Evidence that Ca/calmodulin-dependent protein kinase mediates the modulation of the Ca2+-dependent K+ current,I AHP,by acetylcholine,but not by glutamate,in hippocampal neurons

Muscarinic and metabotropic glutamate receptor agonists increase the excitability of hippocampal and other cortical neurons by suppressing the Ca²⁺-activated K⁺current,I _AHP, which underlies the slow afterhyperpolarization (AHP) and spike frequency adaptation. We have examined the mechanism of action of a muscarinic agonist (carbachol) and a metabotropic glutamate receptor agonist (1-Aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) onI _AHP in hippocampal CA1 neurons in slices, by using highly specific protein kinase inhibitors. We found that inhibition of protein kinase A (PKA) with the adenosine 3,5-cyclic monophosphate (cAMP) analogue Rp-adenosine-3,5-cyclic phosphorothioate Rp-cAMPS, did not prevent the muscarinic and glutamatergic suppression ofI _AHP. In contrast, two specific peptide inhibitors of Ca²⁺/calmodulin-dependent protein kinase II (CaM-K II), each partially blocked the effect of carbachol, but not the effect of t-ACPD onI _AHP. We conclude that CaM-K II, but not PKA, is involved in mediating the muscarinic suppression ofI _AHP, although other pathways may also contribute. In contrast, neither CaM-K II nor PKA seems to mediate the metabotropic glutamate receptor action onI _AHP.     

Direction-selective adaptation in fly visual motion-sensitive neurons is generated by an intrinsic conductance-based mechanism

Motion-sensitive neurons in the blowfly brain present an ideal model system to study the cellular mechanisms and functional significance of adaptation to visual motion. Various adaptation processes have been described, but it is still largely unknown which of these processes are generated in the motion-sensitive neurons themselves and which originate at more peripheral processing stages. By input resistance measurements I demonstrate that direction-selective adaptation is generated by an activity-dependent conductance increase in the motion-sensitive neurons. Based on correlations between dendritic Ca(2+) accumulation and slow hyperpolarizing after-potentials following excitatory stimulation, a regulation of direction-selective adaptation by Ca(2+) has previously been suggested. In the present study, however, adaptation phenomena are not evoked when the cytosolic Ca(2+) concentration is elevated by ultraviolet photolysis of caged Ca(2+) in single neurons rather than by motion stimulation. This result renders it unlikely, that adaptation in fly motion-sensitive neurons is regulated by bulk cytosolic Ca(2+).     

The calcium channel antagonist diltiazem effectively blocks two types of potassium channels in the neuronal membranes

Effect of the Ca²⁺-channel antagonist diltiazem on potential-operated Ca²⁺ and K⁺ currents was studied on isolated edible snail neurons by a two-microelectrode patch-clamp technique. Diltiazem in a concentration of 0.1 mM inhibits Ca²⁺ current, high-threshold Ca²⁺-dependent K⁺ current, and Ca²⁺-independent K⁺ current and has no effect on low-threshold K⁺ current and leakage current. It is suggested that therapeutic effect of diltiazem is mediated through blockade of Ca²⁺ and K⁺ channels. Tranlated fromByulleten' Eksperimental'noi biologii i Meditsiny, Vol. 124, No. 9, pp. 271–274. September, 1997     

Changes to erythrocyte membrane cation permeability induced by a bacterial toxin

Vibrio parahaemolyticus secretes an exotoxin prepared as Kanagawa haemolysin (KH) which causes marked alterations to the function of epithelial, cardiac and other cell types, but whose cellular mode of action is poorly understood. Using human red cells as a model system with radiotracer flux techniques, we have shown that KH (1) markedly elevated the basal leak to K⁺, (2) raised Ca²⁺ influx, and as a consequence of this, (3) stimulated the Ca²⁺-activated K⁺ channel. These results suggest that an important deleterious effect of this toxin is to elevate cation permeability, which will have both direct and indirect actions on the behaviour of a variety of cell typesin vivo.     

A novel synergistic stimulation of Na+-transport across frog skin (Xenopus laevis) by external Cd2+- and Ca2+-ions

Isolated skin of the clawed frogXenopus laevis was mounted in an Ussing-chamber. The transcellular sodiumcurrent (I _Na) was identified either as amiloride-blockable (10^–3 mol/l) short-circuit current (I _SC), or by correctingI _SC for the shunt-current obtained with mucosal Tris. A dose of 10 mmol/l Cd²⁺ applied to the mucosal side increased the current by about 70%. The half-maximal effect was reached at a Cd²⁺-concentration of 2,6 mmol/l (in NaCl-Ringer). The quick and fully reversible effect of Cd²⁺ could not be seen when 10^–3 mol/l amiloride was placed in the outer, Na⁺-containing solution, nor when Na⁺ was replaced by Tris. This suggests that Cd²⁺ stimulatesI _Na. Cd²⁺ intefered with the Na⁺-current self-inhibition, and therefore with the saturation ofI _Na by increasing the apparent Michaelis constant (K _Na) of this process. The I _Na recline after stepping up mucosal [Na⁺] was much reduced in presence of Cd²⁺. Ca²⁺-ions on the mucosal side had an identical effect to Cd²⁺, and 10 mmol/l Ca²⁺ increaseI _Na by about 100%. The half-maximal effect was obtained with 4.4 mmol/l Ca²⁺. The mechanism ofI _Na-stimulation by Ca²⁺ did not seem to differ from that of Cd²⁺. Thus, although of low Na⁺-transport capacity,Xenopus skin appears to be as good a model for Na⁺-transporting epithelia asRanidae skin, with the exception of the calcium effect which, so far, has not been reported forRanidae.     

Capacitative Ca2+ influx and a Ca2u+-dependent nonselective cation pathway are discriminated by genistein in mouse pancreatic acinar cells

We have investigated the effect of genistein on the hormone-stimulated Ca²⁺ influx and on a 28 pS nonselective cation channel in mouse pancreatic acinar cells using the Ca²⁺ indicator fluo-3 and the patch-clamp technique. The identity of the Ca^2u+ influx pathway has not been established in this cell type so far. Therefore we have investigated the Ca²⁺-dependent nonselective cation channel as a potential pathway for Ca²⁺ influx. Capacitative Ca²⁺ entry was induced by depletion of intracellular Ca²⁺ stores with 500 nM acetylcholine or with the Ca²⁺ ATPase inhibitor 2,5-di-tert- butylhydroquinone. In the presence of 100 M genistein, Ca²⁺ release was unimpaired, whereas Ca²⁺ influx was reversibly suppressed. Patch-clamp experiments demonstrated that genistein had no effect on Ca²⁺-activated nonselective cation channels, the activity of which was measured in excised membrane patches (inside/out) or in the whole-cell configuration. Therefore we conclude that this 28 pS nonselective cation channel does not contribute to Ca²⁺ influx into mouse exocrine pancreatic cells. With the exception of genistein and tyrphostin 25, other tyrosine kinase inhibitors such as methyl-2,5-dihydroxycinnamate, lavendustin A, herbimycin A, and tyrphostin B56 were without effect on Ca²⁺ signalling. Thus, the involvement of tyrosine phosphorylation in the activation of the Ca²⁺ entry mechanism in mouse pancreatic acinar cells is unclear.     

Afterhyperpolarization induced by the activation of nicotinic acetylcholine receptors in pelvic ganglion neurons of male rats

The electrophysiological mechanism underlying afterhyperpolarization induced by the activation of the nicotinic acetylcholine receptor (nAChR) in male rat major pelvic ganglion neurons (MPG) was investigated using a gramicidin-perforated patch clamp and microscopic fluorescence measurement system. Acetylcholine (ACh) induced fast depolarization through the activation of nAChR, followed by a sustained hyperpolarization after the removal of ACh in a dose-dependent manner (10 μM to 1 mM). ACh increased both intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺ concentrations ([Na⁺]_i) in MPG neurons. The recovery of [Na⁺]_i after the removal of ACh was markedly delayed by ouabain (100 μM), an inhibitor of Na⁺/K⁺ ATPase. Pretreatment with ouabain blocked ACh-induced hyperpolarization by 67.2 ± 5.4% (n = 7). ACh-induced hyperpolarization was partially attenuated by either the chelation of [Ca²⁺]_i with BAPTA/AM (20 μM) or the blockade of small-conductance Ca²⁺-activated K⁺ channels by apamin (500 nM). Taken together, the activation of nAChR increases [Na⁺]_i and [Ca²⁺]_i, which activates Na⁺/K⁺ ATPase and Ca²⁺-activated K⁺ channels, respectively. Consequently, hyperpolarization occurs after the activation of nAChR in the autonomic pelvic ganglia.     

Repetitive firing properties of putative dopamine-containing neurons in vitro: regulation by an apamin-sensitive Ca2+-activated K+ conductance

Summary Intracellular recording techniques were used to study the effects of apamin (APA), a selective inhibitor of one type of Ca²⁺-activated K⁺ channel, on the electroresponsive properties of dopamine (DA)-containing neurons within the zona compacta of the substantia nigra (SNc) in rat. Bath application of APA (1 M) blocked the slow component of a complex post-spike afterhyperpolarization (AHP_s) without affecting other characteristics of the action potential. Blockade of AHP_s was accompanied by an increase in the number and frequency of action potentials evoked by depolarizing current pulses. However, APA failed to affect the cellular mechanisms underlying spike frequency adaptation or poststimulus inhibitory period. These data indicate that AHP_s can exert a strong influence on the interspike interval but is probably not involved in regulating slower adaptive neuronal responses.     

Modulation of acid-sensing ion channels by Cu(2+) in cultured hypothalamic neurons of the rat

Acid-sensing ion channels (ASICs) are known to distribute throughout the nervous system and serve important roles in various physiological and pathological processes. However, the properties of ASICs in the hypothalamus, an important region of diencephalon, are little known. We herein used whole-cell patch-clamp recordings to characterize proton-induced cation currents in cultured hypothalamic neurons of the rat, and attributed these transient inward currents to ASICs based on their electrophysiological and pharmacological properties. We further examined the effects of Cu(2+), the third most abundant trace element, on ASICs in hypothalamic neurons. Our results showed that this divalent cation reversibly and concentration-dependently inhibited the amplitude of ASIC currents, and slowed down the desensitization of ASIC channels. Our results also displayed that Cu(2+) modulated ASICs independent of change in membrane potential and extracellular protons, suggesting a noncompetitive mechanism. Furthermore, micromolar concentration of Cu(2+) attenuated the acid-induced membrane depolarization. Taken together, our data demonstrate a modulatory effect of Cu(2+) on ASICs in native hypothalamic neurons and suggest a role of this endogenous metal ion in negatively modulating the increased neuronal membrane excitability caused by activation of ASICs.     

Membrane potential oscillations in CA1 hippocampal pyramidal neurons in vitro: intrinsic rhythms and fluctuations entrained by sinusoidal injected current

The mechanisms mediating intrinsic and entrained CA1 pyramidal neuron rhythmic membrane potential oscillations were investigated in rat hippocampal slices. Intrinsic oscillations (6–14 Hz, < 10 mV) were evoked by long duration (2 s), depolarizing current pulses in 42% of the cells. Oscillations were also evoked by imposing sinusoidal transmembrane currents at 2, 7, and 14 Hz, adjusted at 7 Hz to imitate the synaptically mediated in vivo intracellular theta. Slow all-or-none events (40 mV, 55 ms) — reminiscent of the rhythmic, high threshold slow spikes observed in vivo — were evoked and entrained by the sine wave current cycles with large, imposed depolarization in 35% of the cells. Intrinsic oscillations were insensitive to Ca²⁺-free, Co²⁺ (2 mM) and Mn²⁺ (2 mM) solutions, but were blocked by tetrodotoxin (TTX; 5 M), illustrating that they were Na⁺-mediated. Tetraethylammonium (TEA; 15 mM) unmasked slow all-or-none events (40–50 mV, 20–55 ms) and plateau potentials (40–60 mV, 100–700 ms). Plateaus were Co²⁺ and Mn²⁺ resistant and were abolished by TTX, hence suggesting that the underlying persistent conductance was Na⁺-mediated. Plateaus were entrained one-to-one at all sinusoidal current frequencies in Ca²⁺-free, TEA+Co²⁺, or TEA+Mn²⁺ solutions. However, the high threshold Ca²⁺ spikes uncovered in TEA+TTX could only follow sinusoidal currents of less than 7 Hz. In conclusion, the high threshold Ca²⁺ and persistent Na⁺ conductances coexist in CA1 pyramidal cells. The persistent Na⁺ conductance mediated the intrinsic oscillations, and fluctuated at all the sine wave current frequencies used. The more sluggish high-threshold Ca²⁺ conductance exclusively oscillated at frequencies of less than 7 Hz and did not support the intrinsic rhythm. Therefore, the findings suggest that the Na⁺-mediated oscillations may contribute to the high-frequency, type I, hippocampal theta rhythm present in vivo, whereas the high threshold Ca²⁺ conductance may take part in the low-frequency, type II rhythm.     

Relative roles of protein kinase A and protein kinase C in modulation of transient receptor potential vanilloid type 1 receptor responsiveness in rat sensory neurons in vitro and peripheral nociceptors in vivo

The function of the transient receptor potential vanilloid type 1 capsaicin receptor is subject to modulation by phosphorylation catalyzed by various enzymes including protein kinase C and cAMP-dependent protein kinase. The aim of this study was to compare the significance of the basal and stimulated activity of protein kinase C and cAMP-dependent protein kinase in transient receptor potential vanilloid type 1 receptor responsiveness in the rat in vitro by measurement of the intracellular calcium concentration in cultured trigeminal ganglion neurons and in vivo by determination of the behavioral noxious heat threshold. KT5720, a selective inhibitor of cAMP-dependent protein kinase, reduced the calcium transients induced by capsaicin or the other, much more potent transient receptor potential vanilloid type 1 receptor agonist resiniferatoxin in trigeminal sensory neurons and diminished the drop of the noxious heat threshold (heat allodynia) evoked by intraplantar resiniferatoxin injection. Chelerythrine chloride, a selective inhibitor of protein kinase C, failed to alter either of these responses, although it inhibited the effect of phorbol 12-myristate 13-acetate in the in vitro assay. Staurosporine, a rather nonselective protein kinase inhibitor, failed to reduce the capsaicin- and resiniferatoxin-induced calcium transients but inhibited the resiniferatoxin-evoked heat allodynia. Dibutyryl-cAMP and phorbol 12-myristate 13-acetate, activator(s) of cAMP-dependent protein kinase and protein kinase C, respectively, enhanced the effect of capsaicin in the calcium uptake assay while forskolin, an activator of adenylyl cyclase, augmented that of resiniferatoxin in the heat allodynia model. None of the protein kinase inhibitors or activators altered the calcium transients evoked by high potassium, a nonspecific depolarizing stimulus. It is concluded that basal activity of cAMP-dependent protein kinase, unlike protein kinase C, is involved in the maintenance of transient receptor potential vanilloid type 1 receptor function in somata of trigeminal sensory neurons but stimulation of either cAMP-dependent protein kinase or protein kinase C above the resting level can lead to an enhanced transient receptor potential vanilloid type 1 receptor responsiveness. Similar mechanisms are likely to operate in vivo in peripheral terminals of nociceptive dorsal root ganglion neurons.     

Single calcium-activated potassium channel in cultured mammary epithelial cells

The properties of Ca²⁺-activated K⁺ channels in mouse mammary epithelial cells in primary culture were studied by the patch-clamp technique. In cell-attached patches, spontaneous channel openings were sometimes observed; the slope conductance of the currents was about served; the slope conductance of the currents was about 12 pS at negative membrane potentials with a physiological solution (152 mM Na⁺, 5.4 mM K⁺) in the pipette. External application of A23187, a calcium ionophore, activated this channel. In excised inside-out patches, the channel was activated by increasing the internal Ca²⁺ concentration (10^–7 to 10^–6 M). No voltage dependence of the channel activity was observed. Internal Na⁺ blocked the outward K⁺ current in a voltage dependent manner and this block led to the non-linear I–V relationship at positive membrane potentials. The channel was blocked by internal Ba²⁺ (0.1 mM) and tetracthylammonium (TEA⁺, 20–50 mM). Ba²⁺ reduced the open probability but not the single channel conductance, whereas TEA⁺ reduced the single channel conductance. The single channel conductance of this channel, measured from the inward current with a high-K⁺ solution (150 mM K⁺) in the pipette, was large (about 40 pS), and showed inward rectification. These results suggest that this channel is different from the usual small conductance Ca²⁺-activated K⁺ channels observed in many other cells.     

β-Adrenergic modulation of transient inward current in guinea-pig cardiac myocytes

Transient inward current (I_ti) indicating Ca²⁺-release from the sarcoplasmic reticulum and L-type Ca²⁺-current(I_Ca) were studied in atrial and ventricular myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. The increase of I_Ca caused by -adrenergic stimulation using isoprenaline (ISO) or related experimental manoeuvres such as superfusion with forskoline (FORSK) was used as a qualitative monitor of an increase of intracellular cAMP. Changes of I_ti were used to manifest changes of sarcoplasmic Ca²⁺-release. In myocytes dialysed with citrate-based (60 mM) pipette filling solution containing 100 M EGTA spontaneous transient inward currents were recorded at a constant holding potential of –50 mV in the majority of myocytes. Superfusion with a solution containing ISO (5·10^–8M) increased the amplitude of spontaneous I_ti and reduced its time-to-peak. The effects of ISO on I_ti developed in parallel to stimulation of I_Ca. In myocytes which did not show spontaneous cyclic Ca²⁺-release in the above condition, this could be evoked de novo by ISO. Spontaneous I_ti was suppressed in the majority of cells by increasing the concentration of EGTA in the dialysing solution to 200 M. Brief (50 ms) activation of I_Ca by voltage steps from –50 to +10 mV usually failed to trigger Ca²⁺-release from the SR. The increase of I_Ca-amplitude upon administration of ISO went ahead with the induction of Ca²⁺-release by brief activation of I_Ca. The effects of ISO could be mimicked by FORSK or intracellular dialysis with 35-cyclic adenosine monophosphate. The effects on I_Ca and SR Ca²⁺-release were dependent on the concentration of the stimulating substance. In a given cell changing superfusion from a low to a high concentration of ISO or FORSK resulted in an increase of the number of Ca²⁺-release events per number of Ca²⁺-currents elicited and a shortening of time-to-peak of I_ti's. The stimulating effects of ISO or FORSK on Ca²⁺-release were only partially due to an increase of the triggering I_Ca. Ca²⁺-currents too small to trigger Ca²⁺-release before -adrenergic stimulation could evoke Ca²⁺-release after augmentation of intracellular cAMP. Whereas the effects of ISO and FORSK on I_Ca were reversible, the stimulatory effects on Ca²⁺-release persisted after washing out the substances. The results give support to the hypothesis that -adrenoceptor-mediated positive inotropic and arrhythmogenic effects are, at least partly, due to a cyclic AMP-dependent regulatory mechanism modulating sarcoplasmic Ca²⁺-release.This work was supported by the Deutsche Forschungsgemeinschaft (FG Konzell)     

Na+-Ca2+ exchange in the isolated cochlear outer hair cells of the guinea-pig studied by fluorescence image microscopy

The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca²⁺]_i) and sodium concentration ([Na⁺]_i) were measured using fluorescence indicators. Under the resting condition, [Ca²⁺]_i and [Na⁺]_i were 91±9 nM (n = 51) and 110±5 mM (n = 12), respectively. Removal of external Na⁺ by replacing with N-methyl-D-glucamine (NMDG⁺) increased [Ca²⁺]_i by 270±79% (n = 27) and decreased [Na⁺]_i by 23±4 mM (n = 6). Both changes in [Ca²⁺]_i and [Na⁺]_i were totally reversible on returning external Na⁺ to the initial value and were inhibited by addition of 0.1 mM La³⁺ or 100 M amiloride 5-(N,N-dimethyl) hydrochloride. Elevation of external Ca²⁺ ions to 20 mM reversibly decreased [Na⁺]_i by 8±6 mM (n = 5). Moreover, the chelation of the intracellular Ca²⁺ with 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG⁺-induced reduction in [Na⁺]_i. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca²⁺]_i by 290±37% (n = 5), but did not affect the [Ca²⁺]_i elevation induced by the NMDG⁺ solution. The rise in [Ca²⁺]_i induced by the NMDG⁺ solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na⁺]_i. The present results are best explained by the presence of an Na⁺-Ca²⁺ exchanger in cell membrane and indicate that the activity of Na⁺/K⁺ pump is poor in outer hair cells.     

Signaling mechanisms of down-regulation of voltage-activated Ca2+ channels by transient receptor potential vanilloid type 1 stimulation with olvanil in primary sensory neurons

Olvanil ((N-vanillyl)-9-oleamide), a non-pungent transient receptor potential vanilloid type 1 agonist, desensitizes nociceptors and alleviates pain. But its molecular targets and signaling mechanisms are little known. Calcium influx through voltage-activated Ca(2+) channels plays an important role in neurotransmitter release and synaptic transmission. Here we determined the effect of olvanil on voltage-activated Ca(2+) channel currents and the signaling pathways in primary sensory neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated rat dorsal root ganglion neurons. Olvanil (1 microM) elicited a delayed but sustained inward current, and caused a profound inhibition (approximately 60%) of N-, P/Q-, L-, and R-type voltage-activated Ca(2+) channel current. Pretreatment with a specific transient receptor potential vanilloid type 1 antagonist or intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid abolished the inhibitory effect of olvanil on voltage-activated Ca(2+) channel current. Calmodulin antagonists (ophiobolin-A and calmodulin inhibitory peptide) largely blocked the effect of olvanil and capsaicin on voltage-activated Ca(2+) channel current. Furthermore, calcineurin (protein phosphatase 2B) inhibitors (deltamethrin and FK-506) eliminated the effect of olvanil on voltage-activated Ca(2+) channel current. Notably, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin antagonists, and calcineurin inhibitors each alone significantly increased the amplitude of voltage-activated Ca(2+) channel current. In addition, double immunofluorescence labeling revealed that olvanil induced a rapid internalization of Ca(V)2.2 immunoreactivity from the membrane surface of dorsal root ganglion neurons. Collectively, this study suggests that stimulation of non-pungent transient receptor potential vanilloid type 1 inhibits voltage-activated Ca(2+) channels through a biochemical pathway involving intracellular Ca(2+)-calmodulin and calcineurin in nociceptive neurons. This new information is important for our understanding of the signaling mechanisms of desensitization of nociceptors by transient receptor potential vanilloid type 1 analogues and the feedback regulation of intracellular Ca(2+) and voltage-activated Ca(2+) channels in nociceptive sensory neurons.     

Cytoskeletal modulation of the response to mechanical stimulation in human vascular endothelial cells

Possible interactions of cytoskeletal elements with mechanically induced membrane currents and Ca²⁺ signals were studied in human endothelial cells by using a combined patch-clamp and Fura II technique. For mechanical stimulation, cells were exposed to hypotonic solution (HTS). The concomitant cell swelling activates a Cl^– current, releases Ca²⁺ from intracellular stores and activates Ca²⁺ influx. To interfere with the cytoskeleton, cells were loaded either with the F-actin-stabilizing agent phalloidin (10 mol/l), or the F-actin-depolymerizing substance cytochalasin B (50 mol/l). These were administered either in the bath or the pipette solutions. The tubulin structure of the endothelial cells was modulated by taxol (50 mol/l), which supports polymerization of tubulin, or by the depolymerizing agent colcemid (10 mol/l) both applied to the bath. Immunofluorescence experiments show that under the chosen experimental conditions the cytoskeletal modifiers employed disintegrate the F-actin and microtubuli cytoskeleton. Neither of these cytoskeletal modifiers influenced the HTS-induced Cl^– current. Ca²⁺ release was not affected by cytochalasin B, taxol or colcemid, but was suppressed if the cells were loaded with phalloidin. Depletion of intracellular Ca²⁺ stores by thapsigargin renders the intracellular [Ca²⁺] sensitive to the extracellular [Ca²⁺], which is indicative of a Ca²⁺ entry pathway activated by store depletion. Neither cytochalasin B nor phalloidin affected this Ca²⁺ entry. We conclude that F-actin turnover or depolymerization is necessary for Ca²⁺ release by mechanical activation. The tubulin network is not involved. The Ca²⁺ release-activated Ca²⁺ entry is not modulated by the F-actin cytoskeleton.     

Sub-cellular Ca2+ dynamics affected by voltage- and Ca2+-gated K+ channels: Regulation of the soma-growth cone disparity and the quiescent state in Drosophila neurons

Using Drosophila mutants and pharmacological blockers, we provide the first evidence that distinct types of K(+) channels differentially influence sub-cellular Ca(2+) regulation and growth cone morphology during neuronal development. Fura-2-based imaging revealed in cultured embryonic neurons that the loss of either voltage-gated, inactivating Shaker channels or Ca(2+)-gated Slowpoke BK channels led to robust spontaneous Ca(2+) transients that preferentially occurred within the growth cone. In contrast, loss of voltage-gated, non-inactivating Shab channels did not show such a disparity and sometimes produced soma-specific Ca(2+) transients. The fast spontaneous transients in both the soma and growth cone were suppressed by the Na(+) channel blocker tetrodotoxin, indicating that these Ca(2+) fluctuations stemmed from increases in membrane excitability. Similar differences in regional Ca(2+) regulation were observed upon membrane depolarization by high K(+)-containing saline. In particular, Shaker and slowpoke mutations enhanced the size and dynamics of the depolarization-induced Ca(2+) increase in the growth cone. In contrast, Shab mutations greatly prolonged the Ca(2+) increase in the soma. Differential effects of these excitability mutations on neuronal development were indicated by their distinct alterations in growth cone morphology. Loss of Shaker currents increased the size of lamellipodia and the number of filopodia, structures associated with the actin cytoskeleton. Interestingly, loss of Slowpoke currents strongly influenced tubulin regulation, enhancing the number of microtubule loop structures per growth cone. Together, our findings support the idea that individual K(+) channel subunits differentially regulate spontaneous sub-cellular Ca(2+) fluctuations in growing neurons that may influence activity-dependent growth cone formation.