oscillates in the following way. A gradual rise in the ATP/ADP ratio causes gradual progression of depolarization to the threshold for the low-threshold Ca2+ channels, of which the opening causes regenerative depolarization to the plateau potential on which spikes (the L-type Ca2+ channel contributes to spike firing) are superimposed. During the active phase, a fall in the ATP/ADP ratio follows a gradual rise in ATP consumption. Slight repolarization due to the opening of a small fraction of K(ATP) channels triggers regenerative repolarization. With the progress of repolarization, a residual fraction of voltage-gated Ca2+ channels (low-threshold non-inactivating) are deactivated. During the silent phase, a gradual rise in the ATP/ ADP ratio leads to gradual depolarization back to the threshold for the next spike-burst. There are still a diversity of views regarding the mechanism of the initial spike-train. On the basis of observations made in various laboratories including ours, we propose the following working model: At low concentrations of glucose, alpha-cells secret glucagon which induces a rise in cAMP in beta-cells lodged in the same islet. A rise in cAMP itself does not activate the enzymes relevant to glycogenolysis, but merely prepares to activate the enzymes. When extracellular glucose increases, Ca2+ spikes are elicited. Influxed Ca2+ ions, together with cAMP, work to activate the enzymes, resulting in an additional supply of fuel for ATP synthesis. After sometime, the cAMP level falls back to a low level and the additional glucose supply from stored glycogen stops. This reaction sequence may be the mechanism behind the initial spike-train. To substantiate this working model, it may be important to elucidate the dependence of the phosphorylasekinase and glycogenphosphorylase activities on the Ca2+ in beta-cells.

Possible contribution of long open state to noninactivating Ca2+ current in detrusor cells

The whole cell patch-clamp technique was used to measure Ca2+ current in isolated smooth muscle cells from guinea pig urinary bladder. Noniactivating Ca2+ channel current was modeled incorporating the long open state of the Ca2+ channel. When inactivation was examined over a wide voltage range, a completely U-shaped curve was obtained. Lack of inactivation at +80 mV could be attributed to the long open state induced by large depolarization as well as to minimal Ca2+ influx and Ca(2+)-dependent inactivation. Activation parameters were obtained by comparing the amplitudes of conditioned (by +80 mV, 5 s) and unconditioned test potentials. With the use of the activation curve and the U-shaped inactivation curve, a noninactivating current that peaks around +20 mV was obtained. This current is composed of a so-called "window" current and a persistent current brought about by the long open state. Differences in the voltage dependence of the development of the long open state in various smooth muscles, as well as differences in the equilibrium constant between open and inactivated states, could underlie the different patterns of contractile behavior that characterize smooth muscles.

Tetrandrine blocks a slow, large-conductance, Ca(2+)-activated potassium channel besides inhibiting a non-inactivating Ca2+ current in isolated nerve terminals of the rat neurohypophysis.

The effects of tetrandrine, a bis-benzyl-isoquinoline alkaloid, on voltage-gated Ca2+ currents (ICa) and on Ca(2+)-activated K+ current (IK(Ca)) and channels in isolated nerve terminals of the rat neurohypophysis were investigated using patch-clamp techniques. The non-inactivating component of ICa was inhibited by external tetrandrine in a voltage- and dose-dependent manner, with an IC50 = 10.1 microM. IK(Ca) was elicited by depolarizations when approximately 10 microM Ca2+ was present on the cytoplasmic side. Only externally applied tetrandrine, at 1 microM, decreased the amplitude of IK(Ca), whereas the fast inward Na+ current and transient outward K+ current were not affected. Tetrandrine, applied to the extracellular side of outside-out patches excised from the nerve terminals, induced frequent and short closures of single type II, maxi-Ca(2+)-activated K+ channels. Tetrandrine decreased the channel-open probability, within bursts, with an IC50 = 0.21 microM. Kinetic analysis of the channel activity showed that the open-time constant decreased linearly with increasing tetrandrine concentrations (0.01-3 microM), giving an association rate constant of 8.8 x 10(8) M-1 s-1, whereas the arithmetic mean closed time did not change, giving a dissociation rate constant of 136.6 s-1. These results show that tetrandrine is a high-affinity blocker of the type II, maxi-Ca(2+)-activated K+ channel of the rat neurohypophysial terminals.

Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum

The hypothesis of a Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is supported by experiments done in skinned cardiac cells (sarcolemma removed by microdissection). According to this hypothesis, the transsarcolemmal Ca2+ influx does not activate the myofilaments directly but through the induction of a Ca2+ release from the SR. The stimulus gating CICR is not a small change in free Ca2+ concentration (delta[free Ca2+]) outside the SR but a function of the rate of this change (delta[free Ca2+/delta t]). The initial relatively fast component of the transsarcolemmal Ca2+ current would trigger Ca2+ release; the subsequent slow component, perhaps corresponding to noninactivating Ca2+ channels, would load the SR with an amount of Ca2+ available for release during subsequent beats. Inactivation of CICR is caused by the large increase of [free Ca2+] outside the SR resulting from Ca2+ release, which inhibits further release. This negative feedback helps to explain that CICR is not all or none. During relaxation the Ca2+ reaccumulation in the SR is backed up by the Ca2+ efflux across the sarcolemma through Na+-Ca2+ exchange and the sarcolemmal Ca2+ pump. Computations of the Ca2+ buffering in the mammalian ventricular cell and of the systolic transsarcolemmal Ca2+ influx do not support the alternative hypothesis that this influx of Ca2+ is large enough to activate the myofilaments directly. Yet the hypothesis of a CICR can be challenged because of many problems and uncertainties related to the preparations and methods used for skinned cardiac cell experiments.

Transmitter glutamate release from isolated nerve terminals: evidence for biphasic release and triggering by localized Ca2+.

The kinetics of Ca2(+)-dependent release of glutamate from guinea-pig cerebrocortical synaptosomes evoked by KCl or 4-aminopyridine are investigated using a continuous fluorimetric assay. Release by both agents is biphasic, with a rapid phase complete within 2 s followed by a more extensive slow phase with a half-maximal release in 52 s for KCl-evoked release and greater than 120 s for 4-aminopyridine-evoked release. The two phases of glutamate release may reflect a dual localization of releasable vesicles at the active zone and in the bulk cytoplasm. Decreasing depolarization depresses the extent rather than increasing the time for half-maximal Ca2(+)-dependent release. Both the fast and the slow phases of glutamate release require external Ca2+ and cytoplasmic ATP. KCl depolarization produces a transient "spike" of cytoplasmic free Ca2+ [( Ca2+]c), which recovers to a plateau; the major component of glutamate release occurs during this plateau. Predepolarization in the absence of added external Ca2+, to inhibit transient Ca2+ channels, does not affect the subsequent glutamate release evoked by Ca2+ readdition. Thus, release involves primarily noninactivating Ca2+ channels. For a given increase in [Ca2+]c, KCl and 4-aminopyridine cause equal release of glutamate, while ionomycin releases much less glutamate. This lowered efficiency is not due to ATP depletion. It is concluded that glutamate exocytosis is evoked by localized Ca2+ entering through noninactivating voltage-dependent Ca2+ channels and that nonlocalized Ca2+ entry with ionomycin is inefficient.

Synaptic modulation by dopamine of calcium currents in rat pars intermedia

Melanotrophs of the rat pars intermedia are innervated by dopaminergic fibers traveling through the pituitary stalk which inhibit secretion via an action on D-2 receptors. As secretion from the melanotroph has been shown to be calcium (Ca2+) dependent, it is possible that dopamine may have an action to inhibit Ca2+ currents in these cells. This possibility was tested by examining the effects of exogenously applied dopaminergic agonists or synaptically released dopamine upon Ca2+ currents recorded under single electrode voltage clamp in intact rat pars intermedia in vitro. Following blockade of sodium and potassium currents in melanotrophs, Ca2+ spikes were elicited with intracellular injection of depolarizing currents; electrical stimulation of the pituitary stalk caused an inhibition of the Ca2(+)-based action potentials which lasted for several seconds. Using single-electrode voltage-clamp techniques, we recorded inward Ca2+ currents corresponding to the T, N, and L types (see Williams et al., 1990). Stimulation of the pituitary stalk inhibited both the low- and high-threshold peak inward Ca2+ currents elicited from a holding potential of -90 mV. In contrast, when noninactivating Ca2+ currents were elicited from a holding potential of -30 mV, the currents were not altered by stalk stimulation. This pattern of inhibition of the Ca2+ currents was consistent with the preferential inhibition, by stalk stimulation, of the N and T Ca2+ currents, while sparing the L current. We observed that inhibition of Ca2+ currents due to stalk stimulation was completely reversed by bath perfusion of domperidone (1 microM), an antagonist of dopamine at the D-2 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in spontaneous firing patterns of rat hippocampal neurones induced by volatile anaesthetics.

1. The effects of the volatile anaesthetics, halothane, isoflurane and enflurane, on rat hippocampal CA1 and CA3 neurones in in vitro preparations were studied by intracellular recording methods. 2. The three anaesthetics, at concentrations similar to those used clinically (0.2-1.2 mM), initially increased and then subsequently decreased the spontaneous firing of CA1 neurones without affecting the resting membrane properties or the EPSPs evoked by focal stimuli. 3. The anaesthetics at these concentrations depressed both the fast after-hyperpolarization of the soma spike and the post-tetanic hyperpolarization induced by repetitive stimulation. They also decreased the IPSPs evoked by focal stimuli. 4. The threshold for spike generation was gradually elevated by as much as 4-6 mV during application of the anaesthetics at these concentrations. The subthreshold potential oscillations (which are likely to be associated with periodic alterations in non-inactivating Ca2+ and Na+ currents) were enhanced in the low concentrations (0.2-0.5 mM), but were depressed in the high concentrations (0.8-1.2 mM). 5. The results suggest that the transient increase in the firing frequency was caused by a depression of both the spike after-hyperpolarization and the post-tetanic hyperpolarization, and that the reduction of spontaneous firing was mainly due to an elevated threshold for spike generation. 6. The three anaesthetics altered the pattern of spontaneous spike-firing in CA3 neurones from solitary spiking to burst firing without affecting the resting membrane properties. 7. The effects of the anaesthetics on the active membrane properties and the postsynaptic potentials in CA3 neurones were similar to the effects in CA1 neurones. 8. In the majority of CA3 neurones, soma spikes elicited by depolarizing current pulses were followed by a Ca2+-dependent after-depolarization, which was in turn followed by a prolonged after-hyperpolarization (post-burst hyperpolarization). The anaesthetics facilitated the after-depolarizing potential, while they depressed the post-burst hyperpolarization. Combination of the two effects would give rise to the highly stereotyped burst (about 1 Hz in frequency) in the presence of the volatile anaesthetics.

Simulated calcium current can both cause calcium loading in and trigger calcium release from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell.

Skinned canine cardiac Purkinje cells were stimulated by regularly repeated microinjection-aspiration sequences that were programmed to simulate the fast initial component of the transsarcolemmal Ca2+ current and the subsequent slow component corresponding to noninactivating Ca2+ channels. The simulated fast component triggered a tension transient through Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum (SR). The simulated slow component did not affect the tension transient during which it was first introduced but it potentiated the subsequent transients. The potentiation was not observed when the SR function had been destroyed by detergent. The potentiation decreased progressively when the slow component was separated by an increasing time interval from the fast component. The potentiation was progressive over several beats under conditions that decreased the rate of Ca2+ accumulation into the SR (deletion of calmodulin from the solutions; a decrease of the temperature from 22 to 12 degrees C). In the presence of a slow component, an increase of frequency caused a positive staircase, and the introduction of an extrasystole caused a postextrasystolic potentiation. There was a negative staircase and no postextrasystolic potentiation in the absence of a slow component. These results can be explained by a time- and Ca2+-dependent functional separation of the release and accumulation processes of the SR, rather than by Ca2+ circulation between anatomically distinct loading and release compartments. The fast initial component of transsarcolemmal Ca2+ current would trigger Ca2+ release, whereas the slow component would load the SR with an amount of Ca2+ available for release during the subsequent tension transients.

Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum.

The hypothesis of a Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is supported by experiments done in skinned cardiac cells (sarcolemma removed by microdissection). According to this hypothesis, the transsarcolemmal Ca2+ influx does not activate the myofilaments directly but through the induction of a Ca2+ release from the SR. The stimulus gating CICR is not a small change in free Ca2+ concentration (delta[free Ca2+]) outside the SR but a function of the rate of this change (delta[free Ca2+/delta t]). The initial relatively fast component of the transsarcolemmal Ca2+ current would trigger Ca2+ release; the subsequent slow component, perhaps corresponding to noninactivating Ca2+ channels, would load the SR with an amount of Ca2+ available for release during subsequent beats. Inactivation of CICR is caused by the large increase of [free Ca2+] outside the SR resulting from Ca2+ release, which inhibits further release. This negative feedback helps to explain that CICR is not all or none. During relaxation the Ca2+ reaccumulation in the SR is backed up by the Ca2+ efflux across the sarcolemma through Na+-Ca2+ exchange and the sarcolemmal Ca2+ pump. Computations of the Ca2+ buffering in the mammalian ventricular cell and of the systolic transsarcolemmal Ca2+ influx do not support the alternative hypothesis that this influx of Ca2+ is large enough to activate the myofilaments directly. Yet the hypothesis of a CICR can be challenged because of many problems and uncertainties related to the preparations and methods used for skinned cardiac cell experiments.

Temporal characteristics of potassium-stimulated acetylcholine release and inactivation of calcium influx in rat brain synaptosomes.

The time course of Ca2+-dependent [3H]acetylcholine [( 3H]ACh) release and inactivation of 45Ca2+ entry were examined in rat brain synaptosomes depolarized by 45 mM [K+]0. Under conditions where the intrasynaptosomal stores of releasable [3H]ACh were neither exhausted nor replenished in the course of stimulation, the K+-evoked release consisted of a major (40% of the releasable [3H]ACh pool), rapidly terminating phase (t1/2 = 17.8 s), and a subsequent, slow efflux that could be detected only during a prolonged, maintained depolarization. The time course of inactivation of K+-stimulated Ca2+ entry suggests the presence of fast-inactivating, slow-inactivating, and noninactivating, or very slowly inactivating, components. The fast-inactivating component of the K+-stimulated Ca2+ entry into synaptosomes appears to be responsible for the rapidly terminating phase of transmitter release during the first 60 s of K+ stimulus. The noninactivating Ca2+ entry may account for the slow phase of transmitter release. These results indicate that under conditions of maintained depolarization of synaptosomes by high [K+]0 the time course and the amount of transmitter released may be a function of the kinetics of inactivation of the voltage-dependent Ca channels.