Currents In Whole-cell Recordings Research Articles

The role of dopamine D2-like receptors in a “depotentiation-like effect” of deep brain stimulation in kindled rats

The mechanisms involved in the anti-seizure effects of low-frequency stimulation (LFS) have not been completely determined. However, Gi-protein-coupled receptors, including D2-like receptors, may have a role in mediating these effects. In the present study, the role of D2-like receptors in LFS’ anti-seizure action was investigated. Rats were kindled with semi-rapid (6 stimulations per day), electrical stimulation of the hippocampal CA1 area. In LFS-treated groups, subjects received four trials of LFS at 5 min, 6 h, 24 h, and 30 h following the last kindling stimulation. Each LFS set occurred at 5 min intervals, and consisted of 4 trains. Each train contained 200, 0/1 ms long, monophasic square wave pulses at 1 Hz. Haloperidol (D2-like receptors antagonist, 2 µm) and/or bromocriptine (D2-like receptors agonist 2 µg/µlit) were microinjected into the lateral ventricle immediately after the last kindling, before applying LFS. Obtained results showed that applying LFS in fully-kindled subjects led to a depotentiation-like decrease in kindling-induced potentiation and reduced the amplitude and rise slope of excitatory and inhibitory post-synaptic currents in whole-cell recordings from CA1 pyramidal neurons. In addition, LFS restored the kindling-induced, spatial learning and memory impairments in the Barnes maze test. A D2-like receptor antagonist inhibited these effects of LFS, while a D2-like receptor agonist mimicked these effects. In conclusion, a depotentiation-like mechanism may be involved in restoring LFS’ effects on learning and memory, and synaptic plasticity. These effects depend on D2-like receptors activity.

Melatonin activates BKCa channels in cerebral artery myocytes via both direct and MT receptor/PKC-mediated pathway

The pineal hormone melatonin is a neuroendocrine hormone with high membrane permeability that is involved in regulation of circadian rhythm of several biological functions. Large-conductance Ca2+-activated K+ (BKCa) channels are abundantly expressed in vascular smooth muscle cells and play an important role in vascular tone regulation. We investigated the mechanisms through which myocyte BKCa channels mediate effects of melatonin on cerebral arteries (CAs). Arterial contractility measurements showed that melatonin alone did not change vascular tone in CAs; however, it induced concentration-dependent vasodilation of phenylephrine-induced contraction in CAs. In the presence of the potent endothelial oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester, melatonin-elicited relaxation was significantly inhibited by iberiotoxin (BKCa channel blocker). Melatonin significantly increased BKCa currents but not voltage-gated K+ (KV) currents in whole-cell recordings. Melatonin decreased the amplitude of Ca2+ sparks and spontaneous transient outward currents (STOCs), however, a significant increase in open probability of BKCa channels was observed in both inside-out and cell-attached patch-clamp recordings. This melatonin-induced enhancement of BKCa channel activity was significantly suppressed by luzindole (melatonin MT1/MT2 receptor inhibitor), U73122 (phospholipase C (PLC) inhibitor), and Ro31–8220 (protein kinase C (PKC) inhibitor). Melatonin had no significant effects on sarcoplasmic reticulum release of Ca2+. These findings indicate that melatonin-induced vasorelaxation of CAs is partially attributable to direct (passing through the cell membrane) and indirect (via melatonin MT1/MT2 receptors-PLC-PKC pathway) activation of BKCa channels on CA myocytes.

Bradykinin Sensitization of TRPV1 Channels

Bradykinin (BK) and nerve growth factor (NGF) both sensitize TRPV1 channels in dorsal root ganglia (DRG) and trigeminal ganglia (TG) neurons of mammals. BK treatment of dissociated DRG neurons and transiently transfected cells expressing TRPV1 and the B2 receptor for BK (a Gαq-coupled GPCR) confirmed reports in the literature of increased capsaicin-activated currents in whole-cell recordings. Calcium imaging of human embryonic kidney 293 cells (293T) and DRG-neuroblastoma hybrid cells (F-11) show a consistent calcium rise upon treatment with BK, but up to three-day-old dissociated DRG neurons seldom show a calcium rise. Confocal imaging of F11 and 293T cells transfected with a fluorescent diacylglycerol probe (C1-PKC-GFP) shows an increase of DAG in the plasma membrane (PM) upon application of BK, but a fluorescent phosphatidylinositol 4,5-bisphosphate (PIP2) probe (YFP-PLCd1-PH) did not reveal a change in the PIP2 levels on the PM. Calcium imaging of dissociated DRG neurons and F-11 cells expressing NGF-receptors trkA and p75 did not show calcium rise upon application of NGF. F-11 cells expressing NGF receptor, TRPV1 channels and a fluorescent phosphatidylinositol 3,4,5-triphosphate (PIP3) probe (YFP-GRP1-PH) exhibit increased PIP3 levels on the PM upon NGF treatment. F-11 cells expressing the PIP3 probe did not show a change of PIP3 levels on the plasma membrane upon application of BK. These results suggest different pathways for BK and NGF sensitization of TRPV1 channels in cultured cells. BK produces TRPV1 sensitization with no measurable change in PIP2 levels, a rise in calcium and an increase on DAG, suggesting changes of second messengers downstream from phospholipase C (PLC) activation different from PIP2, while NGF produces TRPV1 sensitization with a measurable change of PIP3, and no significant change in calcium suggesting changes of second messengers downstream from phosphatidylinositol 3-kinase (PI3K). Funding provided by Grant EY07031 from the NEI.

Transient receptor potential canonical channels are essential for chemotactic migration of human malignant gliomas

The majority of malignant primary brain tumors are gliomas, derived from glial cells. Grade IV gliomas, Glioblastoma multiforme, are extremely invasive and the clinical prognosis for patients is dismal. Gliomas utilize a number of proteins and pathways to infiltrate the brain parenchyma including ion channels and calcium signaling pathways. In this study, we investigated the localization and functional relevance of transient receptor potential canonical (TRPC) channels in glioma migration. We show that gliomas are attracted in a chemotactic manner to epidermal growth factor (EGF). Stimulation with EGF results in TRPC1 channel localization to the leading edge of migrating D54MG glioma cells. Additionally, TRPC1 channels co-localize with the lipid raft proteins, caveolin-1 and β-cholera toxin, and biochemical assays show TRPC1 in the caveolar raft fraction of the membrane. Chemotaxis toward EGF was lost when TRPC channels were pharmacologically inhibited or by shRNA knockdown of TRPC1 channels, yet without affecting unstimulated cell motility. Moreover, lipid raft integrity was required for gliomas chemotaxis. Disruption of lipid rafts not only impaired chemotaxis but also impaired TRPC currents in whole cell recordings and decreased store-operated calcium entry as revealed by ratiomeric calcium imaging. These data indicated that TRPC1 channel association with lipid rafts is essential for glioma chemotaxis in response to stimuli, such as EGF.

Neuron-glia interactions: P2X7 receptor, the new player of the astrocytic receptor team

Event Abstract Back to Event Neuron-glia interactions: P2X7 receptor, the new player of the astrocytic receptor team Joao Oliveira1* 1 University of Leipzig, Faculty of Medicine, Germany The better understanding of neuron-glia interactions and their influence on synaptic neurotransmission in the prefrontal cortex (PFC) is crucial to address the implication of these interactions for emotional, cognitive and short-term memory processes. Once thought to be only supportive elements allowing optimal nerve communication, astrocytes are now recognized as fully-fledged partners in neuro-neuronal communication. Indeed, recent findings reported the release of glutamate and other neurotransmitters e.g. by the activation of astrocytic purinergic P2X7 receptors in cell culture systems. Astrocyte-released glutamate may be responsible for the differential activation of neuronal regulatory receptors. Functional evidence of P2X7 receptors was sought in astrocytes of rat and mice brain slices containing the PFC, using patch-clamp and immunohistochemical techniques. The application of the P2X7 receptor agonist dibenzoyl-ATP (Bz-ATP) and ATP itself both caused inward currents in whole-cell recordings from those astrocytes. P2X7 receptor-immunoreactivity was co-localized with astrocytic, but not neuronal or microglial markers on cells labelled after electrophysiological measurements with Lucifer Yellow. The inactivity of α,β-methylene ATP, as well as the potency increases of Bz-ATP and ATP in a low divalent cation (X2+) conditions suggested the involvement of P2X7 receptors. This idea was further supported by the inhibition of these current responses by PPADS and selective P2X7 receptor antagonists. The reversal potential of Bz-ATP was near 0 mV, indicating the opening of such cationic receptor-channels. In a low X2+ extracellular medium, ATP induced current responses in PFC astrocytes of wild-type mice, but not of the P2X7-/- knockouts. On the other hand, the activation of astrocytic P2X7 receptors failed to interfere directly with the synaptic transmission between pyramidal neurons of the PFC. In conclusion, cortical astrocytes of rats and mice possess functional P2X7 receptors mediating inward cationic currents near the resting membrane potential. These receptors may most likely participate in necrotic/apoptotic or proliferative reactions after stimulation by large quantities of ATP released by CNS injury, rather than mediate a direct influence in synaptic transmission under physiological states. Conference: 11th Meeting of the Portuguese Society for Neuroscience, Braga, Portugal, 4 Jun - 6 Jun, 2009. Presentation Type: Oral Presentation Topic: Symposium 1– Neuronal Communication Citation: Oliveira J (2009). Neuron-glia interactions: P2X7 receptor, the new player of the astrocytic receptor team. Front. Neurosci. Conference Abstract: 11th Meeting of the Portuguese Society for Neuroscience. doi: 10.3389/conf.neuro.01.2009.11.002 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2009; Published Online: 04 Aug 2009. * Correspondence: Joao Oliveira, University of Leipzig, Faculty of Medicine, Leipzig, Germany, nemoABS01@frontiersin.org Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Joao Oliveira Google Joao Oliveira Google Scholar Joao Oliveira PubMed Joao Oliveira Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Unexpected suppression of neuronal G protein-activated, inwardly rectifying K+ current by common phospholipase C inhibitor

Stabilization of the binding of phosphatidylinositol bisphosphate (PIP(2)) to G protein-coupled inward rectifier K+ (GIRK) channels is essential for their activation, whereas hydrolysis of PIP(2) by phospholipase C (PLC) inhibits channel activity. Apparently inconsistent with this mechanism, we found that the commonly used PLC inhibitor, U73122 (1 microM), produced a significant reduction in the amplitude of baclofen (20 microM)-evoked GIRK currents in whole-cell recordings from acutely isolated rat neocortical pyramidal cells. Also, U73122 reduced the percentage of baclofen-responsive neurons from 100% (n=40) to 56% (n=25). Since NCDC (100 microM), a PLC inhibitor of another molecular class, displayed no effect on GIRK current amplitude or responsiveness (100%, n=6), inhibition of PLC is unlikely to account for the effects of U73122 in our preparation. Lending further support to this notion, the structurally closely related compound, U73343, which does not inhibit PLC, proved to be even more efficient in suppressing GIRK current as compared to U73122. In neurons, in which GIRK channels were irreversibly activated by GTPgammaS (n=10), the depressant action of U71322 was fully preserved. These findings hint at a direct interaction of U73122 with the GIRK channel or a closely associated protein. Caution is therefore warranted when employing this compound to examine the role of PLC and PIP(2) in the regulation of GIRK channel activity.

Mechano‐sensitivity of epithelial sodium channels (ENaCs): laminar shear stress increases ion channel open probability

Epithelial cells are exposed to a variety of mechanical forces, but little is known about the impact of these forces on epithelial ion channels. Here we show that mechanical activation of epithelial sodium channels (ENaCs), which are essential for electrolyte and water balance, occurs via an increased ion channel open probability. ENaC activity of heterologously expressed rat (rENaC) and Xenopus (xENaC) orthologs was measured by whole-cell as well as single-channel recordings. Laminar shear stress (LSS), producing shear forces in physiologically relevant ranges, was used to mechanically stimulate ENaCs and was able to activate ENaC currents in whole-cell recordings. Preceding pharmacological activation of rENaC with Zn2+ and xENaC with gadolinium and glibenclamide largely prevented LSS-activated currents. In contrast, proteolytic cleavage with trypsin potentiated the LSS effect on rENaC whereas the LSS effect on xENaC was reversed (inhibition of xENaC current). Further, we found that exposure of excised outside-out patches to LSS led to an increased ion channel open probability without affecting the number of active channels. We suggest that mechano-sensitivity of ENaC may represent a ubiquitous feature for the physiology of epithelia, providing a putative mechanism for coupling transepithelial Na+ reabsorption to luminal transport.

The development of excitatory capability in Aplysia californica bag cells observed in cohorts

The bag cells of Aplysia release egg laying hormone in sexually mature animals. Bag cells cannot sustain the long-lasting excitatory afterdischarge (AD) required for hormone release prior to sexual maturity (T.A. Nick, L.K. Kaczmarek, T.J. Carew, Ionic currents underlying developmental regulation of repetitive firing in Aplysia bag cell neurons, J. Neurosci. 1996;16:7583–7598; L.A. Fieber, Characterization of Na + and Ca 2+ currents in bag cells of sexually immature Aplysia californica, J. Exp. Biol. 1998;201:745–754). To investigate the development of bag cell excitability, whole-cell voltage-clamp experiments were executed in dissociated bag cells from four cohorts (batches) of hatchery-reared A. californica maintained at 13–15°C. K + current densities, representing the sum of at least four different outward K + currents (Nick et al., 1996), declined significantly as a function of age, beginning at least 2–3 months before sexual maturity. The K + current decreases coincided with the first appearance of Na + and Ca 2+ currents in bag cells, which occurred at ages 6–7 months. Whole cell K + currents were not decreased significantly by a cAMP analog earlier than 1 month prior to the onset of reproductive activity. The frequency of observing Na + currents in whole cell recordings was low for developmental times earlier than sexual maturity. In one winter batch, both control and PMA-treated Na + currents increased significantly with age, and PMA-treated current densities were significantly greater than controls, but the other two batches studied had significant differences in Na + current frequency only at sexual maturity. Ca 2+ currents were reliably measured in more cells than were Na + currents. The Ca 2+ current frequency increased significantly with maturity in one winter batch. Ca 2+ currents were significantly increased by phorbol ester treatment beginning 6–8 weeks before reproductive activity in the two winter batches. These observations support the hypothesis that bag cell excitability is not fully developed until shortly before sexual maturity.

Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons.

Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons. J. Neurophysiol. 80: 2954-2962, 1998. Using tight-seal, whole cell recordings from isolated transverse slices of hamster and rat spinal cord, we investigated the effects of the mu-opioid agonist (-Ala2, N-Me-Phe4,Gly5-ol)-enkephalin (DAMGO) on the membrane potential and conductance of substantia gelatinosa (SG) neurons. We observed that bath application of 1-5 microM DAMGO caused a robust and repeatable hyperpolarization in membrane potential (Vm) and decrease in neuronal input resistance (RN) in 60% (27/45) of hamster neurons and 39% (9/23) of rat neurons, but significantly only when ATP (2 mM) and guanosine 5'-triphosphate (GTP; 100 microM) were included in the patch pipette internal solution. An ED50 of 50 nM was observed for the hyperpolarization in rat SG neurons. Because G-protein mediation of opioid effects has been shown in other systems, we tested if the nucleotide requirement for opioid hyperpolarization in SG neurons was due to G-protein activation. GTP was replaced with the nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S; 100 microM), which enabled DAMGO to activate a nonreversible membrane hyperpolarization. Further, intracellular application of guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S; 500 microM), which blocks G-protein activation, abolished the effects of DAMGO. We conclude that spinal SG neurons are particularly susceptible to dialysis of GTP by whole cell recording techniques. Moreover, the depletion of GTP leads to the inactivation of G-proteins that mediate mu-opioid activation of an inward-rectifying, potassium conductance in these neurons. These results explain the discrepancy between the opioid-activated hyperpolarization in SG neurons observed in previous sharp electrode experiments and the more recent failures to observe these effects with whole cell patch techniques.

Potassium conductance causing hyperpolarization of CA1 hippocampal neurons during hypoxia.

In experiments on slices (from 100- to 150-g Sprague-Dawley rats) kept at 33 degreesC, we studied the effects of brief hypoxia (2-3 min) on CA1 neurons. In whole cell recordings from submerged slices, with electrodes containing only KMeSO4 and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and in the presence of kynurenate and bicuculline (to minimize transmitter actions), hypoxia produced the following changes: under current clamp, 36 cells were hyperpolarized by 2.7 +/- 0.5 (SE) mV and their input resistance (Rin) fell by 23 +/- 2.7%; in 30 cells under voltage clamp, membrane current increased by 114 +/- 22.3 pA and input conductance (Gin) by 4.9 +/- 0.9 nS. These effects are much greater than those seen previously with K gluconate whole cell electrodes, but only half those seen with "sharp" electrodes. The hypoxic hyperpolarizations (or outward currents) were not reduced by intracellular ATP (1-5 mM) or bath-applied glyburide (10 microM): therefore they are unlikely to be mediated by conventional ATP-sensitive K channels. On the other hand, their depression by internally applied ethylene glycol-bis-(beta-aminoethyl ether)-N,N, N',N'-tetraacetic acid (1.1 and 11 mM) and especially 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (11-33 mM) indicated a significant involvement of Ca-dependent K (KCa) channels. The beta-adrenergic agonist isoprenaline (10 microM) reduced hypoxic hyperpolarizations and decreases in Rin (n = 4) (and in another 11 cells corresponding changes in Gin); and comparable but more variable effects were produced by internally applied 3':5'-adenosine cyclic monophosphate (cAMP, 1 mM, n = 6) and bath-applied 8-bromo-cAMP (n = 8). Thus afterhyperpolarization-type KCa channels probably take part in the hypoxic response. A major involvement of G proteins is indicated by the near total suppression of the hypoxic response by guanosine 5'-O-(3-thiotriphosphate) (0. 1-0.3 mM, n = 23) and especially guanosine 5'-O-(2-thiodiphosphate) (0.3 mM, n = 26), both applied internally. The adenosine antagonist 8-(p-sulfophenyl)theophylline (10-50 microM) significantly reduced hypoxic hyperpolarizations and outward currents in whole cell recordings (with KMeSO4 electrodes) from submerged slices but not in intracellular recordings (with KCl electrodes) from slices kept at gas/saline interface. In further intracellular recordings, antagonists of gamma-aminobutyric acid-B or serotonin receptors also had no clear effect. In conclusion, these G-protein-dependent hyperpolarizing changes produced in CA1 neurons by hypoxia are probably initiated by Ca2+ release from internal stores stimulated by enhanced glycolysis and a variable synergistic action of adenosine.

Activity-dependent regulation of inwardly rectifying potassium currents in non-myelinating Schwann cells in mice.

1. Voltage-gated potassium currents were recorded from freshly dissociated non-myelinating Schwann cells of sural and sympathetic nerves from 1- to 12-week-old mice using the whole-cell or a single channel variation of the patch-clamp technique. 2. All sural cells from 2-week-old mice showed inwardly rectifying potassium (Kir+) currents in whole-cell recordings. Kir+ currents were virtually undetectable in sural cells from mice more than 6 weeks old, which also showed depolarization of the resting membrane potential. On the other hand, the magnitude of Kir+ currents increased in cervical sympathetic trunk (CST) cells in parallel with an increase of cell capacitance 1-6 weeks after birth. The density of Kir+ currents in CST cells increased 1-4 weeks after birth and then stayed constant for up to 12 weeks. 3. The unitary conductance of a single Kir+ channel in CST cells was 30 pS 2-12 weeks after birth; this was recorded in a cell-attached configuration with 154 mM K+ in the pipette. The steady-state open channel probability of single Kir+ channels in CST cells decreased with membrane hyperpolarization, but was not markedly changed 2-12 weeks after birth. 4. Conduction block of CST for 5 days induced by local application of tetrodotoxin (TTX) resulted in a significant decrease in both the magnitude and the density of Kir+ currents in whole-cell recordings in CST cells rostral to the sites of TTX block. Similar changes of Kir+ currents in whole-cell recordings were observed in cells in the inferior postganglionic branch of a superior cervical ganglion after 5 days of TTX block of CST. 5. These results suggest that neuronal activity regulates the expression of functional Kir+ channels in non-myelinating Schwann cells in adult nerves. The activity-dependent regulation of the expression of glial potassium channels could play an important role in the regulation of the potassium microenvironment around active axons to maintain impulse conduction in unmyelinated fibres.

Enhancement of N- and L-type calcium channel currents by protein kinase C in frog sympathetic neurons

The effect of protein kinase C (PKC) stimulation on Ca 2+ channels was studied in frog sympathetic neurons. 12,13-Phorbol dibutyrate (PDBu) consistently augmented Ca 2+ channel currents in whole-cell recordings. This enhancement was blocked by staurosporine and PKC(19–31), but not produced by 4α-phorbol 12,13-didecanoate, indicating that PDBu acts via PKC. Both N- and L-type currents, as isolated pharmacologically, were increased. PKC enhancement was independent of the extent of G protein activation, indicating that it was not caused by removal of tonic G protein inhibition. In unitary recordings PDBu produced dramatic increases in single N- and L-type channel activity by sharply decreasing closed time intervals between adjacent openings, but did not alter the unitary current size or mean open time. This up-modulation by PKC may constitute a positive feedback mechanism in the regulation of neuronal Ca 2+ channel activity.

Inhibition of N- and L-type calcium channels by muscarinic receptor activation in rat sympathetic neurons

Modulation of N- and L-type Ca2+ channels by oxotremorine-M (oxo-M) acting on muscarinic receptors and norepinephrine (NE) acting on α-adrenergic receptors was studied in superior cervical ganglion neurons. Oxo-M depresses dihydropyridine-augmented tail currents in whole-cell recordings, whereas NE does not. This modulation of L-type Ca2+ channels by oxo-M is abolished by adding 20 mM BAPTA to the pipette solution. Oxo-M, acting via a diffusible messenger, reduces the probability of opening of single N- and L-type channels recorded in cell-attached patches. We conclude that a diffusible messenger signaling pathway activated by oxo-M inhibits both N- and L-type Ca2+ channels, whereas a membrane-delimited pathway activated by oxo-M and NE inhibits only N-type Ca2+ channels.

Aniracetam reduces glutamate receptor desensitization and slows the decay of fast excitatory synaptic currents in the hippocampus.

Aniracetam is a nootropic drug that has been shown to selectively enhance quisqualate receptor-mediated responses in Xenopus oocytes injected with brain mRNA and in hippocampal pyramidal cells [Ito, I., Tanabe, S., Kohda, A. & Sugiyama, H. (1990) J. Physiol. (London) 424, 533-544]. We have used patch clamp recording techniques in hippocampal slices to elucidate the mechanism for this selective action. We find that aniracetam enhances glutamate-evoked currents in whole-cell recordings and, in outside-out patches, strongly reduces glutamate receptor desensitization. In addition, aniracetam selectively prolongs the time course and increases the peak amplitude of fast synaptic currents. These findings indicate that aniracetam slows the kinetics of fast synaptic transmission and are consistent with the proposal [Trussell, L. O. & Fischbach, G. D. (1989) Neuron 3, 209-218; Tang, C.-M., Dichter, M. & Morad, M. (1989) Science 243, 1474-1477] that receptor desensitization governs the strength of fast excitatory synaptic transmission in the brain.

The Ca2+-activated K+ channel and its functional roles in smooth muscle cells of guinea pig taenia coli.

Currents through single potassium channels were studied in cell-attached or inside-out patches from collagenase-dispersed smooth muscle cells of the guinea pig taenia coli. Under conditions mimicking the physiological state with [K+]i = 135 mM: [K+]o = 5.4 mM, three distinct types of K+ channel were identified with conductances around 0 mV of 147, 94, and 63 pS. The activities of the 94- and 63-pS channel were observed infrequently. The 147-pS channel was most abundant. It has a reversal potential of approximately -75 mV. It is sensitive to [Ca2+]i and to membrane potential. At -30 mV, the probability of a channel being open is at a minimum. At more positive voltages, the probability follows Boltzman distribution. A 10-fold change in [Ca2+]i causes a 25-mV negative shift of the voltage where half of the channels are open; an 11.3-mV change in membrane potential produces an e-fold increase in the probability of the channel being open when P is low. At voltages between -30 and -50 mV, the open probability increases in an anomalous manner because of a large decrease of the channel closed time without much change in the channel open time. This anomalous activity may play a regulatory role in maintaining the resting potential. The histograms of channel open and closed time fit well, respectively, with single and double exponential distributions. Upon step depolarizations by 100-ms pulses, the 147-pS channel opens with a brief delay. The delay shortens and both the number of open channels and the open time increase with increasing positivity of the potential. The averaged currents during the step depolarizations closely resemble the delayed rectifying outward K+ currents in whole-cell recordings.

Ionic channels in mouse astrocytes in culture

We observed Na, K, and Cl voltage-dependent currents in a patch-clamp study of mouse brain astrocytes. In whole-cell recordings, depolarizations activated inward currents that were identified as Na currents since they were blocked by TTX, although complete block required high concentrations (greater than 1 microM). The corresponding single-channel Na currents were observed in outside-out patches. The channels were opened by a depolarizing pulse applied from a holding potential identical to the resting potential (-70 to -80 mV). Therefore, they may be considered functional Na channels. After addition of veratridine and an alpha-scorpion toxin, the decay of Na currents in whole-cell recordings was slower than observed under control conditions. At the single-channel level, the channels appeared to open in bursts. Depolarization did not increase the duration of the bursts, but inside each burst, increased the time spent in the open state. The K currents observed in the whole-cell recording mode were separated into inactivating and noninactivating currents. The inactivating current resembled the A current in its kinetics, its insensitivity to tetraethylammonium, and its sensitivity to 4-aminopyridine. At the single-channel level, at least 3 classes of K channels were observed at steady depolarized potentials. They resembled the K channels found in chromaffin cells by Marty and Neher (1985). Large conductance channels (385 pS) activated around 0 mV were identified as Cl channels.