ATP modulation of large conductance Ca-activated K channels via a functionally associated protein kinase A in CA1 pyramidal neurons from rat hippocampus

Using inside-out configuration of patch clamp techniques, ATP modulation of BK(Ca) channels was studied in hippocampal CA1 pyramidal neurons of adult rat. Intracellular ATP application markedly increased BK(Ca) channel activity, and this ATP-produced increase in BK(Ca) channel activity was characterized by a higher opening frequency with no changes in channel open times. In the presence of specific inhibitor against protein kinase A, H-89, ATP did not induce any increase in the channel activity. Furthermore, adding H-89 after addition of ATP reversed the modulation produced by ATP. In contrast, protein kinase C inhibitor chelerythrine exerted no apparent effects on ATP-induced channel activation. The present study suggests that BK(Ca) channels from hippocampal CA1 pyramidal neurons could be modulated by ATP via a functionally associated protein kinase A-like protein.     

Enhancement in activities of large conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia

It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca(2+)-activated potassium (BK(Ca)) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BK(Ca) channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K(+) in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability (P(o)) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum P(o) was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca(2+)](i) required to half activate BK(Ca) channels was only 1 microM in post-ischemic whereas 2 microM in control neurons, indicating an increase in [Ca(2+)](i) sensitivity after ischemia; and (4) BK(Ca) channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK(Ca) channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP.     

Immunolocalization of BK channels in hippocampal pyramidal neurons

Neurons are highly specialized cells in which the integration and processing of electrical signals critically depends on the precise localization of ion channels. For large-conductance Ca(2+)- activated K(+) (BK) channels, targeting to presynaptic membranes in hippocampal pyramidal cells was reported; however, functional evidence also suggests a somatodendritic localization. Therefore we re-examined the subcellular distribution of BK channels in mouse hippocampus using a panel of independent antibodies in a combined approach of conventional immunocytochemistry on cultured neurons, pre- and postembedding electron microscopy and immunoprecipitation. In cultured murine hippocampal neurons, the colocalization of BK channels with both pre- and postsynaptic marker proteins was observed. Electron microscopy confirmed targeting of BK channels to axonal as well as dendritic membranes of glutamatergic synapses in hippocampus. A postsynaptic localization of BK channels was also supported by the finding that the channel coimmunoprecipitated with PSD95, a protein solely expressed in the postsynaptic compartment. These results thus demonstrate that BK channels reside in both post- and presynaptic compartments of hippocampal pyramidal neurons.     

Transient forebrain ischemia induces persistent hyperactivity of large conductance Ca2+-activated potassium channels via oxidation modulation in rat hippocampal CA1 pyramidal neurons

The present study examined temporal changes in activity of large conductance, Ca2+-activated potassium (BKCa) channels in postischemic CA1 pyramidal neurons at 2, 6, 24 and 48 h after reperfusion. These changes in activity and possible cellular mechanisms were examined using the inside--out configuration of patch clamp. The unitary conductance of postischemic BKCa channels increased transiently to 119% of the control at 2 h after reperfusion, and recovered to the control level thereafter. A persistent increase in [Ca2+]i sensitivity of BKCa channels was observed in postischemic CA1 neurons with the maximal sensitivity to [Ca2+]i at 6 h after reperfusion while channel voltage- dependence showed no obvious changes. Kinetic analyses showed that the postischemic enhancement of BKCa channel activity was due to longer open times and shorter closed times as there was no significant changes in opening frequency after ischemia. Glutathione disulphide markedly increased BKCa channel activity in normal CA1 neurons, while reducing glutathione caused a decrease in BKCa channel activity by reducing the sensitivity of this channel to [Ca2+]i in postischemic CA1 neurons. Similar modulatory effects on postischemic BKCa channels were also observed with another redox couple, DTNB and DTT, suggesting an oxidation modulation of BKCa channel function after ischemia. The present results indicate that a persistent enhancement in activity of BKCa channels, probably via oxidation of channels, in postischemic CA1 pyramidal neurons may account for the decrease in neuronal excitability and increase in fAHP after ischemia. The ischemia-induced augmentation in BKCa channel activity may be also associated with the postischemic neuronal injury.     

Protein kinases A and C are opponents in modulating glial Ca2+ -activated K+ channels.

The modulation of the activity of Ca2+ -activated K+ (BK) channels by activators of protein kinases A and C, respectively, was studied in cell-attached patches on isolated Müller (retinal glial) cells from rabbits. The BK channel activity was stimulated by membrane depolarization and by increasing of the intracellular Ca2+ concentration. Extracellular exposure to dibutyryl-cAMP, known to stimulate the protein kinase A, increased the open probability of the channels. Exposure to a phorbol ester, as an activator of protein kinase C, strongly reduced the channel activity whereas exposure to the protein kinase inhibitor, staurosporine, stimulated the channel activity. As glial BK channels are modulated in an opposite manner by protein kinases A and C, they may act as a cellular focus of integration of the inputs from different signaling pathways.     

Selective blockade of N-type calcium channels by levetiracetam

PURPOSE: We investigated the effect of the new antiepileptic drug (AED) levetiracetam (LEV) on different types of high-voltage-activated (HVA) Ca2+ channels in freshly isolated CA1 hippocampal neurons of rats. METHODS: Patch-clamp recordings of HVA Ca2+ channel activity were obtained from isolated hippocampal CA1 neurons. LEV was applied by gravity flow from a pipette placed near the cell, and solution changes were made by electromicrovalves. Ca2+ channel blockers were used for separation of the channel subtypes. RESULTS: The currents were measured in controls and after application of 1-200 microM LEV. LEV irreversibly inhibited the HVA calcium current by approximately 18% on the average. With a prepulse stimulation protocol, which can eliminate direct inhibition of Ca2+ channels by G proteins, we found that G proteins were not involved in the pathways underlying the LEV inhibitory effect. This suggested that the inhibitory effect arises from a direct action of LEV on the channel molecule. The blocking mechanism of LEV was not related to changes in steady-state activation or inactivation of Ca2+ channels. LEV also did not influence the rundown of the HVA Ca2+ current during experimental protocols lasting approximately 10 min. Finally, LEV at the highest concentration used (200 microM) did not influence the activity of L-, P- or Q-type Ca2+ channels in CA1 neurons, while selectively influencing the activity of N-type calcium channels. The maximal effect on these channels separated from other channel types was approximately 37%. CONCLUSIONS: Our results provide evidence that LEV selectively inhibits N-type Ca2+ channels of CA1 pyramidal hippocampal neurons. These data suggest the existence of a subtype of N-type channels sensitive to LEV, which might be involved in the molecular basis of its antiepileptic action.     

Inhibition of BK channel activity by association with calcineurin in rat brain

Large conductance calcium-activated potassium (BK(Ca)) channels are regulated by a number of different protein kinases and phosphatases. The close association of enzymes and channel have been shown to underlie many examples of modulation. However, only the association of protein kinase A with the BK(Ca) channel has been detailed [Tian et al. (2003)J. Biol. Chem., 278, 8669-8677]. We have found using reciprocal immunoprecipitations that the BK(Ca) channel associates with the calcium/calmodulin-dependent phosphatase calcineurin, in Wistar rat brain. A HA-tagged construct of the carboxyl terminus of rSlo(27), a variant of the BK(Ca) channel that is abundant in the hippocampus [Ha et al. (2000)Eur. J. Biochem., 267, 910-9218], was found to associate only with the B subunit of calcineurin. This data suggests that the majority of the interaction of the BK(Ca) channel with calcineurin is mediated by the B subunit of the phosphatase. This was confirmed by using glutathione-S-transferase (GST) fusion proteins of the linker regions between the S7-S10 hydrophobic domains in the carboxyl terminus of rSlo(27), where only the B subunit of calcineurin interacted with regions between S7 and S9 of the channel. Addition of a constitutively active calcineurin (CaN(420)) to inside-out membrane patches excised from cultured hippocampal neurons resulted in a dramatic reduction in BK(Ca) channel open probability, with only very short-duration events being apparent. These data suggest that BK(Ca) channel activity is inhibited by calcineurin, an effect mediated by the association of the calcineurin B subunit with the carboxyl terminus of the channel.     

MAPK-dependent actin cytoskeletal reorganization underlies BK channel activation by insulin

Numerous brain regions are enriched with insulin and insulin receptors, and several lines of evidence indicate that insulin is an important modulator of neuronal function. Indeed, recent studies have demonstrated that insulin inhibits hippocampal epileptiform-like activity, in part by activating large-conductance Ca2+-activated potassium (BK) channels. Moreover, the mitogen-activated protein kinase (MAPK) signalling cascade has been found to couple insulin to BK channel activation. However, the cellular events downstream of MAPK that underlie this action of insulin are unknown. Here we demonstrate that in hippocampal neurons, BK channel activation by insulin is blocked by actin filament stabilization, suggesting that this process is dependent on the actin cytoskeleton. Stabilizing actin filaments also markedly attenuated the ability of insulin to inhibit the aberrant hippocampal synaptic activity evoked following Mg2+ removal. Insulin also promoted rapid reorganization of fluorescently labelled polymerized actin filaments; an action that was prevented by inhibitors of MAPK activation. Moreover, in parallel studies, insulin increased the level of phospho-MAPK immunostaining in hippocampal neurons. These data are consistent with BK channel activation by insulin involving MAPK-dependent alterations in actin dynamics. This process may have important implications for the role of insulin in regulating hippocampal excitability.     

Glucose and hippocampal neuronal excitability: role of ATP-sensitive potassium channels

Hyperglycemia-related neuronal excitability and epileptic seizures are not uncommon in clinical practice. However, their underlying mechanism remains elusive. ATP-sensitive K(+) (K(ATP)) channels are found in many excitable cells, including cardiac myocytes, pancreatic beta cells, and neurons. These channels provide a link between the electrical activity of cell membranes and cellular metabolism. We investigated the effects of higher extracellular glucose on hippocampal K(ATP) channel activities and neuronal excitability. The cell-attached patch-clamp configuration on cultured hippocampal cells and a novel multielectrode recording system on hippocampal slices were employed. In addition, a simulation modeling hippocampal CA3 pyramidal neurons (Pinsky-Rinzel model) was analyzed to investigate the role of K(ATP) channels in the firing of simulated action potentials. We found that incremental extracellular glucose could attenuate the activities of hippocampal K(ATP) channels. The effect was concentration dependent and involved mainly in open probabilities, not single-channel conductance. Additionally, higher levels of extracellular glucose could enhance neuropropagation; this could be attenuated by diazoxide, a K(ATP) channel agonist. In simulations, high levels of intracellular ATP, used to mimic increased extracellular glucose or reduced conductance of K(ATP) channels, enhanced the firing of action potentials in model neurons. The stochastic increases in intracellular ATP levels also demonstrated an irregular and clustered neuronal firing pattern. This phenomenon of K(ATP) channel attenuation could be one of the underlying mechanisms of glucose-related neuronal hyperexcitability and propagation.     

Rotenone inhibits delayed rectifier K+ current via a protein kinase A-dependent mechanism

Voltage-gated K+ channels (K(V)) regulate cell electrical properties, proliferation, migration, and death. Rotenone is a mitochondrial inhibitor, influencing activity of many channels that potentially participate in cell death processes, but its effect on K(V) channel in neurons remains unclear. This study used whole-cell patch clamp methods and found that rotenone concentration dependently decreased delayed rectifier K+ current (I(DR)) amplitude in cultured ventral mesencephalic neurons, but had no effect on A-type current (I(A)) peak amplitude. Furthermore, the protein kinase A inhibitor H-89 prevented rotenone-induced decrease in I(DR). The inhibition of I(DR) by rotenone was mimicked by extracellular application of forskolin. In summary, our results indicate that rotenone acutely decreased I(DR) currents in cultured mesencephalic neurons via a protein kinase A-dependent mechanism.     

GABAB receptors in neocortical and hippocampal pyramidal neurons are coupled to different potassium channels

Classically, GABA_B receptors are thought to regulate neuronal excitability via G‐protein‐coupled inwardly rectifying potassium (GIRK) channels. Recent data, however, indicate that GABA_B receptors can also activate two‐pore domain potassium channels. Here, we investigate which potassium channels are coupled to GABA_B receptors in rat neocortical layer 5 and hippocampal CA1 pyramidal neurons. Bath application of the non‐specific GIRK channel blocker barium (200 μm ) abolished outward currents evoked by GABA_B receptors in CA1 pyramidal, but only partially blocked GABA_B responses in layer 5 neurons. Layer 5 and CA1 pyramidal neurons also showed differential sensitivity to tertiapin‐Q, a specific GIRK channel blocker. Tertiapin‐Q partially blocked GABA_B responses in CA1 pyramidal neurons, but was ineffective in blocking GABA_B responses in neocortical layer 5 neurons. Consistent with the idea that GABA_B receptors are coupled to two‐pore domain potassium channels, the non‐specific blockers quinidine and bupivacaine partially blocked GABA_B responses in both layer 5 and CA1 neurons. Finally, we show that lowering external pH, as occurs in hypoxia, blocks the component of GABA_B responses mediated by two‐pore domain potassium channels in neocortical layer 5 pyramidal neurons, while at the same time revealing a GIRK channel component. These data indicate that GABA_B receptors in neocortical layer 5 and hippocampal CA1 pyramidal neurons are coupled to different channels, with this coupling pH dependent on neocortical layer 5 pyramidal neurons. This pH dependency may act to maintain constant levels of GABA_B inhibition during hypoxia by enhancing GIRK channel function following a reduction in two‐pore domain potassium channel activity.     

The anti-dementia drug FK960 stimulates glial glutamate release via a PKA pathway

The present study was conducted to understand the mechanism underlying the facilitatory action of FK960, an anti-dementia drug, on hippocampal neurotransmission. FK960 facilitated hippocampal neurotransmission in normal mice, and also in mice lacking the glial glutamate transporter, GLT-1 (glut-1(-/-)), but to a lesser extent. FK960 enhanced glutamate release from cultured hippocampal astrocytes from normal rats and mice, while the drug had no effect on the release from cultured rat hippocampal neurons. The glutamate release was still obtained with cultured hippocampal astrocytes from glut-1(-/-) mice, suggesting that the release is not due to GLT-1-mediated counter transport of glutamate. The FK960 action was inhibited by H-89, a selective inhibitor of cAMP-dependent protein kinase (PKA), bafilomycin A1, an inhibitor of vesicular transport, or BAPTA-AM, a chelator of intracellular Ca(2+). FK960 caused an increase in intracellular Ca(2+) concentrations by stored Ca(2+) release in cultured rat hippocampal astrocytes, and H-89 abolished the increase. Forskolin, a PKA activator, mimicked the effect of FK960 on intracellular Ca(2+) mobilizations. Taken together, it appears that FK960 stimulates glutamate release from astrocytes, likely as a result of raising intracellular Ca(2+) concentrations via a PKA pathway. The FK960 action would increase synaptic glutamate concentrations, in part responsible for the facilitation of hippocampal neurotransmission. The results of the present study may provide a new idea that agents targeting astrocytes could serve as anti-dementia drugs.     

An examination of calcium current function on heterotopic neurons in hippocampal slices from rats exposed to methylazoxymethanol

PURPOSE: To study voltage-dependent calcium currents (VDCCs) on hippocampal heterotopic neurons by using whole-cell patch-clamp techniques in brain slices prepared from methylaxozymethanol (MAM)-exposed rats. METHODS: Whole-cell voltage-clamp recordings were obtained from visually identified neurons in acute brain slices by using an infrared differential interference contrast (IR-DIC) video microscopy system. Heterotopic neurons were compared with normotopic pyramidal cells in hippocampal slices from MAM-exposed rats or CA1 pyramidal neurons in slices from controls. RESULTS: Heterotopic neurons expressed a prominent VDCC, which exhibited a peak current maximum around -30 mV (holding potential, -60 mV) and an inactivation time constant of 48.2 +/- 2.4 ms (n = 91). VDCC peak current and inactivation time constants were similar for normotopic (n = 92) and CA1 pyramidal cells (n = 40). Pharmacologic analysis of VDCC, on heterotopic, normotopic, and CA1 pyramidal cells, revealed an approximately 70% blockade of peak Ca2+ current with nifedipine and amiloride (L- and T-type channel blockers, respectively). Inhibition of VDCC, for all three cell types, also was similar when more specific Ca2+ channel antagonists were used [e.g., omega-conotoxin GVIA (N-type), omega-agatoxin KT (P/Q-type), and sFTX-3.3 (P-type)]. VDCC modulation by norepinephrine (NE) or adrenergic receptor-specific agonists [clonidine (alpha2), isoproterenol (beta), and phenylephrine (alpha1)] was similar for heterotopic and CA1 pyramidal cells. CONCLUSIONS: Heterotopic neurons do not appear to exhibit Ca2+ channel abnormalities that could contribute to the reported hyperexcitability associated with MAM-exposed rats.     

首次发现大鼠海马CA1区神经元的TRP样通道：脑片膜片钳记录及2-氨乙氧二苯基硼酸酯和镧验证

TRP通道最初从果蝇中发现,目前已发展成为具有分子同源性的一个阳离子通道超家族。哺乳动物TRP通道蛋白是具有6个跨膜域和胞浆N、C末端的阳离子通透性通道,并按照其序列相似性,分为TRPC、TRPV、TRPM、TRPA1等亚家族。与TRP通道功能相关的特性还仍然未知,但TRP通道有可能介导阳离子因循其电化学梯度的跨膜流,进而增加细胞内的Ca2+、Na+浓度,并使细胞在多型激活过程中去极化。目前认为,TRP通道与大脑神经元功能的多种特性相关联。我们采用全细胞膜片钳方法,在急性分离的大鼠脑片中,首次记录到了海马CA1神经元的特征性TRP样通道,并且发现2-APB 或镧灌流能够增强该通道电流。     

Different Ca2+ source for slow AHP in completely adapting and repetitive firing pyramidal neurons.

Intracellular recordings in an in vitro neocortical slice preparation from immature rats were used to investigate the Ca2 source for slow afterhyperpolarization (sAHP) generation in pyramidal neurons that exhibit complete spike frequency adaptation (CA neurons). In pyramidal neurons that maintain repetitive firing for long periods of time (RF neurons), N-, P- and Q-type Ca2+ channels supply Ca2+ for sAHP generation. In CA neurons, the sAHP was reduced by only 50% by the combination of antagonists for these Ca2+ channel types and L-type channels. Ryanodine and dantrolene, blockers of Ca2(+)-induced Ca2+ release, reduced the sAHP by approximately 45% in CA neurons, but caused no reduction of the sAHP in RF neurons. Dantrolene application caused CA neurons to fire throughout a 1s suprathreshold current injection (as do RF neurons).     

钾通道阻断剂4-氨基吡啶诱导海马CA1锥体神经元钙瞬变

目的 研究4-氨基吡啶(4-AP)诱导的急性脑片海马CA1锥体神经元钙瞬变现象,探讨钾通道功能与钙瞬变的关系及可能机制.方法 荧光探针标记正常大鼠急性脑片海马神经元.共聚焦显微镜技术进行钙成像,观察不同浓度4-AP及细胞灌流液条件对神经元钙瞬变的影响.结果 低浓度(<15 mmol/L)4-AP诱导的钙瞬变峰值与剂量呈线性相关(r2=0.910,P=0.000),高浓度(20～80 mmol/L)4-AP诱导的钙瞬变峰值随浓度增高而下降.在无钙灌流液条件下,4-AP诱导的钙瞬变峰值水平下降,达峰时间延长,与含钙灌流液比较差异有统计学意义(P<0.05).结论 4-AP可诱导急性脑片海马CA1锥体神经元的钙瞬变,其机制包括细胞外钙内流与钙库钙释放.     

钾通道阻断剂4-氨基吡啶诱导海马CA1锥体神经元钙瞬变

目的 研究4-氨基吡啶(4-AP)诱导的急性脑片海马CA1锥体神经元钙瞬变现象,探讨钾通道功能与钙瞬变的关系及可能机制.方法 荧光探针标记正常大鼠急性脑片海马神经元.共聚焦显微镜技术进行钙成像,观察不同浓度4-AP及细胞灌流液条件对神经元钙瞬变的影响.结果 低浓度(<15 mmol/L)4-AP诱导的钙瞬变峰值与剂量呈线性相关(r2=0.910,P=0.000),高浓度(20～80 mmol/L)4-AP诱导的钙瞬变峰值随浓度增高而下降.在无钙灌流液条件下,4-AP诱导的钙瞬变峰值水平下降,达峰时间延长,与含钙灌流液比较差异有统计学意义(P<0.05).结论 4-AP可诱导急性脑片海马CA1锥体神经元的钙瞬变,其机制包括细胞外钙内流与钙库钙释放.     

钾通道阻断剂4-氨基吡啶诱导海马CA1锥体神经元钙瞬变

目的 研究4-氨基吡啶(4-AP)诱导的急性脑片海马CA1锥体神经元钙瞬变现象,探讨钾通道功能与钙瞬变的关系及可能机制.方法 荧光探针标记正常大鼠急性脑片海马神经元.共聚焦显微镜技术进行钙成像,观察不同浓度4-AP及细胞灌流液条件对神经元钙瞬变的影响.结果 低浓度(<15 mmol/L)4-AP诱导的钙瞬变峰值与剂量呈线性相关(r2=0.910,P=0.000),高浓度(20～80 mmol/L)4-AP诱导的钙瞬变峰值随浓度增高而下降.在无钙灌流液条件下,4-AP诱导的钙瞬变峰值水平下降,达峰时间延长,与含钙灌流液比较差异有统计学意义(P<0.05).结论 4-AP可诱导急性脑片海马CA1锥体神经元的钙瞬变,其机制包括细胞外钙内流与钙库钙释放.

Abstract：

Objective To investigate the calcium transient of CA1 pyramidal neurons induced by potassium blocker 4-aminopyridine (4-AP) in acute hippocampal slices to explore the relation between potassium channel function and calcium transient, and their mechanism. Methods Fluorescent probe was employed to mark the hippocampai neurons in acute brain slices of rats; confocal microscopy was used to perform calcium imaging to observe the influences of different concentrations of 4-AP and perfusate with/without calcium on calcium transient of CA1 pyramidal neurons. Results The response of [Ca2+]I to lower concentration of 4-AP (<15 mmol/L) was in a dose-dependent manner (r2=0.910, P=0.000); the higher the concentration of 4-AP (20-80 mmol/L), the lower the peak level of calcium transient. The latency and amplitude of calcium transient induced by 4-AP were obviously reduced when the extracellular condition was switched to an absence of calcium, which was significantly different as compared with that with calcium (P<0.05). Conclusion Blockade of potassium channels with 4-AP can increase [Ca2+]I in the hippocampal pyramidal neurons of acute slices. The increase of [Ca2+]1 to 4-AP could be ascribe to calcium release from intracellular stores and calcium influx from extracellular matrix.     

Plasma membrane insertion of TRPC5 channels contributes to the cholinergic plateau potential in hippocampal CA1 pyramidal neurons

In cultured hippocampal neurons, transient receptor potential 5 (TRPC5) channels are translocated and inserted into plasma membranes of hippocampal neurons to generate nonselective cation (NSC) currents. We investigated whether TRPC5 channel translocation also contributes to the generation of NSC currents underlying the afterdepolarizations and plateau potentials (PPs) in hippocampal pyramidal cells that are induced by muscarinic receptor activation. Using a biotinylation assay to quantify the change in surface membrane proteins in acute hippocampal slices, we found that muscarinic stimulation significantly enhanced the levels of TRPC5 protein on the membrane surface but not those of TRPC1 or TRPC4 channels. We then investigated the pharmacological sensitivity of the cation current observed during muscarinic stimulation to determine if a component could be due to TRPC5 channels. The TRPC channel antagonists 2‐APB and SKF96365 strongly depressed the generation of PPs, the underlying tail currents (I_tail) and the associated dendritic Ca²⁺ influx induced by muscarinic receptor activation in pyramidal neurons. High intracellular concentrations of ATP, which specifically inhibit TRPC5 channels, depressed I_tail. In addition, pretreatment with the calmodulin (CaM) inhibitor W‐7, which depresses recombinant TRPC5 currents, inhibited both the cation current (I_tail) and the surface insertion of TRPC5 channels. Finally, the phosphatidylinositide 3‐kinase (PI₃K) inhibitor wortmannin, which blocks translocation of TRPC5 channels in cell culture, also inhibited both the I_tail and the surface insertion of TRPC5 channels. Therefore, we conclude that insertion of TRPC5 channels contributes to the generation of the prolonged afterdepolarizations following muscarinic stimulation. This altered plasma membrane expression of TRPC5 channels in pyramidal neurons may play an important role in the generation of prolonged neuronal depolarization and bursting during the epileptiform seizure discharges of epilepsy. © 2010 Wiley‐Liss, Inc.