Evidence for the involvement of more than one mRNA species in controlling the inactivation process of rat and rabbit brain Na channels expressed in Xenopus oocytes

The properties of rat and rabbit brain sodium (Na) channels expressed in Xenopus oocytes following either unfractionated or high-molecular-weight mRNA injections were compared to assess the relative contribution of different size messages to channel function. RNA was size-fractionated on a sucrose gradient and a high-molecular-weight fraction (7-10 kilobase) encoding the alpha-subunit gave rise to functional voltage-dependent Na channels in the oocyte membrane. Single-channel conductance, mean open time, and time to first opening were all similar to the values for channels following injection of unfractionated RNA. In contrast, inactivation properties were markedly different; Na currents from high-molecular-weight RNA inactivated with a several-fold smaller macroscopic inactivation rate and showed a steady-state voltage dependence that was shifted in the depolarizing direction by at least 10 mV relative to that for unfractionated RNA. Single-channel recording revealed that the kinetic difference arose from a greater probability for high-molecular-weight RNA induced channels to reopen during a depolarizing voltage step. Pooling all gradient fractions and injecting this RNA into oocytes led to the appearance of Na channels with inactivation properties indistinguishable from those following injection of unfractionated RNA. These results suggest that mRNA species not present in the high-molecular-weight fraction can influence the inactivation process of rat brain Na channels expressed in Xenopus oocytes. This mRNA may encode beta-subunits or other proteins that are involved in posttranslational processing of voltage-dependent Na channels.     

Voltage-dependent neuromodulation of Na+ channels by D1-like dopamine receptors in rat hippocampal neurons.

Activation of D1-like dopamine (DA) receptors reduces peak Na+ current in acutely isolated hippocampal neurons through phosphorylation of the alpha subunit of the Na+ channel by cAMP-dependent protein kinase (PKA). Here we report that neuromodulation of Na+ currents by DA receptors via PKA is voltage-dependent in the range of -110 to -70 mV and is also sensitive to concurrent activation of protein kinase C (PKC). Depolarization enhanced the ability of D1-like DA receptors to reduce peak Na+ currents via the PKA pathway. Similar voltage-dependent modulation was observed when PKA was activated directly with the membrane-permeant PKA activator DCl-cBIMPS (cBIMPS; 20 microM), indicating that the membrane potential dependence occurs downstream of PKA. PKA activation caused only a small (-2.9 mV) shift in the voltage dependence of steady-state inactivation and had no effect on slow inactivation or on the rates of entry into the fast or slow inactivated states, suggesting that another mechanism is responsible for coupling of membrane potential changes to PKA modulation. Activation of PKC with a low concentration of the membrane-permeant diacylglycerol analog oleylacetyl glycerol also potentiated modulation by SKF 81297 or cBIMPS, and these effects were most striking at hyperpolarized membrane potentials where PKA modulation was not stimulated by membrane depolarization. Thus, activation of D1-like DA receptors causes a strong reduction in Na+ current via the PKA pathway, but it is effective primarily when it is combined with depolarization or activation of PKC. The convergence of these three distinct signaling modalities on the Na+ channel provides an intriguing mechanism for integration of information from multiple signaling pathways in the hippocampus and CNS.     

Molecular mechanism of convergent regulation of brain Na(+) channels by protein kinase C and protein kinase A anchored to AKAP-15

Activation of D1-like dopamine (DA) receptors reduces peak Na(+) current in hippocampal neurons voltage-dependent in a manner via phosphorylation of the alpha subunit. This modulation is dependent upon activation of cAMP-dependent protein kinase (PKA) and requires phosphorylation of serine 573 (S573) in the intracellular loop connecting homologous domains I and II (L(I-II)) by PKA anchored to A kinase anchoring protein-15 (AKAP-15). Activation of protein kinase C (PKC) also reduces peak Na(+) currents and enhances the strength of the PKA modulatory pathway. Here we probe the molecular mechanism responsible for the convergent effects of PKA and PKC on brain Na(v)1.2a channels. Analysis of the interaction of AKAP-15 with the intracellular loops of the Na(v)1.2a channel shows that it binds to L(I-II), thereby targeting PKA directly to its sites of phosphorylation on the Na(+) channel by specific protein-protein interactions. Mutagenesis and expression experiments indicate that reduction of peak Na(+) current by PKC requires S554 and S573 in L(I-II) in addition to S1506 in the inactivation gate. In addition, PKC-dependent phosphorylation of S576 in L(I-II) is necessary for enhancement of PKA modulation of brain Na(+) channels. When S576 is phosphorylated by PKC, the increase in modulation by PKA activation requires phosphorylation of S687 in L(I-II). Thus, the maximal modulation of these Na(+) channels by concurrent activation of PKA and PKC requires phosphorylation at four distinct sites in L(I-II): S554, S573, S576, and S687. This convergent regulation provides a novel mechanism by which information from multiple signaling pathways may be integrated at the cellular level in the hippocampus and throughout the central nervous system.     

Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells.

The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell-specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.     

Serotonergic modulation of persistent sodium currents and membrane excitability via cyclic AMP-protein kinase A cascade in mesencephalic V neurons

In rat mesencephalic trigeminal (Mes V) neurons, persistent sodium currents in conjunction with low-threshold potassium currents are critical for generation of subthreshold membrane oscillations and onset of burst behavior. Here we demonstrate that the cAMP/protein kinase A (PKA) signaling pathway modulates persistent sodium currents. In particular, we show that elevation of cAMP suppresses a low-threshold I(NaP) via a PKA intracellular pathway. Bath application of forskolin (20 microM), a stimulant for the production of cAMP, reduced the peak I(NaP). 1,9-Dideoxy-forskolin (20 microM), an inactive form of forskolin, produced minimal effects on I(NaP), and the membrane-permeable cAMP analogue 8-bromo-cAMP (500 microM) mimicked the effect of forskolin. Additionally, preapplication of H89 (2 microM), a specific PKA inhibitor, suppressed the effect of forskolin, suggesting the involvement of the cAMP/PKA intracellular signaling pathway in this modulation. 5-HT receptor stimulation (20 microM) also mimicked the modulation of I(NaP) by forskolin via the cAMP/PKA-dependent signaling pathway. Current clamp analysis demonstrated that voltage-dependent membrane resonance in response to a ZAP input current at depolarized holding potentials (approximately -50 mV) was specifically suppressed by forskolin or 5-HT. Moreover, drug application enhanced frequency adaptation in response to a 1-sec current pulse. These results indicate that modulation of persistent sodium currents by a cAMP/PKA pathway can significantly alter the membrane excitability and discharge characteristics of Mes V neurons.     

Characterization and regulation of rat microglial Ca(2+) release-activated Ca(2+) (CRAC) channel by protein kinases

We measured the activity of the Ca(2+) release-activated Ca(2+) (CRAC) channel present in cultured rat microglia, using the whole-cell mode of patch clamp technique. When the concentration of divalent cations in external solution was reduced to the micromolar range, and Ca(2+) chelating agent BAPTA was included in the pipette solution, we were able to record Na(+) current through CRAC channels in single-channel levels. The unitary Na(+) conductance through CRAC channel was 42.5 pS, which was similar to that of Jurkat cell. The Na(+) current activated slowly, reaching the maximal current level in about 10 min after whole-cell patches were made. The time required for the half-maximal activation of the current was 205 s (+/-31), while it was reduced to 84.3 s (+/-17.7) by including IP(3) in the pipette solution as well. The peak currents ranged from 320 to 985 pA, which corresponded to 64-197 channels per cell. We studied the regulation of the current by protein kinase A (PKA) and protein kinase C (PKC). The current was enhanced by the addition of membrane-permeant analogue of cAMP, dibutyryl cAMP. Pretreating cells with PKA inhibitor, H-89, prevented the effect of dibutyryl cAMP. By contrast, the addition of PKC activator, PDBu, reduced the current. Staurosporine, a PKC inhibitor, prevented the effect of PDBu. These results suggest that CRAC channel in rat microglia is under the regulation of PKA and PKC in opposite directions.     

Modulation of the voltage-dependent sodium channel by agents affecting G-proteins: a study in Xenopus oocytes injected with brain RNA

The effects of agents known to affect G-proteins on voltage-dependent, tetrodotoxin-sensitive Na+ channels were studied in Xenopus oocytes injected with rat brain RNA, using two-electrode voltage-clamp technique. The non-hydrolysable analogue of GTP, GTP-gamma-S, known to activate G-proteins, inhibited the Na+ current (INa). The decrease in the amplitude of INa was not accompanied by changes in activation or inactivation characteristics of the channel. The non-hydrolysable analogue of GDP, GDP-beta-S, had no effect on INa. The responses to gamma-aminobutyric acid and kainate in the same oocytes were also attenuated by GTP-gamma-S. Pertussis toxin, which inactivates some G-proteins by catalyzing their ADP-ribosylation, enhanced INa, but did not prevent the inhibition of INa by GTP-gamma-S. We conclude that the Na+ channel, and possibly the GABA and kainate receptors and/or channels, are coupled to a G-protein. The activation of the G-protein modulates the channels either directly, or via activation of biochemical cascade possibly involving production of second messengers and channel phosphorylation.     

Changes in ionic conductances induced by cAMP in Helix neurons

Adenosine 3',5'-cyclic monophosphate (cAMP) was injected by a fast and quantitative pressure injection method into voltage-clamped identified Helix neurons. The intracellular elevation of cAMP caused an inward current which was not accompanied by a significant change in membrane conductance in a negative potential range with little activation of voltage-dependent membrane conductances. Near resting potential Na+ ions were the main carrier of the cAMP-induced inward current as measured with ion-selective microelectrodes. TTX did not affect the Na+ influx. K+ and less effective Ca2+ could substitute for Na+ in carrying the inward current. In the presence of Na+, divalent cations such as Ca2+ and Mg2+, and also La3+ exerted an inhibitory influence on the cAMP-induced inward current, and Ca2+ as measured with ion-selective microelectrodes did not contribute significantly to the current. Thus, the inward current was of a non-specific nature. Simultaneously to this cAMP action, the membrane permeability for K+ ions was decreased by cAMP. This effect became particularly obvious when K+ currents were activated by long-lasting, depolarizing voltage steps. In this situation a reduced K+ efflux following cAMP injection was observed by means of K+-selective microelectrodes located near the external membrane surface. Outward K+ currents were less reduced by cAMP if external Ca2+ was replaced by Ni2+. The nearly compensatory increase and decrease of two membrane conductances in the same neuron explained the lack of change in the cell input resistance despite the considerable depolarizing action of intracellularly elevated cAMP.     

PACAP Stimulation of Gonadotropin-II Secretion in Goldfish Pituitary Cells: Mechanisms of Action and Interaction with Gonadotropin Releasing Hormone Signalling

Pituitary adenylate cyclase-activating polypeptide (PACAP) has recently been shown to be a hypophysiotropic factor in the goldfish. In this study, we examined the mechanisms of PACAP action on goldfish maturational gonadotropin (GTH-II) release using primary cultures of pituitary cells. The GTH-II response to mammalian PACAP1-38 (mPACAP) was inhibited by a PACAP receptor antagonist suggesting a receptor-mediated action. Addition of either an adenylate cyclase inhibitor or a protein kinase A (PKA) inhibitor reduced the mPACAP-induced GTH-II release. In addition, when GTH-II release was already stimulated by either forskolin or 8-bromo-cAMP (8Br-cAMP), mPACAP did not further increase GTH-II secretion. These results strongly implicated the involvement of an adenylate cyclase/cAMP/PKA pathway in PACAP-stimulated GTH-II release. Although mPACAP induced a rise in intracellular Ca2+ level in identified gonadotropes, results with voltage-sensitive Ca2+ channel inhibitors indicated that the GTH-II responses to mPACAP, forskolin and 8Br-cAMP did not depend upon Ca2+ entry through these channels. Two protein kinase C (PKC) inhibitors did not affect mPACAP-elicited GTH-II release, and mPACAP further increased GTH-II secretion in the presence of PKC activators. These results indicate that PKC-dependent elements are not essential for the stimulatory action of mPACAP in gonadotropes. Interestingly, while GTH-II responses to a stimulatory concentration of mPACAP were additive to responses elicited by maximal effective concentrations of two endogenous gonadotropin releasing hormones (GnRHs), a subthreshold concentration of mPACAP potentiated GnRH and PKC activator stimulation of GTH-II secretion. Similarly, submaximal concentrations of forskolin potentiated the GTH-II response to the PKC activator, tetradecanoyl phorbol acetate. These data suggest that PACAP and its cAMP-dependent signalling mechanisms provide an alternate stimulatory input to goldfish gonadotropes and may influence the effectiveness of the major neuroendocrine control exerted by the PKC-dependent GnRH signalling pathway.     

Differential modulation of Ca2+-activated K+ channels in ovine pituitary gonadotrophs by GnRH, Ca2+ and cyclic AMP

Patch-clamp techniques were employed to examine the effects of cAMP in relation to gonadotrophin-releasing hormone (GnRH) action on Ca2+-activated K+ channels in pituitary gonadotrophs derived from ovine pars tuberalis. GnRH applied extracellularly increased channel openings in cell-attached patches similar to calcium ionophore (A23187), while raising intracellular cAMP concentration with dibutyryl cAMP or forskolin decreased the number of functional channels (Nf) and the open state probability (Po). Both cAMP and the catalytic subunit of cAMP-dependent protein kinase produced similar results when applied to the cytoplasmic membrane face of inside-out patches, and the effect of cAMP was abolished by the protein kinase inhibitor. Our results suggest that decreased permeability through these channels modulated by cAMP through a phosphorylation-dependent route can modulate luteinizing hormone release.     

Lidocaine stabilizes the open state of CNS voltage-dependent sodium channels

We have previously reported that the lidocaine action is different between CNS and muscle batrachotoxin-modified Na+ channels [Salazar et al., J. Gen. Physiol. 107 (1996) 743-754; Brain Res. 699 (1995) 305-314]. In this study we examined lidocaine action on CNS Na+ currents, to investigate the mechanism of lidocaine action on this channel isoform and to compare it with that proposed for muscle Na+ currents. Na+ currents were measured with the whole cell voltage clamp configuration in stably transfected cells expressing the brain alpha-subunit (type IIA) by itself (alpha-brain) or together with the brain beta(1)-subunit (alphabeta(1)-brain), or the cardiac alpha-subunit (hH1) (alpha-cardiac). Lidocaine (100 microM) produced comparable levels of Na+ current block at positive potentials and of hyperpolarizing shift of the steady-state inactivation curve in alpha-brain and alphabeta(1)-brain Na+ currents. Lidocaine accelerated the rates of activation and inactivation, produced an hyperpolarizing shift in the steady-state activation curve and increased the current magnitude at negative potentials in alpha-brain but not in alphabeta(1)-brain Na+ currents. The lidocaine action in alphabeta(1)-brain resembled that observed in alpha-cardiac Na+ currents. The lidocaine-induced increase in current magnitude at negative potentials and the hyperpolarizing shift in the steady-state activation curve of alpha-brain, are novel effects and suggest that lidocaine treatment does not always lead to current reduction/block when it interacts with Na+ channels. The data are explained by using a modified version of the model proposed by Vedantham and Cannon [J. Gen. Physiol., 113 (1999) 7-16] in which we postulate that the difference in lidocaine action between alpha-brain and alphabeta(1)-brain Na+ currents could be explained by differences in the lidocaine action on the open channel state.     

Interaction of forskolin with voltage-gated K+ channels in PC12 cells

Forskolin (FSK) directly blocks a distinct class of voltage-dependent K+ channels in pheochromocytoma cells. We have studied the biophysical mechanism of FSK action on these channels. The mean open duration decreased linearly with [FSK], indicating that a single molecule of FSK interacts with a single open K+ channel. FSK did not alter the voltage dependence of activation or the latency to first opening. Whole-cell currents in the presence of FSK did not show a rising phase in tail currents, suggesting that FSK-bound channels can close. We used a kinetic scheme in which FSK binds preferentially to the open state of the channel to describe its interaction with the K+ channel. This scheme is analogous to the modulated receptor hypothesis used to describe the interaction of local anesthetics with voltage-dependent Na+ channels.     

AMPA receptor activation induces association of G-beta protein with the alpha subunit of the sodium channel in neurons

Glutamatergic transmission is mediated by ionotropic receptors that directly gate cationic channels and metabotropic receptors that are coupled to second messenger generating systems and to ionic channels via heterotrimeric guanine-nucleotide binding- (G) proteins. This distinction cannot be made for the ionotropic receptor subclass activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), which has been shown to be physically associated with the alpha-subunit of Gi1 protein and activates this G-protein. Here, we report that, in addition to a Ca2+ influx, AMPA induces the mobilization of Ca2+ from the mitochondrial pool by reversing the mitochondrial Na+/Ca2+ exchanger in mouse neurons in primary culture. Both processes required the activation of tetrodotoxin-sensitive Na+ channels. AMPA receptor activation modified the gating properties of the Na+ channel, independently of the AMPA current, suggesting a G-protein-mediated process. Indeed, co-immunoprecipitation experiments indicated that AMPA receptor activation induced the association of Gbeta with the alpha-subunit of the Na+ channel. These results suggest that, in addition to its ionic channel function, the AMPA receptor is coupled to Na+ channels through G-proteins and that this novel metabotropic function is involved in the control of neuronal excitability.     

Inhibition of forskolin-induced sensitisation of frog skin nociceptors by the cyclic AMP-dependent protein kinase A antagonist H-89

The role of cAMP and protein kinase A (PKA) in nociceptor sensitisation has been studied in frog skin in vitro. Multifibre nerve responses during noxious heating were enhanced by adding forskolin, an agent that elevates cAMP. H-89, a PKA antagonist, blocked the increased responses due to forskolin, but did not affect baseline responses. Thus, cAMP appears to act via PKA when sensitising nociceptors in frog skin.     

Metaplasticity of the late-phase of long-term potentiation: a critical role for protein kinase A in synaptic tagging

The late-phase of long-term potentiation (L-LTP) in hippocampal area CA1 requires gene expression and de novo protein synthesis but it is expressed in an input-specific manner. The 'synaptic tag' theory proposes that gene products can only be captured and utilized at synapses that have been 'tagged' by previous activity. The mechanisms underlying synaptic tagging, and its activity dependence, are largely undefined. Previously, we reported that low-frequency stimulation (LFS) decreases the stability of L-LTP in a cell-wide manner by impairing synaptic tagging. We show here that a phosphatase inhibitor, okadaic acid, blocked homosynaptic and heterosynaptic inhibition of L-LTP by prior LFS. In addition, prior LFS homosynaptically and heterosynaptically impaired chemically induced synaptic facilitation elicited by forskolin/3-isobutyl-1-methylxanthine, suggesting that there is a cell-wide dampening of cAMP/protein kinase A (PKA) signaling concurrent with phosphatase activation. We propose that prior LFS impairs expression of L-LTP by inhibiting synaptic tagging through its actions on the cAMP/PKA pathway. In support of this notion, we show that hippocampal slices from transgenic mice that have genetically reduced hippocampal PKA activity display impaired synaptic capture of L-LTP. An inhibitor of PKA, KT-5720, also blocked synaptic capture of L-LTP. Moreover, pharmacological activation of the cAMP/PKA pathway can produce a synaptic tag to capture L-LTP expression, resulting in persistent synaptic facilitation. Collectively, our results show that PKA is critical for synaptic tagging and for input-specific L-LTP. PKA-mediated signaling can be constrained by prior episodes of synaptic activity to regulate subsequent L-LTP expression and perhaps control the integration of multiple synaptic events over time.     

Brevetoxin modulates neuronal sodium channels in two cell lines derived from rat brain

Single Na+ channel currents were recorded from cell-attached membrane patches from two neuronal cell lines derived from rat brain, B50 and B104, and compared before and after exposure of the cells to purified brevetoxin, PbTx-3. B50 and B104 Na+ channels usually exhibited fast activation and inactivation as is typical of TTX-sensitive Na+ channels. PbTx-3 modified channel gating in both cell lines. PbTx-3 caused (1) significant increases in the frequency of channel reopening, indicating a slowing of channel inactivation, (2) a change in the voltage dependence of the channels, promoting channel opening during steady-state voltage clamp of the membrane at voltages throughout the activation range of Na+ currents, but notably near the resting potential of these cells (-60 - -50 mV), and (3) a significant, 6.7 mV hyperpolarized shift in the threshold potential for channel opening. Na+ channel slope conductance did not change in PbTx-3-exposed B50 and B104 neurons. These effects of Pbx-3 may cause hyperexcitability as well as inhibitory effects in intact brain.     

Effect of prolonged hypoxia on Na+ channel mRNA subtypes in the developing rat cortex.

Voltage-gated Na+ channels are regulated in response to oxygen deprivation in the mammalian cortex. Past investigations have demonstrated that Na+ channel protein expression is up-regulated in the immature brain exposed to prolonged hypoxia. Since it is unknown as to which Na+ channel subtype(s) is involved in this regulation, we used RT-PCR to assess the effect of hypoxia on Na+ channel I, II and III alpha-subunit mRNA expression in the developing rat cortex. Na+ channel II mRNA tended to increase during early development, whereas Na+ channel I and III did not change or slightly decreased with age. Hypoxic exposure for 1-day had no effect on Na+ channel expression, while 5-day hypoxia significantly increased Na+ channel III density, with a slight increase in Na+ channel I and no appreciable change in Na+ channel II. These results suggest that Na+ channel subtype expression in the developing cortex is differentially regulated in response to prolonged hypoxic exposure.     

Modulation by cAMP of a slowly activating potassium channel expressed in Xenopus oocytes.

When expressed in the Xenopus oocyte, the minK protein induces a slowly activating voltage-dependent potassium current (Isk). We studied the modulation of this current by altering intracellular cAMP levels and found that the amplitude of Isk is dramatically increased by treatments that raise cAMP levels and decreased by agents that lower cAMP levels. Preinjection of a protein inhibitor of the cAMP-dependent protein kinase blocked the effects of increased cAMP levels. There were no changes in the voltage dependence or kinetics of Isk. Mutations that eliminate a potential phosphorylation site on the minK protein did not block the effects of activating the kinase. In addition, the membrane capacitance of the oocyte increased and decreased in parallel with Isk. Our results fit a mechanism in which channel proteins are selectively inserted into and removed from the plasma membrane in response to changes in kinase activity.     

Calcium current in cortical astrocytes: induction by cAMP and neurotransmitters and permissive effect of serum factors

Voltage-dependent L-type calcium channels were induced in highly purified, cultured, cortical astrocytes by exposure to substances known to increase their intracellular cAMP: 8-bromo-cAMP, forskolin, isoproterenol, and vasoactive intestinal peptide. In untreated control cultures, L-type calcium currents were entirely absent. The induction of this calcium current was specific to cortical astrocytes and a closely related astrocyte type in white matter and did not occur in meningeal cells or oligodendrocytes. The ability of forskolin to induce L-type calcium current in astrocytes depended on previous culture of these cells in a permissive lot of serum for at least 48 hr. In addition, certain lots of sera caused expression of a voltage-dependent T-type calcium channel in untreated control cultures. Several possible mechanisms of calcium channel induction by cAMP are consistent with our data: modification of a silent channel already present in the membrane, indirect effect of cytoskeletal alteration, or insertion of channels from submembrane stores. If the environment in vivo is permissive, regulation of glial ion channels may be under neuronal control.