Afterhyperpolarization current in myenteric neurons of the guinea pig duodenum

Whole cell patch and cell-attached recordings were obtained from neurons in intact ganglia of the myenteric plexus of the guinea pig duodenum. Two classes of neuron were identified electrophysiologically: phasically firing AH neurons that had a pronounced slow afterhyperpolarization (AHP) and tonically firing S neurons that lacked a slow AHP. We investigated the properties of the slow AHP and the underlying current (I(AHP)) to address the roles of Ca(2+) entry and Ca(2+) release in the AHP and the characteristics of the K(+) channels that are activated. AH neurons had a resting potential of -54 mV and the AHP, which followed a volley of three suprathreshold depolarizing current pulses delivered at 50 Hz through the pipette, averaged 11 mV at its peak, which occurred 0.5-1 s following the stimulus. The duration of these AHPs averaged 7 s. Under voltage-clamp conditions, I(AHP)'s were recorded at holding potentials of -50 to -65 mV, following brief depolarization of AH neurons (20-100 ms) to positive potentials (+35 to +50 mV). The null potential of the I(AHP) at its peak was -89 mV. The AHP and I(AHP) were largely blocked by omega-conotoxin GVIA (0.6-1 microM). Both events were markedly decreased by caffeine (2-5 mM) and by ryanodine (10-20 microM) added to the bathing solution. Pharmacological suppression of the I(AHP) with TEA (20 mM) or charybdotoxin (50-100 nM) unmasked an early transient inward current at -55 mV following step depolarization that reversed at -34 mV and was inhibited by niflumic acid (50-100 microM). Mean-variance analysis performed on the decay of the I(AHP) revealed that the AHP K(+) channels have a mean chord conductance of ~10 pS, and there are ~4,000 per AH neuron. Spectral analysis showed that the AHP channels have a mean open dwell time of 2.8 ms. Cell-attached patch recordings from AH neurons confirmed that the channels that open following action currents have a small unitary conductance (10-17 pS) and open with a high probability (相似文献   

Diversity of channels involved in Ca(2+) activation of K(+) channels during the prolonged AHP in guinea-pig sympathetic neurons

The types of Ca(2+)-dependent K(+) channel involved in the prolonged afterhyperpolarization (AHP) in a subgroup of sympathetic neurons have been investigated in guinea pig celiac ganglia in vitro. The conductance underlying the prolonged AHP (gKCa2) was reduced to a variable extent in 100 nM apamin, an antagonist of SK-type Ca(2+)-dependent K(+) channels, and by about 55% in 20 nM iberiotoxin, an antagonist of BK-type Ca(2+)-dependent K(+) channels. The reductions in gKCa2 amplitude by apamin and iberiotoxin were not additive, and a resistant component with an amplitude of nearly 50% of control remained. These data imply that, as well as apamin- and iberiotoxin-sensitive channels, other unknown Ca(2+)-dependent K(+) channels participate in gKCa2. The resistant component of gKCa2 was not abolished by 0.5-10 mM tetraethylammonium, 1 mM 4-aminopyridine, or 5 mM glibenclamide. We also investigated which voltage-gated channels admitted Ca(2+) for the generation of gKCa2. Blockade of Ca(2+) entry through L-type Ca(2+) channels has previously been shown to reduce gKCa2 by about 40%. Blockade of N-type Ca(2+) channels (with 100 nM omega-conotoxin GVIA) and P-type Ca(2+) channels (with 40 nM omega-agatoxin IVA) each reduced the amplitude of gKCa2 by about 35%. Thus Ca(2+) influx through multiple types of voltage-gated Ca(2+) channel can activate the intracellular mechanisms that generate gKCa2. The slow time course of gKCa2 may be explained if activation of multiple K(+) channels results from Ca(2+) influx triggering a kinetically invariant release of Ca(2+) from intracellular stores located close to the membrane.     

Nitric oxide modulates Ca(2+) channels in dorsal root ganglion neurons innervating rat urinary bladder.

The effect of a nitric oxide (NO) donor on high-voltage-activated Ca(2+) channel currents (I(Ca)) was examined using the whole cell patch-clamp technique in L(6)-S(1) dorsal root ganglion (DRG) neurons innervating the urinary bladder. The neurons were labeled by axonal transport of a fluorescent dye, Fast Blue, injected into the bladder wall. Approximately 70% of bladder afferent neurons exhibited tetrodotoxin (TTX)-resistant action potentials (APs), and 93% of these neurons were sensitive to capsaicin, while the remaining neurons had TTX-sensitive spikes and were insensitive to capsaicin. The peak current density of nimodipine-sensitive L-type Ca(2+) channels activated by depolarizing pulses (0 mV) from a holding potential of -60 mV was greater in bladder afferent neurons with TTX-resistant APs (39.2 pA/pF) than in bladder afferent neurons with TTX-sensitive APs (28.9 pA/pF), while the current density of omega-conotoxin GVIA-sensitive N-type Ca(2+) channels was similar (43-45 pA/pF) in both types of neurons. In both types of neurons, the NO donor, S-nitroso-N-acetylpenicillamine (SNAP) (500 microM), reversibly reduced (23.4-26.6%) the amplitude of I(Ca) elicited by depolarizing pulses to 0 mV from a holding potential of -60 mV. SNAP-induced inhibition of I(Ca) was reduced by 90% in the presence of omega-conotoxin GVIA but was unaffected in the presence of nimodipine, indicating that NO-induced inhibition of I(Ca) is mainly confined to N-type Ca(2+) channels. Exposure of the neurons for 30 min to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), an inhibitor of NO-stimulated guanylyl cyclase, prevented the SNAP-induced reduction in I(Ca). Extracellular application of 8-bromo-cGMP (1 mM) mimicked the effects of NO donors by reducing the peak amplitude of I(Ca) (28.6% of reduction). Action potential configuration and firing frequency during depolarizing current pulses were not altered by the application of SNAP (500 microM) in bladder afferent neurons with TTX-resistant and -sensitive APs. These results indicate that NO acting via a cGMP signaling pathway can modulate N-type Ca(2+) channels in DRG neurons innervating the urinary bladder.     

Dendritic voltage-gated ion channels regulate the action potential firing mode of hippocampal CA1 pyramidal neurons.

The role of dendritic voltage-gated ion channels in the generation of action potential bursting was investigated using whole cell patch-clamp recordings from the soma and dendrites of CA1 pyramidal neurons located in hippocampal slices of adult rats. Under control conditions somatic current injections evoked single action potentials that were associated with an afterhyperpolarization (AHP). After localized application of 4-aminopyridine (4-AP) to the distal apical dendritic arborization, the same current injections resulted in the generation of an afterdepolarization (ADP) and multiple action potentials. This burst firing was not observed after localized application of 4-AP to the soma/proximal dendrites. The dendritic 4-AP application allowed large-amplitude Na(+)-dependent action potentials, which were prolonged in duration, to backpropagate into the distal apical dendrites. No change in action potential backpropagation was seen with proximal 4-AP application. Both the ADP and action potential bursting could be inhibited by the bath application of nonspecific concentrations of divalent Ca(2+) channel blockers (NiCl and CdCl). Ca(2+) channel blockade also reduced the dendritic action potential duration without significantly affecting spike amplitude. Low concentrations of TTX (10-50 nM) also reduced the ability of the CA1 neurons to fire in the busting mode. This effect was found to be the result of an inhibition of backpropagating dendritic action potentials and could be overcome through the coordinated injection of transient, large-amplitude depolarizing current into the dendrite. Dendritic current injections were able to restore the burst firing mode (represented as a large ADP) even in the presence of high concentrations of TTX (300-500 microM). These data suggest the role of dendritic Na(+) channels in bursting is to allow somatic/axonal action potentials to backpropagate into the dendrites where they then activate dendritic Ca(2+) channels. Although it appears that most Ca(2+) channel subtypes are important in burst generation, blockade of T- and R-type Ca(2+) channels by NiCl (75 microM) inhibited action potential bursting to a greater extent than L-channel (10 microM nimodipine) or N-, P/Q-type (1 microM omega-conotoxin MVIIC) Ca(2+) channel blockade. This suggest that the Ni-sensitive voltage-gated Ca(2+) channels have the most important role in action potential burst generation. In summary, these data suggest that the activation of dendritic voltage-gated Ca(2+) channels, by large-amplitude backpropagating spikes, provides a prolonged inward current that is capable of generating an ADP and burst of multiple action potentials in the soma of CA1 pyramidal neurons. Dendritic voltage-gated ion channels profoundly regulate the processing and storage of incoming information in CA1 pyramidal neurons by modulating the action potential firing mode from single spiking to burst firing.     

Distinct intracellular calcium profiles following influx through N- versus L-type calcium channels: role of Ca2+-induced Ca2+ release

Selective activation of neuronal functions by Ca(2+) is determined by the kinetic profile of the intracellular calcium ([Ca(2+)](i)) signal in addition to its amplitude. Concurrent electrophysiology and ratiometric calcium imaging were used to measure transmembrane Ca(2+) current and the resulting rise and decay of [Ca(2+)](i) in differentiated pheochromocytoma (PC12) cells. We show that equal amounts of Ca(2+) entering through N-type and L-type voltage-gated Ca(2+) channels result in significantly different [Ca(2+)](i) temporal profiles. When the contribution of N-type channels was reduced by omega-conotoxin MVIIA treatment, a faster [Ca(2+)](i) decay was observed. Conversely, when the contribution of L-type channels was reduced by nifedipine treatment, [Ca(2+)](i) decay was slower. Potentiating L-type current with BayK8644, or inactivating N-type channels by shifting the holding potential to -40 mV, both resulted in a more rapid decay of [Ca(2+)](i). Channel-specific differences in [Ca(2+)](i) decay rates were abolished by depleting intracellular Ca(2+) stores with thapsigargin or by blocking ryanodine receptors with ryanodine, suggesting the involvement of Ca(2+)-induced Ca(2+) release (CICR). Further support for involvement of CICR is provided by the demonstration that caffeine slowed [Ca(2+)](i) decay while ryanodine at high concentrations increased the rate of [Ca(2+)](i) decay. We conclude that Ca(2+) entering through N-type channels is amplified by ryanodine receptor mediated CICR. Channel-specific activation of CICR provides a mechanism whereby the kinetics of intracellular Ca(2+) leaves a fingerprint of the route of entry, potentially encoding the selective activation of a subset of Ca(2+)-sensitive processes within the neuron.     

Characterization of voltage-gated ionic channels in cholinergic amacrine cells in the mouse retina

Recent studies have shown that cholinergic amacrine cells possess unique membrane properties. However, voltage-gated ionic channels in cholinergic amacrine cells have not been characterized systematically. In this study, using electrophysiological and immunohistochemical techniques, we examined voltage-gated ionic channels in a transgenic mouse line the cholinergic amacrine cells of which were selectively labeled with green fluorescent protein (GFP). Voltage-gated K(+) currents contained a 4-aminopyridine-sensitive current (A current) and a tetraethylammonium-sensitive current (delayed rectifier K(+) current). Voltage-gated Ca(2+) currents contained a omega-conotoxin GVIA-sensitive component (N-type) and a omega-Aga IVA-sensitive component (P/Q-type). Tetrodotoxin-sensitive Na(+) currents and dihydropyridine-sensitive Ca(2+) currents (L-type) were not observed. Immunoreactivity for the Na channel subunit (Pan Nav), the K channel subunits (the A-current subunits [Kv. 3.3 and Kv 3.4]) and the Ca channel subunits (alpha1(A) [P/Q-type], alpha1(B) [N-type] and alpha1(C) [L-type]) was detected in the membrane fraction of the mouse retina by Western blot analysis. Immunoreactivity for the Kv. 3.3, Kv 3.4, alpha1(A) [P/Q-type], and alpha1(B) [N-type] was colocalized with the GFP signals. Immunoreactivity for alpha1(C) [L-type] was not colocalized with the GFP signals. Immunoreactivity for Pan Nav did not exist on the membrane surface of the GFP-positive cells. Our findings indicate that signal propagation in cholinergic amacrine cells is mediated by a combination of two types of voltage-gated K(+) currents (the A current and the delayed rectifier K(+) current) and two types of voltage-gated Ca(2+) currents (the P/Q-type and the N-type) in the mouse retina.     

Modulation of Ca(2+) signaling by K(+) channels in a hypothalamic neuronal cell line (GT1-1)

The pulsatile release of gonadotropin releasing hormone (GnRH) is driven by the intrinsic activity of GnRH neurons, which is characterized by bursts of action potentials correlated with oscillatory increases in intracellular Ca(2+). The role of K(+) channels in this spontaneous activity was studied by examining the effects of commonly used K(+) channel blockers on K(+) currents, spontaneous action currents, and spontaneous Ca(2+) signaling. Whole-cell recordings of voltage-gated outward K(+) currents in GT1-1 neurons revealed at least two different components of the current. These included a rapidly activating transient component and a more slowly activating, sustained component. The transient component could be eliminated by a depolarizing prepulse or by bath application of 1.5 mM 4-aminopyridine (4-AP). The sustained component was partially blocked by 2 mM tetraethylammonium (TEA). GT1-1 cells also express inwardly rectifying K(+) currents (I(K(IR))) that were activated by hyperpolarization in the presence of elevated extracellular K(+). These currents were blocked by 100 microM Ba(2+) and unaffected by 2 mM TEA or 1.5 mM 4-AP. TEA and Ba(2+) had distinct effects on the pattern of action current bursts and the resulting Ca(2+) oscillations. TEA increased action current burst duration and increased the amplitude of Ca(2+) oscillations. Ba(2+) caused an increase in the frequency of action current bursts and Ca(2+) oscillations. These results indicate that specific subtypes of K(+) channels in GT1-1 cells can have distinct roles in the amplitude modulation or frequency modulation of Ca(2+) signaling. K(+) current modulation of electrical activity and Ca(2+) signaling may be important in the generation of the patterns of cellular activity responsible for the pulsatile release of GnRH.     

Inhibition of mechanosensitivity in visceral primary afferents by GABAB receptors involves calcium and potassium channels

GABA(B) receptors inhibit mechanosensitivity of visceral afferents. This is associated with reduced triggering of events that lead to gastro-esophageal reflux, with important therapeutic consequences. In other neuronal systems, GABA(B) receptor activation may be linked via G-proteins to reduced N-type Ca(2+) channel opening, increased inward rectifier K(+) channel opening, plus effects on a number of intracellular messengers. Here we aimed to determine the role of Ca(2+) and K(+) channels in the inhibition of vagal afferent mechanoreceptor function by the GABA(B) receptor agonist baclofen. The responses of three types of ferret gastro-esophageal vagal afferents (mucosal, tension and tension mucosal receptors) to graded mechanical stimuli were investigated in vitro. The effects of baclofen (200 microM) alone on these responses were quantified, and the effects of baclofen in the presence of the G-protein-coupled inward rectifier potassium channel blocker Rb(+) (4.7 mM) and/or the N-type calcium channel blocker omega-conotoxin GVIA (0.1 microM). Baclofen inhibition of mucosal receptor mechanosensitivity was abolished by both blockers. Its inhibitory effect on tension mucosal receptors was partly reduced by both. The inhibitory effect of baclofen on tension receptors was unaffected. The data indicate that the inhibitory action of GABA(B) receptors is mediated via different pathways in mucosal, tension and tension mucosal receptors via mechanisms involving both N-type Ca(2+) channels and inwardly rectifying K(+) channels and others.     

Ionic currents underlying fast action potentials in the obliquely striated muscle cells of the octopus arm

The octopus arm provides a unique model for neuromuscular systems of flexible appendages. We previously reported the electrical compactness of the arm muscle cells and their rich excitable properties ranging from fast oscillations to overshooting action potentials. Here we characterize the voltage-activated ionic currents in the muscle cell membrane. We found three depolarization-activated ionic currents: 1) a high-voltage-activated L-type Ca(2+) current, which began activating at approximately -35 mV, was eliminated when Ca(2+) was substituted by Mg(2+), was blocked by nifedipine, and showed Ca(2+)-dependent inactivation. This current had very rapid activation kinetics (peaked within milliseconds) and slow inactivation kinetics (tau in the order of 50 ms). 2) A delayed rectifier K(+) current that was totally blocked by 10 mM TEA and partially blocked by 10 mM 4-aminopyridine (4AP). This current exhibited relatively slow activation kinetics (tau in the order of 15 ms) and inactivated only partially with a time constant of ~150 ms. And 3) a transient A-type K(+) current that was totally blocked by 10 mM 4AP and was partially blocked by 10 mM TEA. This current exhibited very fast activation kinetics (peaked within milliseconds) and inactivated with a time constant in the order of 60 ms. Inactivation of the A-type current was almost complete at -40 mV. No voltage-dependent Na(+) current was found in these cells. The octopus arm muscle cells generate fast (~3 ms) overshooting spikes in physiological conditions that are carried by a slowly inactivating L-type Ca(2+) current.     

Action potential-dependent calcium transients in myenteric S neurons of the guinea-pig ileum.

Simultaneous intracellular microelectrode recording and Fura-2 imaging was used to investigate the relationship between intracellular calcium ion concentration ([Ca2+]i) and excitability of tonic S neurons in intact myenteric plexus of the guinea-pig ileum. S neurons were impaled in myenteric ganglia, at locations near connections with internodal strands. The calcium indicator Fura-2 was loaded via the recording microelectrode. The estimated [Ca2+]i of these neurons was approximately 95 nM (n = 25). Intracellular current injection (200 ms pulses, 0.2 nA, delivered at 0.05 Hz) resulted in action potential firing throughout the stimulus pulse, accompanied by transient increases in [Ca2+]i (to approximately 240 nM, n = 12). Increasing the number of evoked action potentials by increasing stimulus duration (100-500 ms) or intensity (0.05-0.3 nA) produced correspondingly larger [Ca2+]i transients. Single action potentials rarely produced resolvable [Ca2+]i events, while short bursts of action potentials (three to five events) invariably produced resolvable [Ca2+]i increases. Some neurons demonstrated spontaneous action potential firing, which was accompanied by sustained [Ca2+]i increases. Action potential firing and [Ca2+]i increases were also observed by activation of slow synaptic input to these neurons, in cases where the slow depolarization initiated action potential firing. Action potentials (evoked or spontaneous) and associated [Ca2+]i transients were abolished by tetrodotoxin (1 microM). Omega-conotoxin GVIA (100 nM) reduced [Ca2+]i transients by approximately 67%, suggesting that calcium influx through N-type calcium channels contributes to evoked [Ca2+]i increases. The S neurons in this study showed prominent afterhyperpolarizations following bursts of action potential firing. The time-course of afterhyperpolarizations was correlated with the time-course of evoked [Ca2+]i transients. Afterhyperpolarizations were blocked by tetrodotoxin and reduced by omega-conotoxin GVIA, suggesting that calcium influx through N-type channels contributes to these events. The electrical properties of Fura-2-loaded neurons were not significantly different from properties of neurons recorded without Fura-2 injection, suggesting that Fura-2 injection alone does not significantly influence the electrical properties of these cells. These data indicate that myenteric S neurons in situ show prominent, activity-dependent increases in [Ca2+]i. These events can be generated spontaneously, or be evoked by intracellular current injection or synaptic activation. [Ca2+]i transients in these neurons appear to involve action potential-dependent opening of N-type calcium channels, and the elevation in [Ca2+]i increase may underlie afterhyperpolarizations and regulate excitability of these enteric neurons.     

After potentials in nonmyelinated nerve fibers

1. The sucrose-gap technique was employed to examine the different types of after potentials that follow, in desheathed rabbit vagus nerves, a single action potential (AP) elicited by a short (0.4 ms) supramaximal depolarizing pulse. 2. A fast and a slow hyperpolarizing after potential (fHAP and sHAP) as well as a depolarizing after potential (DAP) followed a single spike. Both the fHAP and the sHAP showed a dependence on the K+ electrochemical gradient, indicating that they are due to an outwardly oriented current of K+ ions. 3. The fHAP was sensitive to low concentrations of tetraethylammonium (TEA; 1 mM) and 4-aminopyridine (4-AP; 10 microM) and to millimolar concentrations of Ba2+. We conclude that the fHAP reflects the tail of the delayed rectifier K+ current. 4. The sHAP contained a Ca(2+)-sensitive component that showed a requirement for voltage-dependent Ca2+ entry during the AP. This component was completely blocked by low concentration of TEA (1 mM) and by Cd2+ (1 mM), but unaffected by 4-AP. These observations suggest that it reflects a current flowing through Ca(2+)-activated K+ channels. The remaining, apparently Ca(2+)-insensitive, component was insensitive to 4-AP and could be blocked by TEA only at concentrations greater than 50 mM. 5. The DAP usually appeared when the external concentration of K+ was increased to above approximately 8 mM, but sometimes it was clearly visible even at lower [K+]o. The DAP was TEA insensitive and entirely Ca2+ dependent. This latter property is inconsistent with the widely accepted hypothesis according to which the DAP reflect the accumulation of K+ in the extracellular space during the AP. 6. The origins of both the Ca(2+)-insensitive component of the sHAP and the DAP are not clear. However, in view of the fact that the sucrose-gap technique records not only the membrane potential of the nerve fibers but also of the surrounding glia, there is the possibility that these after potentials reflect changes in the electrical properties of the satellite Schwann cells.     

Voltage-dependent ion channel currents in putative neuroendocrine cells dissociated from the ventral prostate of rat

Prostate neuroendocrine (NE) cells play important roles in the growth and differentiation of the prostate. Following enzymatic digestion of rat ventral prostate, the whole-cell patch-clamp technique was applied to dark, round cells that exhibited chromogranin-A immunoreactivity, a representative marker of NE cells. Under zero current-clamp conditions, putative NE cells showed hyperpolarized resting membrane potentials of some -70 mV, and spontaneous action potentials were induced by an increase in external [K+] or by the injection of current. Using a CsCl pipette solution, step-like depolarization activated high-voltage-activated Ca2+ current (HVA I(Ca)) and tetrodotoxin-resistant voltage-activated Na+ current. The HVA I(Ca) was blocked by nifedipine and omega-conotoxin GVIA, L-type and N-type Ca2+ channel blockers, respectively. Using a KCl pipette solution, the transient outward K+ current (I(to)), Ca2+ -activated K+ currents (I(K,Ca)), the non-inactivating outward current and an inwardly rectifying K+ current (I(Kir)) were identified. I(K,Ca) was suppressed by charybdotoxin (50 nM), iberiotoxin (10 nM) or clotrimazol (1 microM), but not by apamine (100 nM). I(to) was inhibited by 4-aminopyridine (5 mM). I(Kir) was identified as a Ba2+ -sensitive inwardly rectifying current in the presence of a high-K+ bath solution. The voltage- and Ca2+ -activated ion channels could play significant roles in the regulation of neurohormonal secretion in the prostate.     

T-type Ca2+ channels contribute to IBMX/forskolin- and K(+)-induced Ca(2+) transients in porcine olfactory receptor neurons

T-type Ca(2+) channels are low-voltage-activated Ca(2+) channels that control Ca(2+) entry in excitable cells during small depolarization above resting potentials. Using Ca(2+) imaging with a laser scanning confocal microscope we investigated the involvement of T-type Ca(2+) channels in IBMX/forskolin- and sparingly elevated extracellular K(+)-induced Ca(2+) transients in freshly isolated porcine olfactory receptor neurons (ORNs). In the presence of mibefradil (10microM) or Ni(2+) (100microM), the selective T-type Ca(2+) channel inhibitors, IBMX/forskolin-induced Ca(2+) transients in the soma were either strongly (>60%) inhibited or abolished completely. However, the Ca(2+) transients in the knob were only partially (<60%) inhibited. Ca(2+) transients induced by 30mM K(+) were also partially ( approximately 60%) inhibited at both the knob and soma. Furthermore, ORNs responded to as little as a 2.5mM increase in the extracellular K(+) concentration (7.5mM K(+)), and such responses were completely inhibited by mibefradil or Ni(2+). These results reveal functional expression of T-type Ca(2+) channels in porcine ORNs, and suggest a role for these channels in the spread Ca(2+) transients from the knob to the soma during activation of the cAMP cascade following odorant binding to G-protein-coupled receptors on the cilia/knob of ORNs.     

Ionic mechanisms mediating oscillatory membrane potentials in wide-field retinal amacrine cells

Particular types of amacrine cells of the vertebrate retina show oscillatory membrane potentials (OMPs) in response to light stimulation. Historically it has been thought the oscillations arose as a result of circuit properties. In a previous study we found that in some amacrine cells, the ability to oscillate was an intrinsic property of the cell. Here we characterized the ionic mechanisms responsible for the oscillations in wide-field amacrine cells (WFACs) in an effort to better understand the functional properties of the cell. The OMPs were found to be calcium (Ca2+) dependent; blocking voltage-gated Ca2+ channels eliminated the oscillations, whereas elevating extracellular Ca2+ enhanced them. Strong intracellular Ca2+ buffering (10 mM EGTA or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid) eliminated any attenuation in the OMPs as well as a Ca2+-dependent inactivation of the voltage-gated Ca2+ channels. Pharmacological and immunohistochemical characterization revealed that WFACs express L- and N-type voltage-sensitive Ca2+ channels. Block of the L-type channels eliminated the OMPs, but omega-conotoxin GVIA did not, suggesting a different function for the N-type channels. The L-type channels in WFACs are functionally coupled to a set of calcium-dependent potassium (K(Ca)) channels to mediate OMPs. The initiation of OMPs depended on penitrem-A-sensitive (BK) K(Ca) channels, whereas their duration is under apamin-sensitive (SK) K(Ca) channel control. The Ca2+ current is essential to evoke the OMPs and triggering the K(Ca) currents, which here act as resonant currents, enhances the resonance as an amplifying current, influences the filtering characteristics of the cell membrane, and attenuates the OMPs via CDI of the L-type Ca2+ channel.     

Excitatory effect of ATP on acutely dissociated ventromedial hypothalamic neurons of the rat.

The ATP-induced increase in the cytosolic Ca(2+) concentration ([Ca]i) and current in acutely dissociated ventromedial hypothalamic rats neurons were investigated using fura-2 microfluorometry and the nystatin-perforated patch recording method, respectively. The ATP-induced [Ca]i increase was mimicked by dimethyl-thio-ATP and ATPgammaS, and was inhibited by P2 purinoreceptor antagonists. The ATP-induced [Ca]i increase was markedly reduced by removal of external Na(+) or Ca(2+), and by addition of various Ca(2+) channel antagonists. ATP induced a transient inward current exhibiting a strong inward rectification at membrane potentials more positive than -20 mV. The ATP-induced current at a holding potential of -70 mV was concentration-dependent with a half-maximum effective concentration of 26 microM. Increasing the external Ca(2+) concentration to 10 mM shifted the dose-response relationship to the right. ATP induced only a small current and a small increase in [Ca]i, even at 10 mM Ca(2+), when external Na(+) was removed, suggesting the relatively low permeability to Ca(2+) of purinoceptor channels. These results suggest that ATP activates non-selective cation channels by acting on P2X purinoceptors on dissociated ventromedial hypothalamic neurons, which in turn increases [Ca]i by increasing Ca(2+) influx through voltage-dependent Ca(2+) channels.     

Voltage-gated potassium channels activated during action potentials in layer V neocortical pyramidal neurons

To investigate voltage-gated potassium channels underlying action potentials (APs), we simultaneously recorded neuronal APs and single K(+) channel activities, using dual patch-clamp recordings (1 whole cell and 1 cell-attached patch) in single-layer V neocortical pyramidal neurons of rat brain slices. A fast voltage-gated K(+) channel with a conductance of 37 pS (K(f)) opened briefly during AP repolarization. Activation of K(f) channels also was triggered by patch depolarization and did not require Ca(2+) influx. Activation threshold was about -20 mV and inactivation was voltage dependent. Mean duration of channel activities after single APs was 6.1 +/- 0.6 ms (mean +/- SD) at resting membrane potential (-64 mV), 6.7 +/- 0.7 ms at -54 mV, and 62 +/- 15 ms at -24 mV. The activation and inactivation properties suggest that K(f) channels function mainly in AP repolarization but not in regulation of firing. K(f) channels were sensitive to a low concentration of tetraethylammonium (TEA, 1 mM) but not to charybdotoxin (ChTX, 100 nM). Activities of A-type channels (K(A)) also were observed during AP repolarization. K(A) channels were activated by depolarization with a threshold near -45 mV, suggesting that K(A) channels function in both repolarization and timing of APs. Inactivation was voltage dependent with decay time constants of 32 +/- 6 ms at -64 mV (rest), 112 +/- 28 ms at -54 mV, and 367 +/- 34 ms at -24 mV. K(A) channels were localized in clusters and were characterized by steady-state inactivation, multiple subconductance states (36 and 19 pS), and inhibition by 5 mM 4-aminopyridine (4-AP) but not by 1 mM TEA. A delayed rectifier K(+) channel (K(dr)) with a unique conductance of 17 pS was recorded from cell-attached patches with TEA/4-AP-filled pipettes. K(dr) channels were activated by depolarization with a threshold near -25 mV and showed delayed long-lasting activation. K(dr) channels were not activated by single action potentials. Large conductance Ca(2+)-activated K(+) (BK) channels were not triggered by neuronal action potentials in normal slices and only opened as neuronal responses deteriorated (e.g., smaller or absent spikes) and in a spike-independent manner. This study provides direct evidence for different roles of various K(+) channels during action potentials in layer V neocortical pyramidal neurons. K(f) and K(A) channels contribute to AP repolarization, while K(A) channels also regulate repetitive firing. K(dr) channels also may function in regulating repetitive firing, whereas BK channels appear to be activated only in pathological conditions.     

Identical unitary current amplitude and Ca(2+) block of cardiac Na channel before and during beta-adrenergic stimulation

We examined the possibility of Ca(2+) permeation through cardiac Na channels ("slip mode conductance") by an analysis of the voltage-dependent block of Na channels by Ca(2+). A Ca(2+) block of Na channels was evident in rat and guinea pig ventricular myocytes during cell-attached single channel recordings with a physiological ionic environment (140 mM Na(+) and 1 to 10 mM Ca(2+) in the pipette solution). Increasing external Ca(2+) concentration ([Ca(2+)](o)) in the pipette solution reduced the unitary current amplitude predominantly at negative potentials. With [Ca(2+)](o) > 1 mM, unitary current amplitude did not increase at potentials negative to -40 mV in spite of augmented driving forces. The application of 5 microM isoproterenol potentiated the single channel activity elicited by depolarizing pulses from the holding potential of -120 mV, indicating that the channels in the patch under examination were modified by protein kinase A (PKA) stimulation. Increased activity was also confirmed with veratridine-modified Na channels, where channel openings were markedly prolonged. In either case, isoproterenol-induced potentiation neither reduced nor altered the properties of Ca(2+) block of cardiac Na channels, as evidenced by the stable unitary current amplitudes at potential levels from -60 to -20 mV. These results indicate that interactions among Na(+), Ca(2+), and the channel molecule were not modified with respect to permeation properties. They therefore argue against the "slip mode" concept of classical cardiac Na channel if a general concept of ion permeation through "multi-ion pores" is applicable to determine the ionic selectivity of Na channels.     

L-type calcium channel blockade modifies anesthetic actions on aged hippocampal neurons

Previous studies in our laboratory demonstrated a reversal of anesthetic actions on aged neurons by decreasing extracellular [Ca(2+)] in hippocampal slices. Such maneuver indirectly attenuated Ca(2+) influx, hence decreased exogenous intraneuronal Ca(2+) loads during neuronal activity and consequently improved intracellular Ca(2+) concentration homeostasis. Therefore, in the present study we hypothesized that decreasing exogenous Ca(2+) loads by blocking voltage-gated calcium influx in aged neurons would oppose isoflurane actions. Conversely, increasing endogenous Ca(2+) loads by suppressing calcium efflux during forced reversal of Na(+)/Ca(2+) exchanger function would enhance anesthetic effects. Hippocampal slices were prepared from young (2-4 months) and old (24-26 months) Fischer 344 rats. Isoflurane depressed the evoked dendritic field excitatory postsynaptic potentials by approximately 45% in slices taken from old animals. However, application of isoflurane in addition with CoCl(2) or nifedipine opposed the anesthetic actions, which then depressed the evoked dendritic field postsynaptic potentials by only 15%. Selective blockade of the N-type and P/Q-type calcium channels with omega-conotoxin GVIA and omega-conotoxin MVIIC respectively caused rapid but partial depression of synaptic transmission in slices taken from old Fischer 344 rats. However, isoflurane actions in these aged slices were not affected compared with slices perfused only with normal artificial cerebrospinal fluid. Young and aged slices were then exposed to a low sodium perfusate that forces the Na(+)/Ca(2+) exchanger protein into a reverse mode, thus increasing intracellular Ca(2+) concentration. Isoflurane actions under such conditions were profoundly potentiated in aged slices but were not altered in young hippocampi. The current results show that in aged central neurons, selectively blocking L-type calcium channels opposes anesthetic-induced depression of excitatory synaptic transmission. On the contrary, increasing calcium loads in aged neurons potentiates these actions.     

Calcium channels coupled to depolarization-evoked glutamate release in the myenteric plexus of guinea-pig ileum

Glutamate is the major excitatory neurotransmitter in the CNS. The recent characterization of glutamate as a neurotransmitter in the enteric nervous system opened a new line of investigation concerning the role of glutamate in that system. The present study aimed to further characterize the enteric glutamate release and the calcium channels coupled to it. For this study the myenteric plexus-longitudinal muscle of guinea-pig ileum was stimulated with potassium chloride or with electrical pulses. The released glutamate was detected by spectrofluorimetry. Laser scanning confocal microscopy was used for analysis of immunolabeled enteric tissue for co-localization studies of calcium channels (N- and P/Q-type) and glutamate transporters (EAAC1).Here we report the effects of known Ca(2+)-channel blockers on glutamate release evoked by KCl-depolarization or electrical stimulation in the myenteric plexus. We find that N-type Ca(2+) channels control a major portion of evoked glutamate release from this system, with a very small contribution from L-type Ca(2+) channels. Moreover, alpha(1A)-like (P-type Ca(2+) channel) and alpha(1B)-like (N-type Ca(2+ )channel) immunoreactivity co-localized with glutamate transporters in the myenteric plexus. In addition, KCl-evoked or electrically stimulated glutamate release was sensitive to omega-agatoxin IVA, in a frequency-dependent manner, suggesting that P-type channels are also coupled to the release of glutamate. We, thus, conclude that both N-type and P-type Ca(2+) channels control most of the evoked glutamate release from the enteric nervous system, as also occurs in some parts of the CNS.     

Biophysical characterization of whole-cell currents in O2-sensitive neurons from the rat glossopharyngeal nerve

In this study we use nystatin perforated-patch and conventional whole-cell recording to characterize the biophysical properties of neuronal nitric oxide synthase (nNOS)-expressing paraganglion neurons from the rat glossopharyngeal nerve (GPN), that are thought to provide NO-mediated efferent inhibition of carotid body chemoreceptors. These GPN neurons occur in two populations, a proximal one near the bifurcation of the GPN and the carotid sinus nerve, and a more distal one located further along the GPN. Both populations were visualized in whole mounts by vital staining with the styryl pyridinium dye, 4-Di-2-ASP (D289). Following isolation in vitro, proximal and distal neurons had similar input resistances (mean: 1.5 and 1.6 GOmega, respectively), input capacitances (mean: 25.0 and 27.4 pF, respectively), and resting potentials (mean: -53.9 and -53.3 mV, respectively). All neurons had similar voltage-dependent currents composed of: tetrodotoxin (TTX)-sensitive Na+ currents (IC50 approximately 0.2 microM), prolonged and transient Ca2+ currents, and delayed rectifier-type K+ currents. Threshold activation for the Na+ currents was approximately -30 mV and they were inactivated within 10 ms. Inward Ca2+ currents consisted of nifedipine-sensitive L-type, omega-agatoxin IVA-sensitive P/Q-type, omega-conotoxin GVIA-sensitive N-type, SNX-482-sensitive R-type, and Ni2+-sensitive, but SNX-482-insensitive, T-type channels. The voltage-dependent outward K+ currents were sensitive to tetraethylammonium (TEA; 10 mM) and 4-aminopyridine (4-AP; 2 mM). Exposure to a chemosensory stimulus, hypoxia (PO2 range: 80-5 Torr), caused a dose-dependent decrease in K+ current which persisted in the presence of TEA and 4-AP, consistent with the involvement of background K+ channels. Under current clamp, GPN neurons generated TTX-sensitive action potentials, and in spontaneously active neurons, hypoxia caused membrane depolarization and an increase in firing frequency. These properties endow GPN neurons with an exquisite ability to regulate carotid body chemoreceptor function during hypoxia, via voltage-gated Ca2+-entry, activation of nNOS, and release of NO.