Neil S Magoski

Queen's University, Kingston, Ontario, Canada

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Publications (23)104.69 Total impact

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    ABSTRACT: Neuroendocrine secretion often requires prolonged voltage-gated Ca(2+) entry; however, the ability of Ca(2+) from intracellular stores, such as endoplasmic reticulum or mitochondria, to elicit secretion is less clear. We examined this using the bag cell neurons, which trigger ovulation in Aplysia by releasing egg-laying hormone (ELH) peptide. Secretion from cultured bag cell neurons was observed as an increase in plasma membrane capacitance following Ca(2+) influx evoked by a 5-Hz, 1-min train of depolarizing steps under voltage-clamp. The response was similar for step durations of >50 ms, but fell off sharply with shorter stimuli. The capacitance change was attenuated by replacing external Ca(2+) with Ba(2+), blocking Ca(2+) channels, buffering intracellular Ca(2+) with EGTA, disrupting synaptic protein recycling, or genetic knock-down of ELH. Regarding intracellular stores, liberating mitochondrial Ca(2+) with the protonophore, carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP), brought about an EGTA-sensitive elevation of capacitance. Conversely, no change was observed to Ca(2+) released from endoplasmic reticulum or acidic stores. Prior exposure to FCCP lessened the train-induced capacitance increase, suggesting overlap in the pool of releasable vesicles. Employing GTP-γ-S to interfere with endocytosis delayed recovery (presumed membrane retrieval) of the capacitance change following FCCP, but not the train. Finally, secretion was correlated with reproductive behaviour, in that neurons isolated from animals engaged in egg-laying presented a greater train-induced capacitance elevation vs quiescent animals. The bag cell neuron capacitance increase is consistent with peptide secretion requiring high Ca(2+), either from influx or stores, and may reflect the all-or-none nature of reproduction.
    Neuroscience 07/2013; · 3.12 Impact Factor
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    ABSTRACT: Although the contribution of Ca(2+) buffering systems can vary between neuronal types and cellular compartments, it is unknown whether distinct Ca(2+) sources within a neuron have different buffers. As individual Ca(2+) sources can have separate functions, we propose that each is handled by unique systems. Using Aplysia californica bag cell neurons, which initiate reproduction through an afterdischarge involving multiple Ca(2+)-dependent processes, we investigated the role of endoplasmic reticulum (ER) and mitochondrial sequestration, as well as extrusion via the plasma membrane Ca(2+)-ATPase (PMCA) and Na(+)/Ca(2+) exchanger, to the clearance of voltage-gated Ca(2+) influx, Ca(2+)-induced Ca(2+)-release (CICR), and store-operated Ca(2+) influx. Cultured bag cell neurons were filled with the Ca(2+) indicator, fura-PE3, to image Ca(2+) under whole-cell voltage clamp. A 5 Hz, 1 min train of depolarizing voltage steps elicited voltage-gated Ca(2+) influx followed by EGTA-sensitive CICR from the mitochondria. A compartment model of Ca(2+) indicated the effect of EGTA on CICR was due to buffering of released mitochondrial Ca(2+) rather than uptake competition. Removal of voltage-gated Ca(2+) influx was dominated by the mitochondria and PMCA, with no contribution from the Na(+)/Ca(2+) exchanger or sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA). In contrast, CICR recovery was slowed by eliminating the Na(+)/Ca(2+) exchanger and PMCA. Last, store-operated influx, evoked by ER depletion, was removed by the SERCA and depended on the mitochondrial membrane potential. Our results demonstrate that distinct buffering systems are dedicated to particular Ca(2+) sources. In general, this may represent a means to differentially regulate Ca(2+)-dependent processes, and for Aplysia, influence how reproductive behavior is triggered.
    Journal of Neuroscience 04/2013; 33(15):6476-91. · 6.91 Impact Factor
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    ABSTRACT: How aging affects the communication between neurons is poorly understood. To address this question, we have studied the electrophysiological properties of identified neuron R15 of the marine mollusk Aplysia californica. R15 is a bursting neuron in the abdominal ganglia of the central nervous system and is implicated in reproduction, water balance, and heart function. Exposure to acetylcholine (ACh) causes an increase in R15 burst firing. Whole-cell recordings of R15 in the intact ganglia dissected from mature and old Aplysia showed specific changes in burst firing and properties of action potentials induced by ACh. We found that while there were no significant changes in resting membrane potential and latency in response to ACh, the burst number and burst duration is altered during aging. The action potential waveform analysis showed that unlike mature neurons, the duration of depolarization and the repolarization amplitude and duration did not change in old neurons in response to ACh. Furthermore, single neuron quantitative analysis of acetylcholine receptors (AChRs) suggested alteration of expression of specific AChRs in R15 neurons during aging. These results suggest a defect in cholinergic transmission during aging of the R15 neuron.
    PLoS ONE 01/2013; 8(12):e84793. · 3.73 Impact Factor
  • Sean H White, Neil S Magoski
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    ABSTRACT: A brief synaptic input to the bag cell neurons of Aplysia evokes a lengthy afterdischarge and the secretion of peptide hormones that trigger ovulation. The input transmitter is unknown, although prior work has shown that afterdischarges are prevented by strychnine. Because molluscan excitatory cholinergic synapses are blocked by strychnine, we tested the hypothesis that acetylcholine acts on an ionotropic receptor to initiate the afterdischarge. In cultured bag cell neurons, acetylcholine induced a short burst of action potentials followed by either return to near baseline or, like a true afterdischarge, transition to continuous firing. The current underlying the acetylcholine-induced depolarization was dose dependent, associated with increased membrane conductance, and sensitive to the nicotinic antagonists hexamethonium, mecamylamine, and α-conotoxin ImI. Whereas nicotine, choline, carbachol, and glycine did not mimic acetylcholine, tetramethylammonium did produce a similar current. Consistent with an ionotropic receptor, the response was not altered by intracellular dialysis with the G protein blocker guanosine 5'-(β-thio)diphosphate. Recording from the intact bag cell neuron cluster showed acetylcholine to evoke prominent depolarization, which often led to extended bursting, but only in the presence of the acetylcholinesterase inhibitor neostigmine. Extracellular recording confirmed that exogenous acetylcholine caused genuine afterdischarges, which, as per those generated synaptically, rendered the cluster refractory to further stimulation. Finally, treatment with a combination of mecamylamine and α-conotoxin ImI blocked synaptically induced afterdischarges in the intact bag cell neuron cluster. Acetylcholine appears to elicit the afterdischarge through an ionotropic receptor. This represents an expedient means for transient stimulation to elicit prolonged firing in the absence of ongoing synaptic input.
    Journal of Neurophysiology 02/2012; 107(10):2672-85. · 3.30 Impact Factor
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    ABSTRACT: Targeting signalling molecules to ion channels can expedite regulation and assure the proper transition of changes to excitability. In the bag cell neurons of Aplysia, single-channel studies of excised patches have revealed that protein kinase C (PKC) gates a non-selective cation channel through a close, physical association. This channel drives a prolonged afterdischarge and concomitant neuropeptide secretion to provoke reproductive behaviour. However, it is not clear if PKC alters cation channel function and/or the membrane potential at the whole-cell level. Afterdischarge-like depolarizations can be evoked in cultured bag cell neurons by bath-application of Conus textile venom (CtVm), which triggers the cation channel through an apparent intracellular pathway. The present study shows that the CtVm-induced depolarization was reduced by nearly 50% compared to control following dialysis with the G-protein blocker, guanosine-5'-O-2-thiodiphosphate (GDP-β-S), or treatment with either the phospholipase C inhibitor, 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), or the PKC inhibitor, sphinganine. Neurons exposed to the PKC activator, phorbol 12-myristate 13-acetate (PMA), displayed depolarization with accompanying spiking, and were found to be far more responsive to depolarizing current injection versus control. Immunocytochemical staining for the two typical Aplysia PKC isoforms, Apl I and Apl II, revealed that both kinases were present in unstimulated cultured bag cell neurons. However, in CtVm-treated neurons, the staining intensity for PKC Apl I increased, peaking at 10 min post-application. Conversely, the intensity of PKC Apl II staining decreased over the duration of CtVm exposure. Our results suggest that the CtVm-induced depolarization involves PKC activation, and is consistent with prior work showing PKC closely-associating with the cation channel to produce the depolarization necessary for the afterdischarge and species propagation.
    Neuroscience 01/2011; 179:41-55. · 3.12 Impact Factor
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    ABSTRACT: Ion channels may be gated by Ca(2+) entering from the extracellular space or released from intracellular stores--typically the endoplasmic reticulum. The present study examines how Ca(2+) impacts ion channels in the bag cell neurons of Aplysia californica. These neuroendocrine cells trigger ovulation through an afterdischarge involving Ca(2+) influx from Ca(2+) channels and Ca(2+) release from both the mitochondria and endoplasmic reticulum. Liberating mitochondrial Ca(2+) with the protonophore, carbonyl cyanide-4-trifluoromethoxyphenyl-hydrazone (FCCP), depolarized bag cell neurons, whereas depleting endoplasmic reticulum Ca(2+) with the Ca(2+)-ATPase inhibitor, cyclopiazonic acid, did not. In a concentration-dependent manner, FCCP elicited an inward current associated with an increase in conductance and a linear current/voltage relationship that reversed near -40 mV. The reversal potential was unaffected by changing intracellular Cl(-), but left-shifted when extracellular Ca(2+) was removed and right-shifted when intracellular K(+) was decreased. Strong buffering of intracellular Ca(2+) decreased the current, although the response was not altered by blocking Ca(2+)-dependent proteases. Furthermore, fura imaging demonstrated that FCCP elevated intracellular Ca(2+) with a time course similar to the current itself. Inhibiting either the V-type H(+)-ATPase or the ATP synthetase failed to produce a current, ruling out acidic Ca(2+) stores or disruption of ATP production as mechanisms for the FCCP response. Similarly, any involvement of reactive oxygen species potentially produced by mitochondrial depolarization was mitigated by the fact that dialysis with xanthine/xanthine oxidase did not evoke an inward current. However, both the FCCP-induced current and Ca(2+) elevation were diminished by disabling the mitochondrial permeability transition pore with the alkylating agent, N-ethylmaleimide. The data suggest that mitochondrial Ca(2+) gates a voltage-independent, nonselective cation current with the potential to drive the afterdischarge and contribute to reproduction. Employing Ca(2+) from mitochondria, rather than the more common endoplasmic reticulum, represents a diversification of the mechanisms that influence neuronal activity.
    Journal of Neurophysiology 03/2010; 103(3):1543-56. · 3.30 Impact Factor
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    ABSTRACT: Neurons may initiate behavior or store information by translating prior activity into a lengthy change in excitability. For example, brief input to the bag cell neurons of Aplysia results in an approximate 30-min afterdischarge that induces reproduction. Similarly, momentary stimulation of cultured bag cells neurons evokes a prolonged depolarization lasting many minutes. Contributing to this is a voltage-independent cation current activated by Ca(2+) entering during the stimulus. However, the cation current is relatively short-lived, and we hypothesized that a second, voltage-dependent persistent current sustains the prolonged depolarization. In bag cell neurons, the inward voltage-dependent current is carried by Ca(2+); thus we tested for persistent Ca(2+) current in primary culture under voltage clamp. The observed current activated between -40 and -50 mV exhibited a very slow decay, presented a similar magnitude regardless of stimulus duration (10-60 s), and, like the rapid Ca(2+) current, was enhanced when Ba(2+) was the permeant ion. The rapid and persistent Ca(2+) current, but not the cation current, were Ni(2+) sensitive. Consistent with the persistent current contributing to the response, Ni(2+) reduced the amplitude of a prolonged depolarization evoked under current clamp. Finally, protein kinase C activation enhanced the rapid and persistent Ca(2+) current as well as increased the prolonged depolarization when elicited by an action potential-independent stimulus. Thus the prolonged depolarization arises from Ca(2+) influx triggering a cation current, followed by voltage-dependent activation of a persistent Ca(2+) current and is subject to modulation. Such synergy between currents may represent a common means of achieving activity-dependent changes to excitability.
    Journal of Neurophysiology 10/2009; 102(6):3753-65. · 3.30 Impact Factor
  • J E Geiger, C M Hickey, N S Magoski
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    ABSTRACT: Ca(2+) influx through voltage-gated Ca(2+) channels is a fundamental signaling event in neurons; however, non-traditional routes, such as non-selective cation channels, also permit Ca(2+) entry. The present study examines the Ca(2+) permeability of a cation channel that drives an afterdischarge in Aplysia bag cell neurons. The firing of these neurons induces peptide release and reproduction. Single channel-containing inside-out patches excised from cultured bag cell neurons, with the cytoplasmic face bathed in K(+)-aspartate and the extracellular face bathed in artificial seawater (11 mM Ca(2+)), had a reversal potential near +50 mV. In keeping with Ca(2+) permeability, this was right-shifted to approximately +60 mV in high Ca(2+) (substituted for Mg(2+)) and left-shifted to around +40 mV in zero Ca(2+) (replaced with Mg(2+)). The current showed inward rectification between +30 and +90 mV, and a conductance of 29 pS in normal Ca(2+), 30 pS in high Ca(2+), 32 pS in Ba(2+) (substituted for Ca(2+)), but only 21 pS in zero Ca(2+). Despite a greater conductance in Ba(2+), the channel did not display anomalous mol fraction in an equimolar Ca(2+)-Ba(2+) mix. Eliminating internal Mg(2+) lowered activity, but did not alter inward rectification, suggesting intracellular Mg(2+) is a fast, voltage-independent blocker. Imaging bag cell neurons in Mn(2+) saline (substituted for Ca(2+)) revealed enhanced fura-quench following cation channel activation, consistent with Mn(2+) permeating as a Ca(2+) surrogate. Finally, triggering the cation channel while tracking capacitance revealed a Ca(2+)-dependent increase in membrane surface area, consistent with vesicle fusion. Thus, the cation channel not only drives the afterdischarge, but also passes Ca(2+) to potentially initiate secretion. In general, this may represent an alternate means by which neurons elicit neuropeptide release.
    Neuroscience 06/2009; 162(4):1023-38. · 3.12 Impact Factor
  • Kate E Gardam, Neil S Magoski
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    ABSTRACT: Ion channel regulation is key to controlling neuronal excitability. However, the extent that modulators and gating factors interact to regulate channels is less clear. For Aplysia, a nonselective cation channel plays an essential role in reproduction by driving an afterdischarge in the bag cell neurons to elicit egg-laying hormone secretion. We examined the regulation of cation channel voltage and Ca2+ dependence by protein kinase C (PKC) and inositol trisphosphate (IP3)-two prominent afterdischarge signals. In excised, inside-out patches, the channel remained open longer and reopened more often with depolarization from -90 to +30 mV. As previously reported, PKC could closely associate with the channel and increase activity at -60 mV. We now show that, following the effects of PKC, voltage dependence was shifted to the left (essentially enhanced), particularly at more negative voltages. Conversely, the voltage dependence of channels lacking PKC was shifted to the right (essentially suppressed). Predictably, activity was increased at all Ca2+ concentrations following the effects of PKC; nevertheless, Ca2+ dependence was actually shifted to the right. Moreover, whereas IP3 did not alter activity at -60 mV, it drastically shifted Ca2+ dependence to the right-an outcome largely reversed by PKC. With respect to the afterdischarge, these data suggest PKC initially upregulates the channel by direct gating and shifting voltage dependence to the left. Subsequently, PKC and IP3 attenuate the channel by suppressing Ca2+ dependence. This ensures hormone delivery by allowing afterdischarge initiation and maintenance but also prevents interminable bursting. Similar regulatory interactions may be used by other neurons to achieve diverse outputs.
    Journal of Neurophysiology 05/2009; 102(1):259-71. · 3.30 Impact Factor
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    ABSTRACT: Flufenamic acid (FFA) is a nonsteroidal antiinflammatory agent, commonly used to block nonselective cation channels. We previously reported that FFA potentiated, rather than inhibited, a cation current in Aplysia bag cell neurons. Prompted by this paradoxical result, the present study examined the effects of FFA on membrane currents and intracellular Ca2+ in cultured bag cell neurons. Under whole cell voltage clamp, FFA evoked either outward (I out) or inward (I in) currents. I out had a rapid onset, was inhibited by the K+ channel blocker, tetraethylammonium, and was associated with both an increase in membrane conductance and a negative shift in the whole cell current reversal potential. I in developed more slowly, was inhibited by the cation channel blocker, Gd3+, and was concomitant with both an increased conductance and positive shift in reversal potential. FFA also enhanced the use-dependent inactivation and caused a positive-shift in the activation curve of the voltage-dependent Ca2+ current. Furthermore, as measured by ratiometric imaging, FFA produced a rise in intracellular Ca2+ that persisted in the absence of extracellular Ca2+ and was reduced by depleting either the endoplasmic reticulum and/or mitochondrial stores. Ca2+ appeared to be involved in the activation of I in, as strong intracellular Ca2+ buffering effectively eliminated I in but did not alter I out. Finally, the effects of FFA were likely not due to block of cyclooxygenase given that the general cyclooxygenase inhibitor, indomethacin, failed to evoke either current. That FFA influences a number of neuronal properties needs to be taken into consideration when employing it as a cation channel antagonist.
    Journal of Neurophysiology 08/2008; 100(1):38-49. · 3.30 Impact Factor
  • Julia E Geiger, Neil S Magoski
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    ABSTRACT: Intracellular Ca2+ is influenced by both Ca2+ influx and release. We examined intracellular Ca2+ following action potential firing in the bag cell neurons of Aplysia californica. Following brief synaptic input, these neuroendocrine cells undergo an afterdischarge, resulting in elevated Ca2+ and the secretion of neuropeptides to initiate reproduction. Cultured bag cell neurons were injected with the Ca2+ indicator, fura-PE3, and subjected to simultaneous imaging and electrophysiology. Delivery of a 5-Hz, 1-min train of action potentials (mimicking the fast phase of the afterdischarge) produced a Ca2+ rise that markedly outlasted the initial influx, consistent with Ca2+-induced Ca2+ release (CICR). This response was attenuated by about half with ryanodine or depletion of the endoplasmic reticulum (ER) by cyclopiazonic acid. However, depletion of the mitochondria, with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, essentially eliminated CICR. Dual depletion of the ER and mitochondria did not reduce CICR further than depletion of the mitochondria alone. Moreover, tetraphenylphosphonium, a blocker of mitochondrial Ca2+ release, largely prevented CICR. The Ca2+ elevation during and subsequent to a stimulus mimicking the full afterdischarge was prominent and enhanced by protein kinase C activation. Traditionally, the ER is seen as the primary Ca2+ source for CICR. However, bag cell neuron CICR represents a departure from this view in that it relies on store interaction, where Ca2+ released from the mitochondria may in turn liberate Ca2+ from the ER. This unique form of CICR may be used by both bag cell neurons, and other neurons, to initiate secretion, activate channels, or induce gene expression.
    Journal of Neurophysiology 08/2008; 100(1):24-37. · 3.30 Impact Factor
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    Anne Y Hung, Neil S Magoski
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    ABSTRACT: The translation of prior activity into changes in excitability is essential for memory and the initiation of behavior. After brief synaptic input, the bag cell neurons of Aplysia californica undergo a nearly 30-min afterdischarge to release egg-laying hormone. The present study examines a prolonged depolarization in cultured bag cell neurons. A 5-Hz, 10-s action potential train elicited a depolarization of about 10 mV, which lasted </=30 min and was reduced by calmodulin kinase inhibition. Very broad action potentials (resulting from TEA application) decreased prolonged depolarization amplitude, indicating that strong Ca(2+) influx did not necessarily promote the response. The prolonged depolarization current (I(PD)) was recorded after 5-Hz, 10-s trains of square voltage pulses of varying duration (10-150 ms). Despite Ca(2+) influx increasing steadily with pulse duration, I(PD) was most reliably initiated at 100 ms, suggesting a Ca(2+) window or limit exists for triggering I(PD). Consistent with this, modestly broader action potentials, evoked by lengthening the train current-pulse duration, resulted in smaller prolonged depolarizations. With respect to the properties of I(PD), it displayed a linear current-voltage relationship with a reversal potential of about -45 mV that was shifted to approximately -25 mV by lowering internal K(+) or about -56 mV by lowering external Na(+) and Ca(2+). I(PD) was blocked by Gd(3+), but was not antagonized by MDL-123302A, SKF-96365, 2-APB, tetrodotoxin, or flufenamic acid. Optimal Ca(2+) influx may activate calmodulin kinase and a voltage-independent, nonselective cation channel to initiate the prolonged depolarization, thereby contributing to the afterdischarge and reproduction.
    Journal of Neurophysiology 03/2007; 97(3):2465-79. · 3.30 Impact Factor
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    ABSTRACT: Although store-operated Ca(2+) influx has been well-studied in nonneuronal cells, an understanding of its nature in neurons remains poor. In the bag cell neurons of Aplysia californica, prior work has suggested that a Ca(2+) entry pathway can be activated by Ca(2+) store depletion. Using fura-based imaging of intracellular Ca(2+) in cultured bag cell neurons, we now characterize this pathway as store-operated Ca(2+) influx. In the absence of extracellular Ca(2+), the endoplasmic reticulum Ca(2+)-ATPase inhibitors, cyclopiazonic acid (CPA) or thapsigargin, depleted intracellular stores and elevated intracellular free Ca(2+). With the subsequent addition of extracellular Ca(2+), a prominent Ca(2+) influx was observed. The ryanodine receptor agonist, chloroethylphenol (CEP), also increased intracellular Ca(2+) but did not initiate store-operated Ca(2+) influx, despite overlap between CEP- and CPA-sensitive stores. Bafilomycin A, a vesicular H(+)-ATPase inhibitor, liberated intracellular Ca(2+) from acidic stores and attenuated subsequent Ca(2+) influx, presumably by replenishing CPA-depleted stores. Store-operated Ca(2+) influx was partially blocked by low concentrations of La(3+) or BTP2, and strongly inhibited by either 1-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365) or a high concentration of Ni(2+). Regarding IP(3) receptor blockers, 2-aminoethyldiphenyl borate, but not xestospongin C, prevented store-operated Ca(2+) influx. However, jasplakinolide, an actin stabilizer reported to inhibit this pathway in smooth muscle cell lines, was ineffective. The bag cell neurons initiate reproductive behavior through a prolonged afterdischarge associated with intracellular Ca(2+) release and neuropeptide secretion. Store-operated Ca(2+) influx may serve to replenish stores depleted during the afterdischarge or participate in the release of peptide that triggers behavior.
    Journal of Neurophysiology 12/2006; 96(5):2688-98. · 3.30 Impact Factor
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    Derek A Lupinsky, Neil S Magoski
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    ABSTRACT: Ca2+-activated, non-selective cation channels feature prominently in the regulation of neuronal excitability, yet the mechanism of their Ca2+ activation is poorly defined. In the bag cell neurones of Aplysia californica, opening of a voltage-gated, non-selective cation channel initiates a long-lasting afterdischarge that induces egg-laying behaviour. The present study used single-channel recording to investigate Ca2+ activation in this cation channel. Perfusion of Ca2+ onto the cytoplasmic face of channels in excised, inside-out patches yielded a Ca2+ activation EC50 of 10 microm with a Hill coefficient of 0.66. Increasing Ca2+ from 100 nm to 10 microm caused an apparent hyperpolarizing shift in the open probability (Po) versus voltage curve. Beyond 10 microm Ca2+, additional changes in voltage dependence were not evident. Perfusion of Ba2+ onto the cytoplasmic face did not alter Po; moreover, in outside-out recordings, Po was decreased by replacing external Ca2+ with Ba2+ as a charge carrier, suggesting Ca2+ influx through the channel may provide positive feedback. The lack of Ba2+ sensitivity implicated calmodulin in Ca2+ activation. Consistent with this, the application to the cytoplasmic face of calmodulin antagonists, calmidazolium and calmodulin-binding domain, reduced Po, whereas exogenous calmodulin increased Po. Overall, the data indicated that the cation channel is activated by Ca2+ through closely associated calmodulin. Bag cell neurone intracellular Ca2+ rises markedly at the onset of the afterdischarge, which would enhance channel opening and promote bursting to elicit reproduction. Cation channels are essential to nervous system function in many organisms, and closely associated calmodulin may represent a widespread mechanism for their Ca2+ sensitivity.
    The Journal of Physiology 10/2006; 575(Pt 2):491-506. · 4.38 Impact Factor
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    Neil S Magoski, Leonard K Kaczmarek
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    ABSTRACT: Although ion channels are regulated by protein kinases, it has yet to be established whether the behavioral state of an animal may dictate whether or not modulation by a kinase can occur. Here, we describe behaviorally relevant changes in the ability of a nonselective cation channel from Aplysia bag cell neurons to be regulated by protein kinase C (PKC). This channel drives a prolonged afterdischarge that triggers the release of egg-laying hormone and a series of reproductive behaviors. The afterdischarge is followed by a lengthy refractory period, during which additional bursting cannot be elicited. Previously, we reported that, in excised inside-out patches, the cation channel is closely associated with PKC, which increases channel activity. We now show that this channel-kinase association is plastic, because channels excised from certain neurons lack PKC-dependent modulation. Although direct application of PKC-activating phorbol ester to these patches had no effect, exposing the neurons themselves to phorbol ester reinstated modulation, suggesting that an absence of modulation was attributable to a lack of associated kinase. Furthermore, modulation was restored by pretreating neurons with either PP1 [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or SU6656, inhibitors of Src tyrosine kinase, an enzyme whose Src homology 3 domain is required for channel-PKC association. Neurons that were stimulated to afterdischarge and had entered the prolonged refractory period were found to have more phosphotyrosine staining and less channel-PKC association than unstimulated neurons. These findings suggest that Src-dependent regulation of the association between the cation channel and PKC controls both the long-term excitability of these neurons and their ability to induce reproduction.
    Journal of Neuroscience 09/2005; 25(35):8037-47. · 6.91 Impact Factor
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    ABSTRACT: In response to brief synaptic stimulation that activates protein kinase A (PKA), the bag cell neurons of Aplysia trigger the onset of reproductive behaviors by generating a prolonged afterdischarge. In juvenile animals, such afterdischarges are inhibited by a high density of Ca2+ -activated K+ (BK) channels, encoded by the slo gene. An increase in this current also follows an afterdischarge in mature animals, contributing to a subsequent refractory state that limits reproductive behaviors. Using a bag cell cDNA library, we have isolated two alternative transcripts of the slo gene, differing in the presence (slo-a) or absence (slo-b) of a consensus phosphorylation site for PKA. Expression of either isoform in Chinese hamster ovary cells produced Ca2+ - and voltage-dependent channels with macroscopic and unitary properties matching those in bag cell neurons. The isoforms differed, however, in their response to application of the catalytic subunit of PKA, which reduced the open probability of Slo-a, an effect that was reversed by a PKA inhibitor. In contrast, PKA had no effect on Slo-b. By immunocytochemistry, we determined that the PKA-regulated Slo-a subunit is present in adult, but not juvenile, bag cell neurons. Patch clamp recordings from adult and juvenile bag cell neurons confirmed that PKA decreases BK channel activity only in adults. Our findings suggest that a change in the identity of Slo isoforms expressed during development allows mature neurons to generate afterdischarges that are required for reproduction.
    Journal of Biological Chemistry 01/2005; 279(50):52324-30. · 4.65 Impact Factor
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    ABSTRACT: The ability of sodium pyrithione (NaP), an agent that produces delayed neuropathy in some species, to alter neuronal physiology was accessed using ratiometric imaging of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in fura PE-filled cultured Aplysia bag cell neurons. Bath-application of NaP evoked a [Ca(2+)](i) elevation in both somata and neurites with an EC(50) of approximately 300 nM and a Hill coefficient of approximately 1. The response required the presence of external Ca(2+), had an onset of 3-5 min, and generally reached a maximum within 30 min. 2-Methyl-sulfonylpyridine, a metabolite and close structural analog of NaP, did not elevate [Ca(2+)](i). Under whole-cell current-clamp recording, NaP produced a approximately 14 mV depolarization of resting membrane potential that was dependent on external Ca(2+). These data suggested that NaP stimulates Ca(2+) entry across the plasma membrane. To minimize the possibility that a change in cytosolic pH was the basis for NaP-induced Ca(2+) entry, bag cell neuron intracellular pH was estimated with the dye 2',7'-bis(carboxyethyl-5(6)-carboxy-fluorescein acetoxy methylester. Exposure of the neurons to NaP did not alter intracellular pH. The slow onset and sustained nature of the NaP response suggested that a cation exchange mechanism coupled either directly or indirectly to Ca(2+) entry could underlie the phenomenon. However, neither ouabain, a Na(+)/K(+) ATPase inhibitor, nor removal of extracellular Na(+), which eliminates Na(+)/Ca(2+) exchanger activity, altered the NaP-induced [Ca(2+)](i) elevation. Finally, the possibility that NaP gates a Ca(2+)-permeable ion channel in the plasma membrane was examined. NaP did not appear to activate two major forms of bag cell neuron Ca(2+)-permeable ion channels, as Ca(2+) entry was unaffected by inhibition of voltage-gated Ca(2+) channels using nifedipine or by inhibition of a voltage-dependent, nonselective cation channel using a high concentration of tetrodotoxin. In contrast, two potential store-operated Ca(2+) entry current inhibitors, SKF-96365 and Ni(2+), attenuated NaP-induced Ca(2+) entry. We conclude that NaP activates a slow, persistent Ca(2+) influx in Aplysia bag cell neurons.
    Journal of Neurobiology 10/2004; 60(4):411-23. · 3.05 Impact Factor
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    Neil S Magoski
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    ABSTRACT: Ion channel regulation by closely associated kinases or phosphatases has emerged as a key mechanism for orchestrating neuromodulation. An exemplary case is the nonselective cation channel that drives the afterdischarge in Aplysia bag cell neurons. Initial studies showed that this channel is modulated by both a closely associated PKC and a serine/threonine protein phosphatase (PP). In excised, inside-out patches, the addition of ATP (a phosphate source) increases open probability (P(O)) through PKC, and this is reversed by the PP. Previous work also reported that, in certain cases, ATP can decrease cation channel P(O). The present study characterizes and provides a mechanism for this decreased P(O) ATP response. The kinetic change for channels inhibited by ATP was identical to the previously reported effect of exogenously applied protein kinase A (PKA) (i.e., a lengthening of the third closed-state time constant). The decreased P(O) ATP response was blocked by the PKA inhibitor peptide PKA(6-22), and its reversal was prevented by the PP inhibitor microcystin-LR. Furthermore, PKA(6-22) did not alter the increased P(O) ATP response. This suggests that both PKA and a PP are closely associated with these cation channels, but PKA and PKC are not simultaneously targeted. After an afterdischarge, the bag cell neurons are refractory and fail to respond to subsequent stimulation. The association of PKA with the cation channel may contribute to this decrease in excitability. Altering the constituents of a regulatory complex, such as exchanging PKA for PKC, may represent a general mechanism to precisely control ion channel function and excitability.
    Journal of Neuroscience 08/2004; 24(30):6833-41. · 6.91 Impact Factor
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    Yalan Zhang, Neil S Magoski, Leonard K Kaczmarek
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    ABSTRACT: Stimulation of the bag cell neurons of Aplysia activates several biochemical pathways, including protein kinase C (PKC), and alters their excitability for many hours. After an approximately 30 min afterdischarge, these neurons enter an approximately 18 hr inhibited state during which additional stimulation fails to evoke discharges. In vivo, this refractory period limits the frequency of reproductive behaviors associated with egg laying. We have now examined the role of Ca2+-activated K+ (BK) currents in the refractory period. Outward currents gated by both intracellular Ca2+ and depolarization, with pharmacological characteristics of BK currents, were recorded in isolated bag cell neurons. These currents were enhanced by the BK channel activators phloretin and 1,3-dihydro-1-[2-hydroxy-5-(trifluoro-methyl)phenyl]-5-trifluoromethyl-2H-benzimidazol-2-one and inhibited by the BK blocker paxilline. The BK component of K+ current was enhanced by 12-O-tetradecanoyl-phorbol-13-acetate, an activator of PKC, and this effect was blocked by sphinganine and PKC(19-36), inhibitors of PKC in bag cell neurons. To test whether the BK current is altered during the refractory period, intact clusters were stimulated to afterdischarge, and neurons were isolated after the clusters had entered the refractory period. Compared with unstimulated cells, current density was almost doubled in refractory neurons. This increase in current was inhibited by preincubating clusters in sphinganine. Treatment of refractory clusters with paxilline significantly restored the ability of stimulation to evoke afterdischarges. Conversely, application of phloretin to previously unstimulated clusters inhibited the onset of afterdischarges. These results indicate that a prolonged increase in BK channel activity contributes to the prolonged refractory period of the bag cell neurons.
    Journal of Neuroscience 01/2003; 22(23):10134-41. · 6.91 Impact Factor
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    ABSTRACT: Accumulating evidence suggests that many ion channels reside within a multiprotein complex that contains kinases and other signaling molecules. The role of the adaptor proteins that physically link these complexes together for the purposes of ion channel modulation, however, has been little explored. Here, we examine the protein-protein interactions required for regulation of an Aplysia bag cell neuron cation channel by a closely associated protein kinase C (PKC). In inside-out patches, the PKC-dependent enhancement of cation channel open probability could be prevented by the src homology 3 (SH3) domain, presumably by disrupting a link between the channel and the kinase. SH3 and PDZ domains from other proteins were ineffective. Modulation was also prevented by an SH3 motif peptide that preferentially binds the SH3 domain of src. Furthermore, whole-cell depolarizations elicited by cation channel activation were decreased by the src SH3 domain. These data suggest that the cation channel-PKC association may require SH3 domain-mediated interactions to bring about modulation, promote membrane depolarization, and initiate prolonged changes in bag cell neuron excitability. In general, protein-protein interactions between ion channels and protein kinases may be a prominent mechanism underlying neuromodulation.
    Journal of Neuroscience 02/2002; 22(1):1-9. · 6.91 Impact Factor

Publication Stats

345 Citations
104.69 Total Impact Points

Institutions

  • 2003–2013
    • Queen's University
      • • Department of Biomedical and Molecular Sciences
      • • Department of Physiology
      Kingston, Ontario, Canada
  • 1998–2005
    • Yale University
      • Department of Pharmacology
      New Haven, CT, United States
  • 2000
    • Yale-New Haven Hospital
      New Haven, Connecticut, United States