Separation of the sodium and potassium channels in the surface membrane of mollusk nerve cells]

Characteristics of transmembrane ionic currents under controlled changes in ionic composition of extra-and intracellular medium were studied by means of intracellular dialysis and voltage clamp in isolated neurons from the molluscs Helix pomatia and Limnea stagnalis. The outward potassium currents were eliminated by replacement of intracellular potassium by Tris and the pure inward current could be measured. Replacement of the Ringer solution by NA-free or Ca-free solutions in the extracellular medium made it possible to separate the inward current into additive components, one of which is carried by sodium ions, and the other, by calcium ions. The sodium and calcium inward currents are shown to have different kinetics and potential dependence: taumNa = 1+/-0.5 ms, taumCa = = 3+/-1 ms, tauhNa = 8+/-2 ms, tauhCa = 115+/-10 ms when Vm = 0, GNa = 0.5 when Vm==-21+/-2 mV, GCa = 0.5 when Vm=-8+/-2 mV. Both currents were not altered by tetrodoxin (TTX), however calcium current is specifically blocked by externally applied calcium ions (2 X 10(-3) M), verapamil, D = 600 as well as by fluoride while introduced inside a cell. These data prove the existence of separate systems of sodium and calcium ion-conducting channels in the somatic membrane.     

Blocking of the Ca-dependent inward current in the somatic membrane of mollusk neurons by an elevated intracellular pH

The action of elevated intracellular pHi (pHi) on the transmembrane ionic currents in the somatic membrane was studied in intracellularly perfused nerve cells from Helix pomatia. Following a change in pHi from 7.3 to 9.0 the amplitude of potassium outward current recorded simultaneously with the calcium inward current was significantly reduced. This was accompanied by a shift of its I-V curve to more positive membrane potential values. In case of the calcium inward current blocking by external Cd2+ ions no reduction of the outward current was observed. Only a shift of its I-V curve along the potential axis remained. The calcium inward current was practically the same. It is suggested that the elevated pHi selectively blocks the Ca-dependent component of the potassium outward current.     

Activation of calcium channel currents in rat sensory neurons by large depolarizations: effect of Guanine nucleotides and (-)-baclofen

Calcium channel currents have been recorded from cultured rat sensory neurons at clamp potentials of between -30 and +120 mV. At large depolarizing potentials between +50 and +120 mV, the current was outward. This outward current was shown to be largely due to ions passing through calcium channels, because it was substantially although generally incompletely blocked by Cd2+ (1 mM) and omega-conotoxin (1 microM). Internal GTP-gamma-S (100 microM) and to a lesser extent GTP (1 mM) reduced the amplitude and slowed the activation of the outward, as well as the inward calcium channel current. Baclofen (100 microM) reversibly inhibited both the inward and outward currents. These results suggest that the effect of baclofen and G protein activation on calcium channel currents is not due to a shift in the voltage-dependence of channel availability.     

Separation of potassium and calcium channels in the nerve cell soma membrane]

Calcium inward and potassium outward currents were studied on internally dialysed isolated neurons of the snail Helix pomatia. Different sensitivity of the corresponding channels to changes in external pH was found. This difference was used for separation of their activation regions on the potential axis so that the characteristics of the inward and outward currents could be studied with minimal overlap. It is shown that the outward current channels possess a definite permeability to Tris ions (PTris :PK=0.05). This explains the impossibility to switch off this current by substituting Tris for internal potassium. The channels for the inward calcium current inactivate slowly with a first order kinetic; their instantaneous current-voltage characteristic reveals considerable Goldman-type rectification. The selectivity of the calcium channels to other bivallent cations is Ba:Sr:Ca:Mg=2.8:2.6:1.0:0.2.     

The Secretion of α40-MSH from Xenopus Melanotropes Involves Calcium Influx through ω-Conotoxin-Sensitive Voltage-Operated Calcium Channels

The secretory activity of endocrine cells largely depends on the concentration of free cytosolic calcium. We have studied the mechanisms that are involved in supplying the calcium necessary for the secretion of α-melanophore-stimulating hormone (α-MSH) from melanotrope cells in the pituitary intermediate lobe of the amphibian Xenopus laevis. Using whole-cell voltage clamp, high-voltage activated calcium currents were observed, with a peak current between 0 and +20 mV. Two types of Ca^{2 +}-currents appeared, depending on the experimental setup. An inactivating current, which was observed after a 10 msec depolarizing prepulse, resembled currents through N-type channels as it was clearly inhibited by 1 μM ω-conotoxin. The second type was a non-inactivating current, which was blocked up to 50% by 1 μM nifedipine, indicating its L-type nature. Only a small component of this inactivating current could be blocked by ω-conotoxin. No evidence was found for the presence of transient, low-voltage activated currents. The spontaneous secretion of α-MSH from superfused neurointermediate lobes was dependent on extracellular calcium, as low calcium conditions (10^?4-10^?8 M) rapidly inhibited this process. Under these conditions, secretion was not affected by depolarizing concentrations of potassium chloride. The calcium ionophore A23187 increased secretion under low calcium conditions, but had no effect on spontaneous α-MSH release. Treatment with CoCI₂, a blocker of calcium channels, strongly inhibited the secretory process. These results suggest that spontaneous α-MSH release depends on influx of calcium through voltage-operated calcium channels. Nifedipine did not affect spontaneous secretion from lobes, nor did it affect potassium-induced α-MSH secretion from dispersed melanotropes. Also BAY-K8644, a specific agonist of L-type channels, did not influence α-MSH release, neither under normal nor under low calcium conditions. On the other hand, ω-conotoxin dose-dependently inhibited α-MSH release, to a maximum of 65% at a concentration of 5 μM, and inhibited potassiuminduced secretion by 40%. Thapsigargin, an agent that mobilizes calcium ions from intracellular stores, had no effect on spontaneous α-MSH release under normal or low calcium conditions. From these results it is concluded that the spontaneous release of α-MSH by melanotropes of X. laevis is effectuated by calcium influx through ω-conotoxin-sensitive, voltage-operated N-type calcium channels and that mobilization of calcium from intracellular stores does not play a major role in the regulation of this release.     

Pharmacologic and metabolic dependence of the delayed inactivated potassium outward current in the somatic membrane of snail neurons

Peculiarities of pharmacological and metabolic sensitivities of delayed potassium outward current depending on extracellular calcium ions (IK(Ca(out)) have been studied in experiments on isolated intracellularly perfused Helix neurons. It is shown that verapamil depresses the amplitude and accelerates the inactivation of this current. Blocking effect of verapamil increases with extracellular Ca2+ concentration. Functioning of IK(Ca(out)) channels depends on the intracellular metabolic processes. The current amplitude decreases during the neuron perfusion. Lowering of the intracellular solution temperature to +10 degrees C brings about the analogous result. Addition of ATP (2 mmol/l) and Mg2+ (3 mmol/l) to the intracellular perfusate prevents a decrease of potassium current; intracellular introduction of the exogenous protein kinase C restores the amplitude of this current. Polymyxin B (10(-4) mol/l), a blocker of protein kinase C, depresses the potassium current sensitive to extracellular calcium ions. The possible mechanism of Ca2+ action on IK(Ca(out)) through phosphatidyl-inositol metabolism is discussed.     

Inactivation of potassium outward current depending on the extracellular calcium ions

Inactivation of the potassium outward current depending on the extracellular calcium ions was studied in voltage clamp experiments on nonidentified intracellularly perfused neurons of the snail Helix pomatia. The decay of this current can be approximated by two exponents with time constants of 50-70 ms and 220-300 ms, respectively. The steady-state inactivation depended on the intracellular concentration of K ions. With a decrease of the latter to 20 mmol/l the current was inactivated completely. The inactivation degree was independent of the level of depolarizing shifts of the membrane potential and reduced with a rise of the extracellular K ions concentration. Addition of 5-10 mmol/l K+ to K+-free extracellular solution induced a slow-down of the fast component of the decay (tau = 167 ms) and acceleration of deinactivation. The possible mechanism of inactivation of the investigated current is discussed.     

The effect of intracellular cAMP injections on stationary membrane conductance and voltage- and time-dependent ionic currents in identified snail neurons

3'5'-cAMP injected iontophoretically into identified neurons of the snail, Helix pomatia, induced depolarization of the membrane. Clamping the membrane potential revealed the appearance of a simultaneous inward transmembrane current ('cAMP-current') followed by a weaker outward current. External application of theophylline increased the amplitude of cAMP-current. Imidazole had an opposite effect on this current. Tolbutamide and lowering of temperature largely reduced its rate of rise and, correspondingly, its amplitude. Simultaneous removal of Na+ ions from external solution and addition of Cd2+ ions resulted in complete disappearance of the inward cAMP-current. Analysis of current-voltage characteristics of the cAMP-current at varying external concentrations of Na+, K+, Cl and Ca2+-ions has shown that the cAMP-current is due to an increase of membrane conductance to Na+, K+ and Ca2+ ions; a late component of the cAMP-current is associated with an increase of potassium conductance of the neuronal membrane induced, probably by Ca2+, influx. Besides the induction of stationary currents, cAMP injection also decreased the voltage- and time-dependent calcium currents reducing the maximum calcium conductance. After the end of injection, calcium currents restored their initial value in 1-2 min. An analogous decrease of the calcium current could be evoked by prolonged external application of theophylline. Possible mechanisms of intracellular effects of cAMP on electrical characteristics of the neuronal membrane are discussed.     

Regulation of bradykinin- and ATP-activated Ca(2+)-permeable channels in rat pheochromocytoma (PC12) cells.

Membrane currents activated by bradykinin (500 nM) and by extracellular ATP (50 microM) were studied in voltage-clamped, NGF-treated rat pheochromocytoma (PC12) cells. Under quasiphysiological ionic conditions, both substances caused an outward current due to opening of Ca(2+)-activated K+ channels. Bradykinin caused an additional inward current that could be studied after blockade by internal Cs+ of the initial transient outward current. The inward current became larger when the extracellular Ca2+ concentration was increased. Neither inositol-1,4,5-trisphosphate, dioctanoylglycerol, phorbol 12-myristat 13-acetate, forskolin, GTP, GTP-gamma-S, or pretreatment with pertussis toxin affected this current component. Increasing the internal Ca buffer concentration [EGTA or bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid] from 1 to 10 mM had no effect on the inward current as long as the free [Ca2+]i was kept constant. However, it was modulated by the resting free [Ca2+]i. Elevation of [Ca2+]i from nominally 0 to 60 or to 180 nM increased the bradykinin-induced average peak current density from 0.14 to 1.04 or to 2.29 pA/pF, respectively. This regulation may depend on a calmodulin-dependent pathway, since CGS 9343B, a calmodulin inhibitor, blocked the effect of elevated [Ca2+]i. With ATP as an agonist, outward current was preceded by a large inward current that was partially blocked by extracellular Ca2+ in the millimolar range. Extracellular Ca2+ was also found to reduce the single-channel conductance estimated from outside-out patches treated with ATP.     

Calcium and calcium-activated potassium currents of motor nerve endings in the frog

Evoked electrical responses of nerve endings were recorded in experiments on frog cutaneous-pectoris muscle under visual control. During superfusion by Ca2+-free solution with tubocurarine a late inward current was found in the evoked response of nerve endings recorded by CaCl2 filled electrode. With addition of the external solution of 4-aminopyridine the late current was changed into an outward one. This outward current depended on Ca2+ concentration in the electrode, decreased after local increase of K+ concentration and was abolished by Co2+ ions. Local ionophoretic application of tetraethyl-ammonium eliminated the outward current and revealed a strong and prolonged inward current. Similar currents were recorded by microelectrodes filled with SrCl2, BaCl2 and MgCl2. It is concluded that the late inward current is carried through potential-dependent calcium channels, while the late outward one--through Ca2+-activated K+ channels. The ratio of calcium and Ca2+-activated potassium currents in motor nerve endings and their role in the transmitter release are discussed.     

2 types of calcium-dependent outward potassium currents in the somatic membrane of Helix pomatia neurons

Peculiarities of delayed potassium outward current were studied in voltage clamp experiments on nonidentified intracellularly perfused neurons of the snail Helix pomatia. Together with voltage operated potassium currents which depend on the intracellular Ca2+ ions (IK(Cain], another peculiar calcium-dependent potassium current was shown to exist. This current increases with an increase of the external Ca2+ concentration (IK(Caout] and is insensitive to intracellular administration of EGTA and fluoride. It is blocked by extracellular application of cobalt ions (1.5 mmol/l). As distinct from (IK(Cain), IK(Caout] rapidly reaches a maximum and then inactivates to a steady-state level.     

Effect of ethanol on subthreshold currents of Aplysia pacemaker neurons

The subthreshold currents in bursting pacemaker neurons of the Aplysia abdominal ganglion were individually studied with the voltage clamp technique for sensitivity to 4% ethanol. The most prevalent effect of ethanol on unclamped bursting neurons was a hyperpolarization. This was shown to be due to a decrease of a voltage independent inward leakage current. Direct measurement of the Na-dependent slow inward current showed that this current was eliminated by 4% ethanol. Direct measurement of the Ca-dependent slow inward current showed that this current was substantially reduced by 4% ethanol. Injection of EGTA into cell bodies did not eliminate the ethanol-induced block of the slow inward calcium current. Thus, ethanol cannot be reducing the Ca-dependent slow inward current solely by an increase of internal calcium concentration. The effect of ethanol on voltage dependent outward current was measured by blockage of all inward current. The peak outward current was increased by ethanol. The rate of inactivation of this outward current was also increased. Calcium activated potassium current (IK(Ca)) is particularly complicated in its response to ethanol because it is dependent on both Ca and voltage for its activation. The level of IK(Ca) elicited in response to constant Ca injection was increased by ethanol treatment. The level of this current as activated by voltage clamp pulses was either increased or decreased depending on the neuron type. Ca2+ activated potassium conductance increased e-fold for a 26 mV depolarization in membrane holding potential. Ethanol decreased this voltage dependence to e-fold for a 55 mV change in potential. This result was interpreted to mean that ethanol shifted an effective Ca2+ binding site of these channels from about halfway through the membrane field to one quarter of the way across. The same theoretical approach allowed the further conclusion that ethanol caused an increased internal free calcium concentration probably by decreasing calcium binding by intracellular buffers.     

Single-channel analysis of four distinct classes of potassium channels in Drosophila muscle

A number of mutations have been shown to affect potassium channels in Drosophila muscle. Single-channel analysis of the effects of mutations will prove a powerful approach for studying the molecular mechanisms of ion channel gating. As an initial step towards studying the effects of mutations at the single-channel level, we have characterized wild-type potassium channels in cultured embryonic myotubes using whole-cell, cell-attached, inside-out, and outside-out configurations of the patch-clamp technique. The myotubes differentiate in vitro from primary cultures of late-gastrula stage embryos of Drosophila. The whole-cell outward currents develop in a characteristic sequence. At 8 hr after plating a small delayed outward current is present. Between 10 and 12 hr after plating an A-type outward current develops, followed, between 13 and 16 hr, by a large increase in the delayed current. The A-type current is absent at all developmental stages in myotubes homozygous for the mutant ShKS133. At least 4 different types of potassium channels contribute to the whole-cell outward currents: a fast transient 14 pS A-type potassium channel (A1), a slowly inactivating 14 pS potassium channel (KD), a 40 pS potassium channel that does not inactivate during voltage pulses up to 2.4 sec in duration (KO), and a 90 pS potassium channel that is strongly activated by membrane stretch (KST). Channels indistinguishable from the KD and KST channels were also observed in patch-clamp studies on larval body wall muscle fibers. A1 channels were also present in intact dorsal longitudinal flight muscles. The A1 channel underlies the rapidly inactivating component of the whole-cell current. It inactivates with a similar time course and voltage dependence to the A-current and is similarly blocked by 5 mM 4-aminopyridine. The KD channel underlies a large fraction of the delayed component of the whole-cell current. Ensemble averages of single KD channels inactivate with the same time course as the delayed current. The KO channel represents a smaller fraction of the whole-cell delayed outward current. Its increase in open probability with voltage is due primarily to a voltage dependence of its closed times. The KST channel is voltage and calcium independent and would therefore only contribute to the leak whole-cell current.     

Membrane currents in identified lactotrophs of rat anterior pituitary

Qualitative features of the primary inward and outward current components of identified lactotrophs of the rat anterior pituitary were examined. Identification of lactotrophs in heterogeneous dissociated anterior pituitary cultures was accomplished by application of the reverse hemolytic plaque assay. Currents in lactotrophs were subsequently examined using whole-cell or patch recording techniques. Two components of inward calcium current were observed: a transient component and a sustained component. The transient component activated at voltages as negative as -50 mV and was the major contributor to total lactotroph calcium current. The sustained component activated at voltages above about -10 mV. The 2 currents could be qualitatively separated by differences in inactivation properties and in sensitivity to cadmium. At least 3 components of outward current were distinguished. Either 30 mM TEA or 0 calcium eliminated a major portion of sustained outward current. This is likely to represent primarily calcium- and voltage-activated potassium current. The remaining current could be further differentiated into a transient current component that could be inactivated with conditioning potentials above -60 mV. A slowly activating and deactivating potassium current remained following inactivation of the transient current. Although the time course of the transient current is reminiscent of "A" current, activation of this current required potentials above -30 mV. Candidates for the single-channel currents that underlie the whole-cell outward currents were observed in cell-attached recordings. When combined with patch-clamp electrophysiological methods, the reverse hemolytic plaque assay promises to be a powerful technique for the electrophysiological characterization of specific cell subtypes in heterogeneous dissociated cell populations.     

Blocking action of calmidazolium and chlorpromazine on the potassium outward current which depends on intracellular calcium ions

Effects of calmidazolium (R 24571) and chlorpromazine on the delayed potassium outward current in the somatic membrane were studied on nonidentified intracellularly perfused neurons of the snail Helix pomatia. Extracellular application of these substance evoked depression of the outward current. Inhibition of IK occurs at concentrations of calmodulin inhibitors 10(-9)-10(-8) mol/l. These agents inhibit primarily a component of the potassium current depending on the intracellular Ca2+ ions (IK(Ca in)). The inhibitory effect of these drugs can be explained by calmodulin-like structures of the receptor for intracellular calcium, providing modulation of IK(Ca in).     

Intracellular Ca2+ suppressed a transient potassium current in hippocampal neurons

The effects of intracellular Ca2+ (Ca2+i) on K+ currents in hippocampal cells were examined using acutely isolated cells obtained from adult guinea pigs. Whole-cell voltage-clamp recordings were carried out in a configuration that allowed a continuous perfusion of the intracellular medium. Recording media were made to block inward currents and allowed selective activation of K(+)-dependent outward currents. Voltage-dependent outward currents consisted of an initial rapidly decaying component followed by a sustained component. The time constant of decay of the transient current was about 25 msec, and previous studies (Numann et al., 1987) showed that the kinetic and pharmacological properties of this current closely resembled the A current recorded in invertebrate neurons (Connor and Stevens, 1971; Thompson, 1982). Intracellular perfusion of hippocampal cells with a solution containing elevated Ca2+ (about 4.5 x 10(-4) M) elicited outward currents at the holding potential (-45 to -55 mV) and produced changes in voltage-dependent K+ currents. The transient outward current (IA) activated by depolarization was suppressed with increases in Ca2+i. Delayed, sustained K+ currents were greatly potentiated. Data also showed that, among the 3 effects elicited by Ca2+i, suppression of IA was most sensitive to Ca2+i elevation. Previous results (Numann et al., 1987) showed that IA had a lower threshold (about -45 mV) than sustained currents (about -40 mV). By using low levels of depolarization (-40 mV), IA can be selectively activated, and the suppressive effect of Ca2+i on IA was confirmed on the kinetically isolated IA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nystatin-perforated patch recordings disclose NMDA-induced outward currents in cultured neocortical neurons

Excitatory amino acid-induced responses were studied in cultured rat neocortical neurons using two types of whole-cell patch-clamp recordings. Conventional recording methods, using either KCl or CsCl in the patch pipet, showed N-methyl-D-aspartate (NMDA) currents to be biphasic, consisting of peak and steady-state currents with similar reversal potentials. Recordings obtained with the nystatin-perforated patch method and KCl as the principal intracellular cation disclosed an NMDA-evoked outward current. Outward currents were not seen with either recording method in response to kainate or quisqualate, nor in response to NMDA when CsCl was the major intracellular cation. The NMDA-evoked outward current is attributed to activation of a potassium current by calcium entering through the NMDA channel. This outward current may serve to limit neuronal depolarization during excessive NMDA-mediated excitation.     

Inhibition of delayed rectifier K+ conductance in cultured rat cerebellar granule neurons by activation of calcium-permeable AMPA receptors

Activation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors in cerebellar granule cells during perforated-patch whole-cell recordings activated an inward current at negative voltages which was followed, after a delay, by the inhibition of an outward potassium current at voltages positive to -20 mV. The activated inward current was inwardly rectifying suggesting that the AMPA receptors were Ca2+-permeable. This was confirmed by direct measurements of intracellular calcium where Ca2+ rises were seen following AMPA receptor activation in Na+-free external solution. Ca2+ rises were equally large in the presence of 100 microM Cd2+ to block voltage-gated Ca2+ channels. Specific voltage-protocols, allowing selective activation of the delayed rectifier potassium current (KV) and the transient A current (KA), showed that kainate inhibited KV, but not to any great extent KA. The inhibition of KV was blocked by the AMPA receptor antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and was no longer observed when the KV current was abolished with high concentrations of Ba2+. The responses to kainate were not altered by pre-treating the cells with pertussis toxin, suggesting that the AMPA receptor stimulation of the G-protein Gi cannot account for the effects observed. Replacing extracellular Na+ with choline did not alter the inhibition of KV by kainate, however, removing extracellular Ca2+ reduced the kainate response. The inhibition of KV by kainate was unaffected by the presence of 100 microM Cd2+. The guanylyl cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), did not alter kainate inhibition of KV. It is concluded that ion influx (particularly Ca2+ ions) through AMPA receptor channels following receptor activation leads to an inhibition of KV currents in cerebellar granule neurons.     

Effect of TEA on light emission from aequorin-injected Aplysia central neurons

Aplysia central neurons were injected with the calcium-sensitive photoprotein aequorin and stimulated with trains of identical depolarizing voltage-clamp pulses. The light emissions grew and the outward currents declined in successive pulses.Tetraethylammonium (TEA) enhanced the light emissions to single depolarizing pulses and suppressed the outward current. The remaining net inward current is carried primarily by calcium ions and does not facilitate. The aequorin emissions were larger at all amplitudes of depolarizing pulses that elicited emissions, and the facilitation of emissions in a train of pulses was reduced. The effect of TEA on outward current was nearly maximal when sodium ions were partially replaced with 0.1 M TEA, while the aequorin emissions were further enhanced by increasing the TEA concentration to 0.459 M. TEA enhanced the aequorin emissions at all voltages. These observations suggest that the action of TEA on aequorin emissions is not strictly a consequence of its better known outward current blocking action. The effects of TEA could be partly due to the lowered sodium concentration of these solutions. Replacement of sodium by Tris, sucrose or mannose, however, all produced no enhancement of emissions. Tetramethylammonium (TMA) replacement of sodium had effects similar to those of TEA. Thus TEA and TMA appear to have a specific effect.Part of the enhancement of light emissions by TEA is due to the removal of a series resistance error in the voltage clamp, and this may also account partly for the reduced facilitation of aequorin emissions in TEA. The remainder of the action of TEA on aequorin emissions evidently reflects a specific but previously unrecognized action on the cellular metabolism of calcium ions or on the voltage-dependent calcium channels.     

Ionic mechanism of the outward current induced by intracellular injection of inositol trisphosphate into Aplysia neurons

Inositol 1,4,5-trisphosphate (InsP3) has been proposed to be the intracellular second messenger in the mobilization of Ca2+ from intracellular stores in a variety of cell types. The ionic mechanism of the effect of intracellularly injected InsP3 on the membrane of identified neurons (R9-R12) of Aplysia kurodai was investigated with conventional voltage-clamp, pressure-injection, and ion-substitution techniques. Brief pressure injection of InsP3 into a neuron voltage-clamped at -40 mV reproducibly induced an outward current (10-60 sec in duration, 20-60 nA in amplitude) associated with a conductance increase. The current was increased by depolarization and decreased by hyperpolarization up to -80 mV, where it disappeared. Extracellular application of tetraethylammonium (TEA; 5 mM) blocked the InsP3-induced outward current, and the current was not affected by the presence of bath-applied 4-aminopyridine (4-AP; 5 mM). The InsP3-induced outward current recorded at a holding potential of -40 mV increased in amplitude in low-K+ solutions and decreased in amplitude in high-K+ solutions. Alteration of [Cl-]0, as well as perfusion with Ca2+ free plus 2 mM EGTA solution, did not affect the outward current. The InsP3-induced outward current was found to disappear when the neuron was injected with the Ca2+ chelator EGTA. The outward current evoked by repeated InsP3 injection at low doses exhibited summation and facilitation and, at high doses, was shown to desensitize. The calmodulin inhibitor N-(6-amino-hexyl)-5-chloro-1-naphthalene sulfonamide (W-7; 20-50 microM), inhibited both the InsP3-induced and the Ca2+-activated outward currents. An intracellular pressure injection of Ca2+ ions into the same identified neuron was shown to produce an outward current associated with a K+ conductance increase similar to the InsP3-induced current, and the current was blocked by bath-applied TEA (5mM). These results suggest that brief pressure injection of InsP3 into certain identified neurons of Aplysia induces a 4-AP-resistant, TEA-sensitive K+ current activated by increased intracellular free Ca2+ concentration, and this increase might be the result of the mobilization of Ca2+ from intracellular stores by InsP3.