Phosphorylation enhances inactivation of N-type calcium channel current in bullfrog sympathetic neurons

We have investigated the effects of phosphatase and protein kinase inhibitors on calcium channel currents of bullfrog sympathetic neurons using the whole cell configuration of the patch clamp technique. Intracellular dialysis with the phosphatase inhibitors okadaic acid and calyculin A markedly enhanced the decline of inward current during a depolarizing voltage step. Tail current analysis demonstrated that this was genuine inactivation of calcium channel current, not activation of an outward current. The rapidly inactivating current is N-type calcium current (blocked by -conotoxin and resistant to nifedipine). Staurosporine, a nonselective protein kinase inhibitor, prevented the action of okadaic acid, suggesting that protein phosphorylation is involved. Under control conditions, the time course of inactivation could be described by the sum of two exponentials (= 150 ms and 1200 ms), plus a constant (apparently noninactivating) component, during depolarizations lasting 2 s. Okadaic acid induced a rapid inactivation process (=15 ms) that was absent or negligible under control conditions, without obvious effect on the two slower time constants. As in control cells, inactivation in okadaic-acid-treated cells was strongest near –20 mV, with less inactivation at more positive voltages. However, inactivation did not depend on calcium influx. Modulation of calcium channel activity by phosphorylation may underly the spontaneous shift between inactivating and noninactivating modes recently observed for N-type calcium channels. Differences in basal phosphorylation levels could also explain why N-type calcium channels, originally described as rapidly and completely inactivating, inactivate slowly and incompletely in many neurons.     

Modulation of N-type Ca2+ channel current kinetics by PMA in rat sympathetic neurons

The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) has been used extensively in studies of G protein modulation of Ca2+ channels. PMA has been shown to be a powerful tool for inducing phosphorylation and interrupting G-protein-mediated signaling pathways. Here we re-examine the effects of PMA on whole-cell N-type Ca2+-channel currents in rat sympathetic neurons. We found that, along with an increase in the current amplitude previously reported by others, PMA pretreatment leads to alterations in current activation and inactivation kinetics. These alterations in current kinetics are voltage-dependent and are not reproduced by internal dialysis with the G protein inhibitor GDPbetaS. Alterations in current kinetics by PMA may therefore indicate the existence of a modulated state, presumably phosphorylated, of N-type Ca2+ channels. We propose that the increase in current amplitude is due primarily to alterations in current kinetics rather than to removal of tonic inhibition.     

Domain III regulates N-type (CaV2.2) calcium channel closing kinetics

Ca(V)2.2 (N-type) and Ca(V)1.2 (L-type) calcium channels gate differently in response to membrane depolarization, which is critical to the unique physiological functions mediated by these channels. We wondered if the source for these differences could be identified. As a first step, we examined the effect of domain exchange between N-type and L-type channels on activation-deactivation kinetics, which were significantly different between these channels. Kinetic analysis of chimeric channels revealed N-channel-like deactivation for all chimeric channels containing N-channel domain III, while activation appeared to be a more distributed function across domains. This led us to hypothesize that domain III was an important regulator of N-channel closing. This idea was further examined with R-roscovitine, which is a trisubstituted purine that slows N-channel deactivation by exclusively binding to activated N-channels. L-channels lack this response to roscovitine, which allowed us to use N-L chimeras to test the role of domain III in roscovitine modulation of N-channel deactivation. In support of our hypothesis, all chimeric channels containing the N-channel domain III responded to roscovitine with slowed deactivation, while those chimeric channels with L-channel domain III did not. Thus a combination of kinetic and pharmacological evidence supports the hypothesis that domain III is an important regulator of N-channel closing. Our results support specialization of gating functions among calcium channel domains.     

omega-conotoxin GVIA alters gating charge movement of N-type (CaV2.2) calcium channels

omega-conotoxin GVIA (omegaCTX) is a specific blocker of N-type calcium (CaV2.2) channels that inhibits neuropathic pain. While the toxin appears to be an open channel blocker, we show that N-channel gating charge movement is modulated. Gating currents were recorded from N-channels expressed along with beta2a and alpha2delta subunits in HEK293 cells in external solutions containing either lanthanum and magnesium (La-Mg) or 5 mM Ca2+ plus omegaCTX (omegaCTX-Ca). A comparison showed that omegaCTX induced a 10-mV right shift in the gating charge versus voltage (Q-V) relationship, smaller off-gating current time constant (tau Q(Off)), a lower tau Q(Off) voltage dependence, and smaller on-gating current (Q(On)) tau. We also examined gating current in La-Mg plus omegaCTX and found no significant difference from that in omegaCTX-Ca; this demonstrates that the modulation was induced by the toxin. A model with strongly reduced open-state occupancy reproduced the omegaCTX effect on gating current and showed that the gating modulation alone would inhibit N-current by 50%. This mechanism of N-channel inhibition could be exploited to develop novel analgesics that induce only a partial block of N-current, which may limit some of the side effects associated with the toxin analgesic currently approved for human use (i.e., Prialt).     

Ca2+-dependent inactivation of Ca2+-induced Ca2+ release in bullfrog sympathetic neurons

We studied inactivation of Ca(2+)-induced Ca(2+) release (CICR) via ryanodine receptors (RyRs) in bullfrog sympathetic neurons. The rate of rise in [Ca(2+)](i) due to CICR evoked by a depolarizing pulse decreased markedly within 10-20 ms to a much slower rate despite persistent Ca(2+) entry and little depletion of Ca(2+) stores. The Ca(2+) entry elicited by the subsequent pulse within 50 ms, during which the [Ca(2+)](i) level remained unchanged, did not generate a distinct [Ca(2+)](i) rise. This mode of [Ca(2+)](i) rise was unaffected by a mitochondrial uncoupler, carbonyl cyanide p-trifluromethoxy-phenylhydrazone (FCCP, 1 microm). Paired pulses of varying interval and duration revealed that recovery from inactivation became distinct >or= 50 ms after depolarization and depended on [Ca(2+)](i). The inactivation was prevented by BAPTA (>or= 100 microm) but not by EGTA (相似文献   

Cholinergic control of firing pattern and neurotransmission in rat neostriatal projection neurons: role of CaV2.1 and CaV2.2 Ca2+ channels

Besides a reduction of L-type Ca2+-currents (Ca(V)1), muscarine and the peptidic M1-selective agonist, MT-1, reduced currents through Ca(V)2.1 (P/Q) and Ca(V)2.2 (N) Ca2+ channel types. This modulation was strongly blocked by the peptide MT-7, a specific muscarinic M1-type receptor antagonist but not significantly reduced by the peptide MT-3, a specific muscarinic M4-type receptor antagonist. Accordingly, MT-7, but not MT-3, blocked a muscarinic reduction of the afterhyperpolarizing potential (AHP) and decreased the GABAergic inhibitory postsynaptic currents (IPSCs) produced by axon collaterals that interconnect spiny neurons. Both these functions are known to be dependent on P/Q and N types Ca2+ channels. The action on the AHP had an important effect in increasing firing frequency. The action on the IPSCs was shown to be caused presynaptically as it coursed with an increase in the paired-pulse ratio. These results show: first, that muscarinic M1-type receptor activation is the main cholinergic mechanism that modulates Ca2+ entry through voltage-dependent Ca2+ channels in spiny neurons. Second, this muscarinic modulation produces a postsynaptic facilitation of discharge together with a presynaptic inhibition of the GABAergic control mediated by axon collaterals. Together, both effects would tend to recruit more spiny neurons for the same task.     

Nociceptin is a potent inhibitor of N-type Ca(2+) channels in rat sympathetic ganglion neurons

Nociceptin, an endogenous agonist of the opioid receptor-like(1) (ORL(1)) receptor, is implicated in a wide range of physiological functions including cardiovascular control. However, the effect of nociceptin on peripheral sympathetic ganglion neurons has not been studied. Whole-cell voltage clamp was used to study Ca(2+) currents on freshly dissociated sympathetic superior cervical ganglion neurons from juvenile rats. Nociceptin (1 microM) caused a fast inhibition of the peak currents by 69+/-3% in all neurons. Strong positive prepulses counteracted the inhibition of the peak current by 64% and no effect of nociceptin was observed when the cells were pre-incubated with Pertussis toxin. The inhibition was reversible and dose-dependent with an EC(50) of 508+/-50 pM. Blockade of N-type channels by 1 microM omega-conotoxin GVIA reduced the peak currents by 83+/-1% and abolished the action of nociceptin. Naloxone could not prevent the inhibition by nociceptin and [D-Ala(2), N-Me-Phe(4), Gly(5)-ol] enkephalin (DAMGO) only depressed a small proportion of the current in 1/7 neurons. These data suggests that nociceptin inhibits transmitter release from sympathetic neurons by a selective blockade of N-type channels, which may be of importance for its depressive effect on the cardiovascular system.     

Effect of high Ba(2+) on norepinephrine-induced inhibition of N-type calcium current in bullfrog sympathetic neurons

The voltage-dependent inhibition of N-type calcium current by neurotransmitters is the best-understood example of neuronal calcium channel inhibition. One of the mechanisms by which this pathway is thought to inhibit the calcium current is by reducing the permeation of divalent cations through the channel. In this study one prediction of this hypothesis was examined, that high concentrations of divalent cations reduce the maximum neurotransmitter-induced inhibition. Norepinephrine (NE)-induced inhibition was compared in external solutions containing either 2 or 100 mM Ba(2+). Initially, NE dose-response curves were generated by averaging data from many neurons, and it was found that the relationship was right shifted in the high-Ba(2+) external solution without an effect on maximum inhibition. The IC(50) was 0.6 and 3 microM in 2 and 100 mM Ba(2+), respectively. This shift was verified by comparing the effect of NE on single neurons exposed to both 2 and 100 mM Ba(2+). The inhibition induced by 1 microM NE was reduced in 100 mM Ba(2+) compared with that in 2 mM Ba(2+). However, the response to 100 microM NE was identical between high and low Ba(2+). Thus, divalent cations appear to act as a competitive inhibitor of NE binding, which likely results from these ions' interacting with negatively charged amino acids that are important for catecholamine binding to adrenergic receptors. Because the maximum inhibition induced by NE was similar in low and high Ba(2+), the effect of inhibition on single N-type calcium channels was not altered by the divalent cation concentration.     

Fluorophore-assisted light inactivation produces both targeted and collateral effects on N-type calcium channel modulation in rat sympathetic neurons

The accumulation of amyloid β-protein (Aβ) in brain regions serving memory and cognition is a central pathogenic feature of Alzheimer's disease (AD). We have shown that small soluble oligomers of human Aβ that are naturally secreted by cultured cells inhibit hippocampal long-term potentiation (LTP) in vitro and in vivo and transiently impair the recall of a complex learned behaviour in rats. These results support the hypothesis that diffusible oligomers of Aβ initiate a synaptic dysfunction that may be an early event in AD. We now report detailed electrophysiological analyses that define conditions under which acute application of soluble Aβ inhibits hippocampal synaptic plasticity in wild-type mice. To ascertain which Aβ assemblies contribute to the impairment of LTP, we fractionated oligomers by size-exclusion chromatography and found that Aβ trimers fully inhibit LTP, whereas dimers and tetramers have an intermediate potency. Natural Aβ oligomers are sensitive to heat denaturation, primarily inhibit the induction phase of LTP, and cause a sustained impairment of LTP even after extensive washout. We observed no effects of Aβ oligomers on presynaptic vesicle release. LTP in juvenile mice is resistant to the effects of Aβ oligomers, as is brain-derived-neurotrophic-factor-induced LTP in adult hippocampus. We conclude that specific assemblies, particularly timers, of naturally secreted Aβ oligomers are potent and selective inhibitors of certain forms of hippocampal LTP.     

Microscopic heterogeneity in unitary N-type calcium currents in rat sympathetic neurons.

1. Single N-type calcium (Ca2+) channels in rat superior cervical ganglion neurons display complex patterns of activity in both inactivating and non-inactivating gating modes. Unitary currents were elicited by holding the patch at -90 mV and stepping to +30 mV for 740 ms. Barium (110 mM) was used as the charge carrier. The dihydropyridine agonist (+)-202-791 was included in the bath to ensure that single channel recordings showed no L-type Ca2+ channel mode 2 activity. Using this protocol, we characterized three additional patterns of N-type Ca2+ channel activity named: (1) LLP for large unitary current amplitude (i = -0.92 pA) and low open probability (Po = 0.26); (2) SLP for small unitary current amplitude (i = -0.77 pA) and low open probability (Po = 0.25); and (3) SHP for its small unitary current (i = -0.77 pA) and higher open probability (Po = 0.39). 2. Transitions among these patterns of activity occur more slowly than transitions between closed and open states, resulting in significant clustering of like sweeps. Thus, the complicated gating of single N-type Ca2+ channels can be dissected into multiple, independent modes, each with the same reproducible pattern of activity. 3. This heterogeneous activity is not unique to sympathetic neurons, for inactivating (4), non-inactivating (4), SLP (4) and SHP (3 patches) gating modes were also observed in cell-attached patch recordings (n = 4) of single N-type Ca2+ channels in differentiated phaeochromocytoma (PC12) cells. 4. The 1568 sweeps from four single N-type Ca2+ channel recordings that used the same voltage protocol were categorized by mode to determine the frequency of occurrence of each. Of the 54% of sweeps that showed activity, 42% were inactivating and 58% were non-inactivating. The contribution by each mode to the sustained current was estimated using the equation: I = NPoi, where N is the frequency of occurrence of each mode and Po and i are the mean values of open probability and unitary current amplitude respectively. The LLP mode contributed 18%, the SLP mode 16%, and the SHP mode 66% of the sustained whole cell N-type Ba2+ current. 5. The variability in the incidence among these modes in other cell types may resolve some of the controversy surrounding the characterization of N- and L-type whole cell Ca2+ current components in peripheral neurons. In addition, the number of different modes provides a source of plasticity that may be a target of modulation by neurotransmitters and cellular signals.     

N-arachidonoyl L-serine, a putative endocannabinoid, alters the activation of N-type Ca2+ channels in sympathetic neurons

The effect of N-arachidonoyl L-serine (ARA-S), a recently discovered lipoamino acid found in the CNS, on N-type Ca²⁺ channels of rat sympathetic ganglion neurons was determined using whole cell patch clamp. Application of ARA-S produced a rapid and reversible augmentation of Ca²⁺ current that was voltage dependent and resulted from a hyperpolarizing shift in the activation curve. ARA-S did not influence G protein modulation of Ca²⁺ channels and appeared to act independently of G-protein-coupled receptors. These findings provide a foundation for investigating possible roles for ARA-S in nervous system function.     

A reconfiguration of CaV2 Ca2+ channel current and its dopaminergic D2 modulation in developing neostriatal neurons

The modulatory effect of D(2) dopamine receptor activation on calcium currents was studied in neostriatal projection neurons at two stages of rat development: postnatal day (PD)14 and PD40. D(2)-class receptor agonists reduced whole cell calcium currents by about 35% at both stages, and this effect was blocked by the D(2) receptor antagonist sulpiride. Nitrendipine partially occluded this modulation at both stages, indicating that modulation of Ca(V)1 channels was present throughout this developmental interval. Nevertheless, modulation of Ca(V)1 channels was significantly larger in PD40 neurons. omega-Conotoxin GVIA occluded most of the Ca(2+) current modulation in PD14 neurons. However, this occlusion was greatly decreased in PD40 neurons. omega-Agatoxin TK occluded a great part of the modulation in PD40 neurons but had a negligible effect in PD14 neurons. The data indicate that dopaminergic D(2)-mediated modulation undergoes a change in target during development: from Ca(V)2.2 to Ca(V)2.1 Ca(2+) channels. This change occurred while Ca(V)2.2 channels were being down-regulated and Ca(V)2.1 channels were being up-regulated. Presynaptic modulation mediated by D(2) receptors reflected these changes; Ca(V)2.2 type channels were used for release in young animals but very little in mature animals, suggesting that changes took place simultaneously at the somatodendritic and the synaptic membranes.     

A novel molecular inactivation determinant of voltage-gated CaV1.2 L-type Ca2+ channel

The inactivation of voltage-gated L-type Ca(2+) channels (Ca(V)1) regulates Ca(2+) entry and controls intracellular Ca(2+) levels that are essential for cellular activity. The molecular entities implicated in L-channel (Ca(V)1.2) inactivation are not fully identified. Here we show for the first time the functional impact of one of the two highly conserved clusters of six negatively charged glutamates and aspartate (802-807; poly ED motif) at the II-III loop of the alpha 1 subunits of rabbit of Ca(v)1.2, alpha(1)1.2 and alpha(1)1.2 DeltaN60-Delta1733) on voltage-dependent inactivation. Mutation of the poly ED motif to alanine or glutamine/asparagine greatly enhanced voltage-dependent inactivation, shifting the voltage dependence to negative potentials by >50 mV and conferring a neuronal like inactivation kinetics onto Ca(V)1.2. The large shift in the midpoint of inactivation of the steady-state inactivation kinetics was observed also in Ca(2+) or Ba(2+) and was not altered by the beta2A subunit. Missing from the fast inactivating neuronal P/Q (Ca(V)2.1)-, N (Ca(V)2.2)- or R (Ca(V)2.3)-type channels and modulating Ca(V)1.2 inactivation kinetics, the poly ED motif is likely to be a specific L-type Ca(2+) channels inactivating domain. Our results fit a model in which the poly ED either by itself or as part of a larger inactivating motif acts as Ca(V)1.2 specific built-in "stopper." In this model, Ca(V)1 accomplishes a large Ca(2+) influx during depolarization, possibly by the poly ED hindering occlusion at the pore. Furthermore, the selective designed poly ED perhaps clarifies major inactivation differences between L- and non-L-type calcium channels.     

Comparative analysis of inactivated-state block of N-type (Cav2.2) calcium channels

Objective

The aim of this study was to compare a diverse set of peptide and small-molecule calcium channel blockers for inactivated-state block of native and recombinant N-type calcium channels using fluorescence-based and automated patch-clamp electrophysiology assays.

Methods

The pharmacology of calcium channel blockers was determined at N-type channels in IMR-32 cells and in HEK cells overexpressing the inward rectifying K⁺ channel Kir2.1. N-type channels were opened by increasing extracellular KCl. In the Kir2.1/N-type cell line the membrane potential could be modulated by adjusting the extracellular KCl, allowing determination of resting and inactivated-state block of N-type calcium channels. The potency and degree of state-dependent inhibition of these blockers were also determined by automated patch-clamp electrophysiology.

Results

N-type-mediated calcium influx in IMR-32 cells was determined for a panel of blockers with IC₅₀ values of 0.001?C7???M and this positively correlated with inactivated-state block of recombinant channels measured using electrophysiology. The potency of several compounds was markedly weaker in the state-dependent fluorescence-based assay compared to the electrophysiology assay, although the degree of state-dependent blockade was comparable.

Conclusions

The present data demonstrate that fluorescence-based assays are suitable for assessing the ability of blockers to selectively interact with the inactivated state of the N-type channel.     

Cyclic AMP selectively reduces the N-type calcium current component of mouse sensory neurons in culture by enhancing inactivation

1. The single-electrode voltage-clamp technique was used to assess the effect of elevated intracellular cyclic AMP levels on the three calcium current components of mouse dorsal root ganglion (DRG) neurons in culture. 2. Neither forskolin, cholera toxin, nor 8-Br-cyclic AMP affected the isolated transient low-threshold (T) calcium current. 3. When calcium currents were evoked at clamp potentials (Vc) positive to -20 mV from holding potentials (Vh) near the resting membrane potential, the calcium current consisted primarily of the transient high-threshold (N) and the slowly inactivating high-threshold (L) calcium current components. Under these conditions forskolin, cholera toxin, and 8-Br-cyclic AMP reduced the peak calcium current but had little or no effect on the late (greater than or equal to 300 ms) calcium current. When calcium currents were evoked from very negative Vh, however, there was no effect of these compounds. 4. Forskolin had no effect on the voltage-dependence of the current-voltage relation, nor on the rate of recovery of the calcium current from inactivation. 5. In other experiments, current traces were fitted using a multiexponential curve-fitting program that determined the amplitudes and inactivation time constants (tau i) of the three calcium current components. Forskolin selectively reduced the magnitude of the (curve-fitted) N current, and reduced its tau i. 6. Forskolin also enhanced steady-state inactivation of the N current, producing a -7.5 mV shift in the steady-state inactivation curve. 7. Cholera toxin, forskolin, and 8-Br-cyclic AMP had similar effects on calcium currents in mouse DRG neurons in culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

M(1) and M(2) muscarinic acetylcholine receptor subtypes mediate Ca(2+) channel current inhibition in rat sympathetic stellate ganglion neurons

Muscarinic acetylcholine receptors (mAChRs) are known to mediate the acetylcholine inhibition of Ca(2+) channels in central and peripheral neurons. Stellate ganglion (SG) neurons provide the main sympathetic input to the heart and contribute to the regulation of heart rate and myocardial contractility. Little information is available regarding mAChR regulation of Ca(2+) channels in SG neurons. The purpose of this study was to identify the mAChR subtypes that modulate Ca(2+) channel currents in rat SG neurons innervating heart muscle. Accordingly, the modulation of Ca(2+) channel currents by the muscarinic cholinergic agonist, oxotremorine-methiodide (Oxo-M), and mAChR blockers was examined. Oxo-M-mediated mAChR stimulation led to inhibition of Ca(2+) currents through voltage-dependent (VD) and voltage-independent (VI) pathways. Pre-exposure of SG neurons to the M(1) receptor blocker, M(1)-toxin, resulted in VD inhibition of Ca(2+) currents after Oxo-M application. On the other hand, VI modulation of Ca(2+) currents was observed after pretreatment of cells with methoctramine (M(2) mAChR blocker). The Oxo-M-mediated inhibition was nearly eliminated in the presence of both M(1) and M(2) mAChR blockers but was unaltered when SG neurons were exposed to the M(4) mAChR toxin, M(4)-toxin. Finally, the results from single-cell RT-PCR and immunofluorescence assays indicated that M(1) and M(2) receptors are expressed and located on the surface of SG neurons. Overall, the results indicate that SG neurons that innervate cardiac muscle express M(1) and M(2) mAChR, and activation of these receptors leads to inhibition of Ca(2+) channel currents through VI and VD pathways, respectively.     

Modulation of N-type Ca2+ channels by intracellular pH in chick sensory neurons

Both physiological and pathological neuronal events, many of which elevate intracellular [Ca2+], can produce changes in intracellular pH of between 0.15 and 0.5 U, between pH 7.4 and 6.8. N-type Ca2+ channels, which are intimately involved in exocytosis and other excitable cell processes, are sensitive to intracellular pH changes. However, the pH range over which N-type Ca2+ channels are sensitive, and the sensitivity of N-type Ca2+ channels to small changes in intracellular pH, are unknown. We studied the influence of intracellular pH changes on N-type calcium channel currents in dorsal root ganglion neurons, acutely isolated from 14-day-old chick embryos. Intracellular pH was monitored in patch-clamp recordings with the fluorescent dye, BCECF, and manipulated in both the acidic and basic direction by extracellular application of NH4+ in the presence and absence of intracellular NH4+. Changes in intracellular pH between 6.6 and 7.5 produced a graded change in Ca2+ current magnitude with no apparent shift in activation potential. Intracellular acidification from pH 7.3 to 7.0 reversibly inhibited Ca2+ currents by 40%. Acidification from pH 7.3 to pH 6.6 reversibly inhibited Ca2+ currents by 65%. Alkalinization from pH 7.3 to 7.5 potentiated Ca2+ currents by approximately 40%. Channels were sensitive to pHi changes with high intracellular concentrations of the Ca2+ chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, which indicates that the effects of pHi did not involve a Ca2+-dependent mechanism. These data indicate that N-type Ca2+ channel currents are extremely sensitive to small changes in pHi in the range produced by both physiological and pathological events. Furthermore, these data suggest that modulation of N-type Ca2+ channels by pHi may play an important role in physiological processes that produce small changes in pHi and a protective role in pathological mechanisms that produce larger changes in pHi.     

E(f)-current contributes to whole-cell calcium current in low calcium in frog sympathetic neurons

Because Ca(2+) plays diverse roles in intracellular signaling in neurons, several types of calcium channels are employed to control Ca(2+) influx in these cells. Our experiments focus on resolving the paradox of why whole-cell current has not been observed under typical recording conditions for one type of calcium channel that is highly expressed in frog sympathetic neurons. These channels, referred to as E(f)-channels, are present in the membrane at a density greater than the channels that carry approximately 90% of whole-cell current in low Ba(2+); but, E(f)-current has not been detected in low Ba(2+). Using Ca(2+) instead of Ba(2+) as the charge carrier, we recorded a possible E-type current in frog sympathetic neurons. The current was resistant to specific blockers of N-, L-, and P/Q-type calcium channels but was more sensitive to Ni(2+) block than was N- or L-current. Current amplitude in Ca(2+) is slightly greater than that in Ba(2+). In 3 mM Ca(2+), the current contributed approximately 12% of total current at peak voltage and increased at voltages more hyperpolarized to the peak, reaching approximately 40% at -30 mV, where whole-cell current starts to activate. The presence of E(f)-current in 3 mM Ca(2+) suggests a potential role for E(f)-channels in regulating calcium influx into sympathetic neurons.