Mechanism of action of tubocurarine on nicotinic cholinoreceptors of neurons of the sympathetic ganglia of rats

Tubocurarine (Tc) effect on membrane currents elicited by acetylcholine (ACh) was studied in isolated superior cervical ganglion neurons of rat using patch-clamp method in the whole-cell recording mode. The "use-dependent" block of ACh current by Tc was revealed in the experiments with ACh applications, indicating that Tc blocked the channels opened by ACh. Mean lifetime of Tc-open channel complex, tau, was found to be 9.8 +/- 0.5 s (n = 7) at -50 mV and 20-24 degrees C. tau exponentially increased with membrane hyperpolarization (e-fold change in tau corresponded to the membrane potential shift by 61 mV). Inhibition of the ACh-induced current by Tc (3-30 microM/1) was completely abolished by membrane depolarization to the level of 80-100 mV. Inhibition of ACh-induced current was augmented at increased ACh doses. It is concluded that the open channel block produced by Tc is likely to be the only mechanism for Tc action on nicotinic acetylcholine receptors in superior cervical ganglion neurons of rat.     

Modulation of alpha2beta4 neuronal nicotinic acetylcholine receptors by zinc

A study was made of the modulation of nicotinic acetylcholine receptors by the divalent cation zinc. Rat neuronal nicotinic receptors (alpha2beta4) were expressed in Xenopus oocytes and membrane currents evoked by acetylcholine (ACh currents) were recorded using a two microelectrode voltage clamp. In non-injected oocytes, or in oocytes expressing alpha2beta4 receptors, Zn2+ by itself (1 microM-4 mM) generated only very small membrane currents. In contrast, in oocytes expressing alpha2beta4 receptors, Zn2+ greatly and reversibly increased the ACh current, without affecting considerably its time course. The ACh current potentiation by Zn2+ was weakly dependent on the membrane potential (2.33+/-0.10 times the control current at -100 mV vs 2.04+/-0.06 at -60 mV, suggesting that Zn2+ interacts with the receptor in the vestibule of the ion channel or at an external domain of the protein. The inward rectification of control and Zn2+-potentiated ACh-currents was similar. We conclude that Zn2+ positively and reversibly modulates neuronal nicotinic receptors in a practically voltage-independent manner and without affecting their rate of desensitization. These results will help to understand better the roles played by Zn2+ in brain functions.     

Physostigmine and acetylcholine differentially activate nicotinic receptor subpopulations in Locusta migratoria neurons

The effects of acetylcholine (ACh) and physostigmine (PHY) on thoracic ganglion neurons of Locusta migratoria were investigated using whole-cell and cell-attached voltage clamp. ACh activated whole-cell currents with variable amplitudes, time course and ion channel block between cells, suggesting differential expression of nicotinic acetylcholine receptor (nAChR) subtypes. This was supported by selective block of the peak of the currents by the α7-specific α-conotoxin ImI. PHY at 100 μM evoked smaller whole-cell currents with variable amplitudes and marginal desensitization. The PHY/ACh amplitude ratio varied between cells, and was positively related to the time constant of decay of the ACh response. EC₅₀ values for the peak amplitude of the ACh- and PHY-induced currents were 50 μM and 3 μM, respectively. Both agonists activated nAChR, indicated by equal voltage-dependence and reversal potentials and the same pharmacological properties of ACh and PHY responses. In addition, PHY and ACh induced ion channel block. Co-application and cross-desensitization experiments showed that ACh and PHY activate the same nAChR subpopulations. Both agonists activated nicotinic single channels with three conductance levels, which were equal for ACh and PHY, indicating activation of the same nAChR subtypes by both agonists. However, for all levels PHY displayed a lower open probability than ACh. Taken together, different whole-cell responses appear to originate from differential activation, desensitization and ion channel block by ACh and PHY of distinct nAChR populations.     

Acetylcholine-activated currents in mouse neuroblastoma cells

The nicotinic and muscarinic responses of differentiated mouse neuroblastoma cells from the clonal line N1E 115 to applied cholinergic agents were recorded using single channel and whole cell patch clamp techniques. An inward macroscopic current induced by acetylcholine (ACh) at the resting potential was blocked by curare; cell-attached recordings revealed a single channel conductance of 18 pS and a lifetime of 36 ms at 30°C, with 200 nM ACh. The zero current potential was close to 0 mV. The kinetics of these nicotinic currents were described by multiexponential functions for both the open and closed time distributions. An outward single channel current, present at resting and slightly depolarized potentials, was also observed and has been tentatively described as being dependent on muscarinic receptor activation, as it was usually blocked by atropine. Under our conditions of whole cell clamp, no macroscopic outward current sensitive to ACh was observed.     

Fluoxetine prevents acetylcholine‐induced excitotoxicity blocking human endplate acetylcholine receptor

Introduction: Fluoxetine is an open channel blocker of fetal muscle acetylcholine (ACh) receptor (AChR) and slow‐channel mutant AChRs. It is used commonly to treat patients with slow‐channel congenital myasthenic syndromes. Fluoxetine effects on adult wild‐type endplate AChR are less characterized, although muscle AChR isoforms are differentially modulated by some drugs. Methods: Excitotoxicity assays and patch clamp recordings were performed in human embryonic kidney 293 (HEK) cells expressing wild‐type or slow‐channel mutant human AChRs. Results: Fluoxetine (2–10 μM) abolished ACh‐induced death and decreased ACh‐activated whole‐cell currents in cells expressing all AChR types. In outside‐out patches, fluoxetine rapidly curtailed ACh evoked unitary activity and macroscopic currents. The effect was increased if fluoxetine was applied before ACh. Conclusions: Fluoxetine is an open channel blocker, but it also affects AChR in the closed state. AChR blockade likely underlies the rescue of HEK cells from ACh‐induced death. Muscle Nerve 49 : 90–97, 2014     

Short-term depression and long-term enhancement of ACh-gated channel currents induced by linoleic and linolenic acid

The effects of cis-unsaturated free fatty acids such as linoleic and linolenic acid on ACh-evoked currents were examined using normal and mutant nicotinic acetylcholine (ACh) receptors lacking protein kinase C (PKC) phosphorylation sites on the α and δ subunits expressed in Xenopus oocytes. These free fatty acids reduced ACh-gated channel currents during treatment and to a greater extent in Ca²⁺-free extracellular solution. After treatment, the currents were enhanced as the drug was washed out, but this effect was not observed in the absence of extracellular Ca²⁺. Linolenic acid was more potent of the current enhancement (300% of the control) than linoleic acid (190% of the control). The current enhancement induced by these free fatty acids was inhibited by the selective PKC inhibitor, GF109203X, while the current depression was not affected. Furthermore, these lipids decreased ACh-evoked currents in mutant ACh receptors to the same extent as in normal ACh receptors, but never enhanced the currents. These results indicate that linoleic and linolenic acid have biphasic actions on ACh receptor currents; a short-term depression and a long-term enhancement. The short-term depression may be due to an interaction with the ACh receptor channels, presumably at Ca²⁺ binding sites. The long-lasting enhancement appears to result from Ca²⁺-dependent PKC activation followed by PKC phosphorylation of the ACh receptors. © 1997 Elsevier Science B.V. All rights reserved.     

Different GTP-binding proteins mediate regulation of calcium channels by acetylcholine and noradrenaline in rat sympathetic neurons

In dissociated neurons of rat superior cervical ganglion (SCG), noradrenaline (NA) and acetylcholine (ACh) suppressed Ca2+ currents elicited by depolarizations to 0 mV from -60 mV. With GTP-gamma-S in patch electrodes, ACh and NA caused persistent inhibition of Ca2+ currents. Pretreatment of SCG cells with pertussis toxin abolished the action of ACh but not of NA. The results suggest that ACh and NA reduce the Ca2+ currents in SCG cells through different G proteins.     

Zinc (Zn2+) blocks voltage gated calcium channels in cultured rat dorsal root ganglion cells.

Dorsal root ganglion cells (DRGs) exhibit 3 types of voltage-dependent calcium channels. We have cultured DRGs from 2- to 4-day-old rat pups and obtained whole-cell patch-clamp recordings of calcium-channel currents after 1-5 days in culture. The calcium-channel currents (carried by barium) were recorded with tetrodotoxin (TTX) in the external solution. A cesium-based solution containing Na-ATP, HEPES and EGTA was used in the recording pipette. Cells were held at -80 mV and calcium channel currents were evoked by stepping to depolarized voltages. The divalent cation zinc (Zn2+) blocked sustained and transient voltage sensitive calcium channel currents. Onset of the blockade was fast and a steady-state was reached within 5-15 min, depending upon the concentration used. The IC50 for inhibition of the peak current evoked by a step depolarization from -80 mV to 0 mV (N plus L channels) for 80 ms was 69 microM Zn2+ and the Hill slope about 1. The calcium current evoked by a voltage step from -80 mV to voltages between -40 mV and -15 mV (T-type current) was more sensitive (> 80% block with 20 microM Zn2+). During wash the effect was only partly reversible in 50% of the neurons. Thus, Zn2+ is a potent blocker of voltage dependent calcium currents in mammalian neurons, especially of T-type currents.     

Characterization of depolarization-evoked ERG K+ currents in interstitial cells of Cajal

Interstitial cells of Cajal (ICC) harbour the ether-a-go-go related gene (ERG) channel as shown by its characteristic rapidly deactivating current upon hyperpolarization. This property, however, does not explain the marked increase in cell excitability by ERG channel blockers, namely an increase in slow wave plateau duration and action potential generation. The objective of the present study was to characterize the depolarization-activated, E4031-sensitive ERG currents in murine ICC within a range of physiologically relevant membrane potentials. Whole cell currents were recorded from ICC isolated from murine neonatal jejunum, superfused with a physiological salt solution and with high intracellular Cs(+) to block most other K(+) currents. Upon depolarizing the cell from the resting membrane potential (approximately -60 mV) towards the region of the slow wave plateau (approximately -30 mV), significant sustained (window) current was generated between the potentials of -40 to 0 mV (maximal at -30 mV) and inhibited by the ERG specific blocker E4031. Channel activation followed by rapid inactivation produced a steady state conductance at -30 mV which was 51.6 +/- 11% of the hyperpolarization-evoked peak conductance value at -100 mV. When the cell repolarized from -30 mV, again, significant currents were generated, indicating recovery from inactivation, a typical characteristic of ERG channels. These data provide evidence that the ERG channel is of significance in the regulation of ICC excitability and provide the mechanism by which ERG channel blockade increases the slow wave duration.     

Voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671

A characterization of the properties of voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671 is presented. Membrane currents were recorded under voltage clamp conditions using the patch clamp technique in both the whole-cell and the excised-patch configurations. Macroscopic sodium currents display a typical transient time course with a sigmoidal rise to a peak followed by an exponential decay. The rates of early activation and subsequent inactivation accelerate and approach a maximum in response to test potentials, V, of greater depolarization. The magnitude of peak sodium current increased from negligible values below V = -50 mV and reached a maximum at V = -3.6 mV +/- 2.7 mV (mean +/- S.E.M., n = 12). Sodium currents reversed at V = + 70 mV, near the predicted Nernst equilibrium potential for a Na+ selective channel. The peak sodium conductance, gpeak increased with depolarizing voltages to a maximum at V = approximately 0 mV, exhibiting half-activation voltage at V approximately equal to -36.8 mV and an e-fold change in gpeak/9.5 mV. The Hodgkin-Huxley inactivation parameter h infinity indicates that at V = -73.6 mV half of the sodium currents were inactivated. Single channel current recordings demonstrated the occurrence of discrete events: the latency for first opening was shorter as the depolarizing pulse became more positive. The single-channel current amplitude was ohmic with a slope conductance, gamma = 17.13 pS +/- 0.66 pS. Sodium channel currents were reversibly blocked by tetrodotoxin (TTX).(ABSTRACT TRUNCATED AT 250 WORDS)     

N-Ethylmaleimide modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

The effects of N-ethylmaleimide (NEM), an alkylating reagent to protein sulfhydryl groups, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. When currents were evoked by step depolarizations to 0 mV from a holding potential of −80 mV NEM decreased the amplitude of TTX-S sodium current, but exerted little or no effect on that of TTX-R sodium current. The inhibitory effect of NEM on TTX-S sodium channel was mainly due to the shift of the steady-state inactivation curve in the hyperpolarizing direction. NEM did not affect the voltage-dependence of the activation of TTX-S sodium channel. The steady-state inactivation curve for TTX-R sodium channel was shifted by NEM in the hyperpolarizing direction as that for TTX-S sodium channel. NEM caused a change in the voltage-dependence of the activation of TTX-R sodium channel unlike TTX-S sodium channel. After NEM treatment, the amplitudes of TTX-R sodium currents at test voltages below −10 mV were increased, but those at more positive voltages were not affected. This was explained by the shift in the conductance–voltage curve for TTX-R sodium channels in the hyperpolarizing direction after NEM treatment.     

MK-801 inhibition of nicotinic acetylcholine receptor channels.

MK-801 is a potent inhibitor of the NMDA subtype of glutamate receptors. Single-channel and macroscopic currents indicate that MK-801 also inhibits nicotinic acetylcholine receptors (nAChRs). MK-801 does not significantly increase desensitization of the nAChRs or compete for the ACh binding site. Although there is a slight inhibition of the closed nAChR, the main action of MK-801 is to enter and block the open channel. The voltage dependence for block is consistent with a single binding site within the channel that is 50% of the way through the membrane field. The IC50 for block is 3 microM at -70 mV for currents induced by 0.5 microM ACh. The data from both single-channel and macroscopic currents can be used to estimate a Kd (0) of 7 microM, which is about 40 times higher than the Kd (0) for MK-801 binding to the NMDA receptor. The relative potency of tricyclic compounds like MK-801 for various neurotransmitter systems points out that the pharmacologic action of these drugs could involve complicated interactions in vivo.     

Mechanism of action of the epileptogenic drug pentylenetetrazol on a cloned neuronal potassium channel

The action of the epileptogenic agent pentylenetetrazol (PTZ) on a cloned potassium channel of the rat brain was studied. The Kv1.1 channel was expressed in oocytes ofXenopus laevis and potassium currents were investigated in outside-out and inside-out membrane patches. The results show that PTZ increased the multi-channel potassium currents at strongly negative potentials and decreased them at potentials positive to −35 mV both in outside-out and inside-out membrane patches. The extent and manner of PTZ action, the concentration dependence as well as the onset and time course of the PTZ effect were the same both in outside-out and inside-out membrane patches. The single-channel potassium currents showed an increase in open probability and frequency of opening and a decrease in close time at −50 mV and vice versa at 0 mV with application of PTZ. The amplitude of single-channel current, the open time and the latency to the first channel opening remained almost unchanged under PTZ. The results indicate that PTZ acts via the cell membrane and influences the membrane-associated part of the potassium channel. Thereby, PTZ accelerates the transition from the inactivated to the open state of the channel at strongly negative potentials and reduces it at slightly negative and positive potentials. This mechanism may be the basis for a gate function which is in favour of the development of epileptic discharges.     

Two components of calcium channel current in embryonic chick skeletal muscle cells developing in culture

The properties of the Ca channel currents in chick skeletal muscle cells (myoballs) in culture were studied using a suction pipette technique which allows internal perfusion and voltage clamp. The Ca channel currents as carried by Ba ions were recorded, after suppression of currents through ordinary Na, K and Cl channels by absence of Na, K and Cl ions, by external TEA, by internal EGTA and by observing the Ba currents instead of the Ca currents. Two components of Ba current could be distinguished. One was present only if the myoballs were held at relatively negative holding potentials below -50 mV. This component first became detectable at clamp potentials of about -50 mV and reached a maximum between -10 and -20 mV. During long clamp steps, it became inactivated completely. The inactivation process of this component at a clamp potential of -30 mV was well fitted to a single exponential with a time constant of about -20 ms. Half-maximal steady-state inactivation was observed at -63 mV. The other component persisted even at relatively positive holding potentials above -40 mV, was observed during clamp pulses to -20 mV and above, and reached a maximum between +10 and +20 mV. This component inactivated very little; a substantial fraction of this component remained at the end of clamp pulses lasting 1 s. The inactivation process of this component at a clamp potential of -10 mV apparently followed a single exponential with a time constant of about 1 s. Half-maximal steady-state inactivation was attained at -33 mV. Both components of Ba current were blocked by Co ions, but organic Ca channel blocker D600 preferentially blocked the high-threshold, slowly inactivating component. The relationship between the current amplitude and the concentration of the external Ba ions was different between the two components. Furthermore, the two components of Ba current also differed in their developmental profile. These findings demonstrate the existence of two distinct types of Ca channels in the early stages of chick muscle cell development.     

Capsaicin differentially modulates voltage-activated calcium channel currents in dorsal root ganglion neurones of rats

It is discussed whether capsaicin, an agonist of the pain mediating TRPV1 receptor, decreases or increases voltage-activated calcium channel (VACC) currents (I(Ca(V))). I(Ca(V)) were isolated in cultured dorsal root ganglion (DRG) neurones of rats using the whole cell patch clamp method and Ba2+ as charge carrier. In large diameter neurones (>35 micorm), a concentration of 50 microM was needed to reduce I(Ca(V)) (activated by depolarizations to 0 mV) by 80%, while in small diameter neurones (< or =30 microm), the IC50 was 0.36 microM. This effect was concentration dependent with a threshold below 0.025 microM and maximal blockade (>80%) at 5 microM. The current-voltage relation was shifted to the hyperpolarized direction with an increase of the current between -40 and -10 mV and a decrease between 0 and +50 mV. Isolation of L-, N-, and T-type calcium channels resulted in differential effects when 0.1 microM capsaicin was applied. While T-type channel currents were equally reduced over the voltage range, L-type channel currents were additionally shifted to the hyperpolarized direction by 10 to 20 mV. N-type channel currents expressed either a shift (3 cells) or a reduction of the current (4 cells) or both (3 cells). Thus, capsaicin increases I(Ca(V)) at negative and decreases I(Ca(V)) at positive voltages by differentially affecting L-, N-, and T-type calcium channels. These effects of capsaicin on different VACCs in small DRG neurones, which most likely express the TRPV1 receptor, may represent another mechanism of action of the pungent substance capsaicin in addition to opening of TRPV1.     

Low-voltage-activated calcium channels in human retinoblastoma cells

Retinoblastoma cells represent pluripotent neural-progenitor cells which, if induced to differentiate, express many features of mature human retinal neurons. Ca channel currents were recorded from isolated, undifferentiated human retinoblastoma Y79 cells in a bath solution containing 20 mM BaCl2 using whole-cell patch-clamp pipettes containing CsCl. The transient, macroscopic currents inactivated with a time constant of about 20 ms at -20 mV and had other properties similar to low-voltage-activated calcium channels described in other cell types: Activation curves fit by the Boltzmann relation had a midpoint of -32 mV and a slope factor of 6.8 mV (-80 mV holding potential) and inactivation curves had a midpoint of -40 mV and a slope factor of 3.7 mV. Non-stationary fluctuation analysis of the currents performed over a 2 kHz bandwidth indicated that each channel contributed 0.44 pA to the macroscopic transient current at -20 mV, suggesting a unitary conductance of about 7 pS.     

Characteristics of IA currents in adult rabbit cerebellar Purkinje cells

Classical conditioning the rabbit nictitating membrane involves changes in synaptic and intrinsic membrane properties of cerebellar Purkinje cell dendrites, and a 4-aminopyridine (4-AP)-sensitive potassium channel underlies these membrane properties. We characterized I(A) currents in adult, rabbit Purkinje cells to determine whether I(A) is the target channel involved in learning. Whole-cell recordings of Purkinje cell somas and dendrites revealed a fast activating and inactivating current with half maximal activation at -27.08 +/- 3.48 mV and -25.51 +/- 1.15 mV in somas and dendrites, respectively; half maximal inactivation at -58.91 +/- 2.34 mV and -49.90 +/- 2.58 mV; and a recovery time constant of 22.81 +/- 1.92 ms and 16.60 +/- 4.26 ms. Outside-out patch recordings from cerebellar Purkinje cell somas confirmed these 4-AP-sensitive currents with half maximal activation at -13.85 +/- 1.17 mV and half maximal inactivation at -55.07 +/- 5.54 mV. More importantly, there was an overlap of activation and incomplete inactivation at potentials from -60 to -40 mV, suggesting a "window" current that was responsible for subthreshold variations of membrane potential and might underlie conditioning-specific increases in Purkinje cell excitability. The potassium current was inhibited by 4-AP and by Heteropodatoxin, a specific blocker of Kv4.2 and Kv4.3 channels, but not by Stromatoxin, a blocker of Kv4.2 channels. Mouse monoclonal antibody labeling identified both Kv4.3 and Kv4.2 subunits in the granule cell layer but only Kv4.3 subunits in the molecular layer. This is the first demonstration of A-type currents in adult, rabbit Purkinje cells that may play a role in regulating membrane potential and firing frequency and comprise the target channel mediating conditioning-specific changes of excitability in rabbit Purkinje cell dendrites.     

Fast excitatory postsynaptic currents in voltage-clamped mammalian sympathetic ganglion neurones.

Fast excitatory postsynaptic currents (EPSCs) were recorded in voltage-clamped neurones of isolated superior cervical ganglion of the rabbit. The rise time, decay time and whole duration of EPSC, as well as miniature EPSC, were shorter than those of corresponding postsynaptic potentials. Characteristic impedance for EPSC was 5.5 +/- 1.1 M omega, and was a few times lower than for current evoked by iontophoretic application of ACh. The rise time of EPSC was 2.0 +/- 0.2 msec, the time constant of decay was 3.6 +/- 0.5 msec, and the mean amplitude of EPSC was -5.5 +/- 1.0 nA at the resting potential level (-53.8 +/- 1.4 mV) and at 36 degrees C. Amplitude of EPSC varied with membrane potential almost linearly at negative potentials, non-linearly at positive potentials, and nullified at -8.9 +/- 1.8 mV. The decay of EPSC was exponential over the most of its time course and the rate constant of decay (alpha) varied exponentially with membrane potential according to the relationship alpha(V) = B exp(AV), with A = 0.00716 +/- 0.00101 mV-1 and B = 0.46 +/- 0.07 msec-1. The voltage sensitivity of EPSC decay is interpreted in terms of voltage sensitivity in ionic channel lifetimes.     

Zinc (Zn) blocks voltage gated calcium channels in cultured rat dorsal root ganglion cells

Dorsal root ganglion cells (DRGs) exhibit 3 types of voltage-dependent calcium channels. We have cultured DRGs from 2- to 4-day-old rat pups and obtained whole-cell patch-clamp recordings of calcium-channel currents after 1–5 days in culture. The calcium-channel currents (carried by barium) were recorded with tetrodotoxin (TTX) in the external solution. A cesium-based solution containing Na-ATP, HEPES and EGTA was used in the recording pipette. Cells were held at −80 mV and calcium channel currents were evoked by stepping to depolarized voltages. The divalent cation zinc (Zn²⁺) blocked sustained and transient voltage sensitive calcium channel currents. Onset of the blockade was fast and a steady-state was reached within 5–15 min, depending upin the concentration used. The IC₅₀ for inhibition of the peak current evoked by a step depolarization from −80 mV to 0 mV (N plus L channels) for 80 ms was 69 μM Zn²⁺ and the Hill slope about 1. The calcium current evoked by a voltage step from −80 mV to voltages between −40 mV and −15 mV (T-type current) was more sensitive (> 80% block with 20 μM Zn²⁺). During wash the effect was only partly reversible in 50% of the neurons. Thus, Zn²⁺ is a potent blocker of voltage dependent calcium currents in mammalian neurons, especially of T-type currents.     

Voltage-gated ionic currents in an identified modulatory cell type controlling molluscan feeding

An important modulatory cell type, found in all molluscan feeding networks, was investigated using two-electrode voltage- and current-clamp methods. In the cerebral giant cells of Lymnaea, a transient inward Na+ current was identified with activation at -58 +/- 2 mV. It was sensitive to tetrodotoxin only in high concentrations (approximately 50% block at 100 microm), a characteristic of Na+ channels in many molluscan neurons. A much smaller low-threshold persistent Na+ current (activation at < -90 mV) was also identified. Two purely voltage-sensitive outward K+ currents were also found: (i) a transient A-current type which was activated at -59 +/- 4 mV and blocked by 4-aminopyridine; (ii) a sustained tetraethylammonium-sensitive delayed rectifier current which was activated at -47 +/- 2 mV. There was also evidence that a third, Ca2+-activated, K+ channel made a contribution to the total outward current. No inwardly rectifying currents were found. Two Ca2+ currents were characterized: (i) a transient low-voltage (-65 +/- 2 mV) activated T-type current, which was blocked in NiCl2 (2 mm) and was completely inactivated at approximately -50 mV; (ii) A sustained high voltage (-40 +/- 1 mV) activated current, which was blocked in CdCl2 (100 microm) but not in omega-conotoxin GVIA (10 microm), omega-agatoxin IVA (500 nm) or nifedipine (10 microm). This current was enhanced in Ba2+ saline. Current-clamp experiments revealed how these different current types could define the membrane potential and firing properties of the cerebral giant cells, which are important in shaping the wide-acting modulatory influence of this neuron on the rest of the feeding network.