bluuebunny/crossref_metadata_2025_split huggingface dataset:
The sulphonylurea receptor SUR1 regulates ATP‐sensitive mouse Kir6.2 K+ channels linked to the green fluorescent protein in human embryonic kidney cells (HEK 293)
Authors: John Scott A. , Monck Jonathan R. , Weiss James N. , Ribalet Bernard 
Published: September 2004

Using a chimeric protein comprising the green fluorescent protein (GFP) linked to the C‐terminus of the K+ channel protein mouse Kir6.2 (Kir6.2‐C‐GFP), the interactions between the sulphonylurea receptor SUR1 and Kir6.2 were investigated in transfected human embryonic kidney cells (HEK 293) by combined imaging and patch clamp techniques.
HEK 293 cells transfected with mouse Kir6.2‐C‐GFP and wild‐type Kir6.2 exhibited functional K+ channels independently of SUR1. These channels were inhibited by ATP (IC50= 150 μM), but were not responsive to stimulation by ADP or inhibition by sulphonylureas. Typically, 15 ± 7 active channels were found in an excised patch.
The distribution of Kir6.2‐C‐GFP protein was investigated by imaging of GFP fluorescence. There was a lamellar pattern of fluorescence labelling inside the cytoplasm (presumably associated with the endoplasmic reticulum and the Golgi apparatus) and intense punctate labelling near the cell membrane, but little fluorescence was associated with the plasma membrane.
In contrast, cells co‐transfected with Kir6.2‐C‐GFP and SUR1 exhibited intense uniform plasma membrane labelling, and the lamellar and punctate labelling seen without SUR1 was no longer prominent.
In cells co‐transfected with Kir6.2‐C‐GFP and SUR1, strong membrane labelling was associated with very high channel activity, with 484 ± 311 active channels per excised patch. These K+ channels were sensitive to inhibition by ATP (IC50= 17 μM), stimulated by ADP and inhibited by sulphonylureas.
We conclude that co‐expression of SUR1 and Kir6.2 generates channels with the properties of native KATP channels. In addition, SUR1 promotes uniform insertion of Kir6.2‐C‐GFP into the plasma membrane and a 35‐fold increase in channel activity, suggesting that SUR1 facilitates protein trafficking of Kir6.2 into the plasma membrane.
KATP channel formation by the sulphonylurea receptors SUR1 with Kir6.2 subunits in rat dorsal vagal neurons in situ
Authors: Karschin Andreas , Brockhaus Johannes , Ballanyi Klaus 
Published: September 2004

Functional and molecular properties of ATP‐sensitive K+ (KATP) channels were studied in dorsal vagal neurons (DVNs) of rat brainstem slices using patch‐clamp and single‐cell antisense RNA amplification‐polymerase chain reaction (PCR) techniques.
In the cell‐attached configuration, 1 mM cyanide resulted in block of spontaneous firing and concomitant opening of single channels with a mean single open time of 2‐3 ms and a burst duration of up to several hundred milliseconds. Inhibition of such single‐channel activity with 200 μM tolbutamide led to the reappearance of spontaneous discharge.
Whole‐cell recordings during anoxia revealed a hyperpolarization of the DVNs. Harvesting of cytoplasm, antisense RNA amplification and subsequent PCR showed coexpression for single DVNs of mRNA for the sulphonylurea receptor SUR1 isoform and for the inwardly rectifying K+ channel subunit Kir6.2, but not for the SUR2 or Kir6.1 isoforms of these channel/receptor subclasses.
Upon anoxia, a stable depolarization by less than 10 mV was observed in non‐excitable cells in the dorsal vagal nucleus. These cells, which expressed glial fibrillary acidic protein (GFAP), showed a high level of mRNA for Kir6.2, a weak signal for SUR1, whereas SUR2 or Kir6.1 were not detected.
The results suggest that functional KATP channels in DVNs are constituted by the formation of Kir6.2 subunits with SUR1 receptors.
The effect of overexpression of auxiliary Ca2+ channel subunits on native Ca2+ channel currents in undifferentiated mammalian NG108–15 cells
Authors: Wyatt Christopher N. , Page Karen M. , Berrow Nicholas S. , Brice Nicola L. , Dolphin Annette C. 
Published: September 2004

High voltage activated (HVA) Ca2+ channels are composed of a pore‐forming α1 subunit and the accessory β and α2‐δ subunits. However, the subunit composition of low voltage activated (LVA), or T‐type, Ca2+ channels has yet to be elucidated. We have examined whether native calcium channels in NG108–15 mouse neuroblastoma × rat glioma hybrid cells, which express predominantly LVA currents when undifferentiated, are modulated by overexpression of accessory calcium channel subunits.
Endogenous α1A, B, C, D and E, and low levels of β and α2‐δ subunit protein were demonstrated in undifferentiated NG108–15 cells.
The α2‐δ, β2a or β1b accessory subunits were overexpressed by transfection of the cDNAs into these cells, and the effect examined on the endogenous Ca2+ channel currents. Heterologous expression, particularly of α2‐δ but also of β2a subunits clearly affected the profile of these currents. Both subunits induced a sustained component in the currents evoked by depolarizing voltages above −30 mV, and α2‐δ additionally caused a depolarization in the voltage dependence of current activation, suggesting that it also affected the native T‐type currents. In contrast, β1b overexpression had no effect on the endogenous Ca2+ currents, despite immunocytochemical evidence for its expression in the transfected cells.
These results suggest that in NG108–15 cells, overexpression of the Ca2+ channel accessory subunits α2‐δ and β2a induce a sustained component of HVA current, and α2‐δ also influences the voltage dependence of activation of the LVA current. It is possible that native T‐type α1 subunits are not associated with β subunits.
Chronic hypoxia reduces adenosine A2A receptor‐mediated inhibition of calcium current in rat PC12 cells via downregulation of protein kinase A
Authors: Kobayashi Shuichi , Beitner‐Johnson Dana , Conforti Laura , Millhorn David E. 
Published: September 2004

Adenosine has been shown to decrease Ca2+ current (ICa) and attenuate the hypoxia‐induced enhancement of intracellular free Ca2+ ([Ca2+]i) in oxygen‐sensitive rat phaeochromocytoma (PC12) cells. These effects are mediated via the adenosine A2A receptor and protein kinase A (PKA). The current study was undertaken to determine the effects of adenosine on Ca2+ current and hypoxia‐induced change in [Ca2+]i during chronic hypoxia.
Whole cell patch‐clamp studies revealed that the effect of adenosine on ICa was significantly reduced when PC12 cells were exposed to hypoxia (10 % O2) for 24 and 48 h.
Ca2+ imaging studies using fura‐2 revealed that the anoxia‐induced increase in [Ca2+]i was significantly enhanced when PC12 cells were exposed to 10 % O2 for up to 48 h. In contrast, the inhibitory effects of adenosine on anoxia‐induced elevation of [Ca2+]i was significantly blunted in PC12 cells exposed to hypoxia for 48 h.
Northern blot analysis revealed that mRNA for the A2A receptor, which is the only adenosine receptor subtype expressed in PC12 cells, was significantly upregulated by hypoxia. Radioligand binding analysis with [3H]CGS21680, a selective A2A receptor ligand, showed that the number of adenosine A2A receptor binding sites was similarly increased during exposure to 10 % O2 for 48 h.
PKA enzyme activity was significantly inhibited when PC12 cells were exposed to 10 % O2 for 24 and 48 h. However, we found that hypoxia failed to induce change in adenosine‐ and forskolin‐stimulated adenylate cyclase enzyme activity. Chronic hypoxia also did not alter the immunoreactivity level of the G protein Gsα, an effector of the A2 signalling pathway.
Whole cell patch‐clamp analysis showed that the effect of 8‐bromo‐cAMP, an activator of PKA, on ICa was significantly attenuated during 48 h exposure to 10 % O2.
We conclude therefore that the reduced effect of adenosine on ICa and [Ca2+]i in PC12 cells exposed to chronic hypoxia is due to hypoxia‐induced downregulation of PKA. This mechanism may serve to reduce the negative feedback on ICa and [Ca2+]i by adenosine and therefore maintain enhanced membrane excitability of PC12 cells during long‐term hypoxia.
Synaptic interactions between smooth and spiny neurones in layer 4 of cat visual cortex in vitro
Authors: Tarczy‐Hornoch K. , Martin K. A. C. , Jack J. J. B. , Stratford K. J. 
Published: September 2004

Dual intracellular recording was used to examine the interactions between neighbouring spiny (excitatory) and smooth (inhibitory) neurones in layer 4 of cat visual cortex in vitro. Synaptic connections were found in seventeen excitatory‐inhibitory neurone pairs, along with one inhibitory‐inhibitory connection.
Fast excitatory inputs onto smooth neurones (basket cells) from spiny cells (spiny stellate or pyramidal cells) (n= 6) produce large excitatory postsynaptic potentials (EPSPs) of up to 4 mV mean amplitude, whereas basket cells evoke slower inhibitory postsynaptic potentials (IPSPs) in their postsynaptic targets (n= 17), of smaller amplitude (up to 1.6 mV at membrane potentials of ‐60 mV).
Both types of PSP appear to be multiquantal, and both may exhibit depression of up to 60 % during short trains of presynaptic spikes. This depression can involve presynaptic and/or postsynaptic factors.
One‐third (n= 5) of the spiny cell‐smooth cell pairs tested were reciprocally connected, and in the one pair for which the suprathreshold interactions were comprehensively investigated, the pattern of basket cell firing was strongly influenced by the activity in the connected excitatory neurone. The basket cell was only effective in inhibiting spiny cell firing when the excitatory neurone was weakly driven.
Involvement of tyrosine kinases, Ca2+ and PKC in activation of mitogen‐activated protein (MAP) kinase in human polymorphonuclear neutrophils
Authors: Zhang Hong , Garlichs Christoph D. , Mügge Andreas , Daniel Werner G. 
Published: September 2004

Activation of mitogen‐activated protein (MAP) kinase is an early response to a wide variety of stimuli and plays an important role in the regulation of cellular functions. In the present study we investigated the activation of MAP kinase in human polymorphonuclear neutrophils (PMNs).
Activity of MAP kinase and protein kinase C (PKC) was measured radiometrically from the rate of phosphorylation of specific peptide substrates. Protein phosphorylation was measured by immunoprecipitation and Western blot analysis.
N‐Formyl‐Met‐Leu‐Phe (fMLP), phorbol 12‐myristate, 13‐acetate (PMA) and the Ca2+‐ATPase inhibitors thapsigargin (Tg) and cyclopiazonic acid (CPA) increased MAP kinase activity significantly. The tyrosine kinase inhibitors erbstatin and herbimycin A partially inhibited the effects of fMLP and PMA, and completely abolished the effects of both Tg and CPA. The specific PKC inhibitor calphostin C suppressed activation of MAP kinase produced by fMLP and PMA, but had no effect on that produced by Tg and CPA. Tg and CPA were without effect on PKC activity.
Immunoprecipitation and Western blot analysis indicated that the 42 and 44 kDa tyrosine‐phosphorylated proteins found after stimulation of PMNs were both members of the MAP kinase family. Pretreatment of PMNs with staurosporine, EGTA or erbstatin significantly reduced the tyrosine phosphorylation of MAP kinase(s).
These results suggest that in human PMNs, MAP kinase can be stimulated in both a PKC‐dependent and a PKC‐independent manner. The Ca2+ signal leads to activation of tyrosine kinases, which contribute to the activation of MAP kinase. However, a PMA‐sensitive Ca2+‐independent pathway also exists. Mobilization of Ca2+ and activation of PKC synergistically induce maximal MAP kinase activation and tyrosine phosphorylation.
Facilitation of currents through rat Ca2+‐permeable AMPA receptor channels by activity‐dependent relief from polyamine block
Authors: Rozov Andrei , Zilberter Yuri , Wollmuth Lonnie P. , Burnashev Nail 
Published: September 2004

In outside‐out patches excised from human embryonic kidney (HEK) 293 cells expressing Ca2+‐permeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate receptor (AMPAR) channels, currents activated by 1 ms glutamate pulses at negative membrane potentials facilitated during and following a repetitive (2 to 100 Hz) agonist application. The degree of facilitation depended on subunit type, membrane potential and stimulation frequency being antagonized by a slow recovery from desensitization.
Activity‐dependent current facilitation occurred in Ca2+‐permeable but not in Ca2+‐impermeable AMPAR channels. Current facilitation, however, does not depend on Ca2+ flux. Rather it reflects a relief from the block of Ca2+‐permeable AMPARs by intracellular polyamines since facilitation occurred only in the presence of polyamines and since facilitated currents had a nearly linear current‐voltage relation (I‐V).
Relief from polyamine block was use dependent and occurred mainly in open channels. The relief mechanism was determined primarily by membrane potential rather than by current flow.
In closed channels the degree of polyamine block was independent of membrane potential. The voltage dependence of the rate of relief from the block in open channels rather than the voltage dependence of the block underlies the inwardly rectifying shape of the I‐V at negative potentials.
Currents through native Ca2+‐permeable AMPAR channels in outside‐out or nucleated patches from either hippocampal basket cells or a subtype of neocortical layer II nonpyramidal cells also showed facilitation.
It is concluded that a use‐dependent relief from polyamine block during consecutive AMPAR channel openings underlies current facilitation. This polyamine‐AMPAR interaction may represent a new activity‐dependent postsynaptic mechanism for control of synaptic signalling.
Ca2+ influx through voltage‐gated Ca2+ channels regulates 5‐HT3 receptor channel desensitization in rat glioma × mouse neuroblastoma hybrid NG108–15 cells
Authors: Jones Susan , Yakel Jerrel L. 
Published: September 2004

The kinetics of desensitization of the 5‐HT3 receptor (5‐HT3R)‐gated ion channel were investigated using whole‐cell and perforated‐patch recording techniques in NG108–15 cells.
Rapid application of 5‐HT (50 μM) elicited a 5‐HT3R‐mediated inward current response that desensitized completely in the continued presence of agonist. In the whole‐cell recording configuration (holding potential of −70 mV) while buffering internal calcium (Ca2+i) with 5 mM EGTA (0.5 mM added Ca2+; with an estimated free [Ca2+] of 30 nM), the rate of desensitization was initially rapid (with a half‐time of ≈230 ms), but dramatically slowed with time by 1120 ± 160 %.
This slowing in the rate of desensitization was reduced by stronger Ca2+ buffering (20 mM BAPTA, without added Ca2+), or by the bath application of cadmium (100 μM) to block voltage‐gated Ca2+ channels. The rate of desensitization was also dependent on membrane potential.
In perforated‐patch recordings, the rate of desensitization remained constant. However, a slowing in the desensitization rate could be induced by depolarizing cells immediately prior to the application of 5‐HT.
The depolarization‐induced slowing was blocked by incubating cells with BAPTA‐AM (a membrane‐permeant analogue of BAPTA) or by the bath application of cadmium.
These data suggest that Ca2+ influx through a cadmium‐sensitive voltage‐gated Ca2+ channel increases the cytoplasmic Ca2+ concentration ([Ca2+]i) and induces a dramatic slowing in the kinetics of desensitization of the 5‐HT3R channel. These data provide evidence for cross‐talk between voltage‐gated Ca2+ channels and 5‐HT3Rs in NG108–15 cells.
Contrasting Ca2+ channel subtypes at cell bodies and synaptic terminals of rat anterioventral cochlear bushy neurones
Authors: Doughty Joanne M. , Barnes‐Davies Margaret , Rusznák Zoltan , Harasztosi Csaba , Forsythe Ian D. 
Published: September 2004

Whole‐cell patch clamp recordings were made from bushy cells of the anterioventral cochlear nucleus (aVCN) and their synaptic terminals (calyx of Held) in the medial nucleus of the trapezoid body (MNTB).
Both high voltage‐activated (HVA) and low voltage‐activated (LVA) calcium currents were present in acutely dissociated aVCN neurones and in identified bushy neurones from a cochlear nucleus slice.
The transient LVA calcium current activated rapidly on depolarization (half‐activation, −59 mV) and inactivated during maintained depolarization (half‐inactivation, −89 mV). This T‐type current was observed in somatic recordings but was absent from presynaptic terminals.
On the basis of their pharmacological sensitivity, P/Q‐type Ca2+ channels accounted for only 6 % of the somatic HVA, while L‐, N‐ and R‐type Ca2+ channels each accounted for around one‐third of the somatic calcium current.
The divalent permeabilities of these native calcium channels were compared. The Ba2+/Ca2+ conductance ratios of the somatic HVA and LVA channels were 1.4 and 0.7, respectively. The conductance ratio of the presynaptic HVA current was 0.9, significantly lower that that of the somatic HVA current.
We conclude that LVA currents are expressed in the bushy cell body, but are not localized to the excitatory synaptic terminal. All of the HVA current subtypes are expressed in bushy cells, but there is a strong polarity to their localization; P‐type contribute little to somatic currents but predominate at the synaptic terminal; L‐, N‐ and R‐types dominate at the soma, but contribute negligibly to calcium currents in the terminal.
Near‐visible ultraviolet light induces a novel ubiquitous calcium‐permeable cation current in mammalian cell lines
Authors: Mendez Francisco , Penner Reinhold 
Published: September 2004

We studied the immediate and short‐term effects of UV light in the near‐visible range at the cellular and membrane level using the whole‐cell patch‐clamp technique in combination with digital fluorescence imaging.
Illumination with monochromatic UVA light (340‐380 nm) induced a sustained non‐saturable increase in membrane conductance dependent on wavelength and light intensity in several different mammalian cell types including RBL, mast, HEK, PC12 and 3T3 cells.
The current was non‐selective for cations and permeable to Ca2+, but was inhibited by trivalent cations and was not due to the activation of an endogenous ion channel. We termed this novel current ILiNC for light‐induced non‐selective cation current.
A similar current was evoked by chemical peroxidants such as hydrogen peroxide and tert‐butylhydroperoxide, but not by cytosolic oxidized glutathione.
The free‐radical scavengers tocopherol (vitamin E) and ascorbic acid (vitamin C) significantly reduced the UV light effect.
The generation of the current was membrane delimited since it could be induced by the same UVA treatment in cell‐free membrane patches showing a similar wavelength dependence.
These results suggest that ILiNC is activated by UVA light‐induced generation of free radicals acting through lipid or protein peroxidation, and may represent a ubiquitous mechanism by which Na+ and Ca2+ can enter cells after phototoxic or free radical‐induced membrane damage.
Calcineurin involvement in the regulation of high‐threshold Ca2+ channels in NG108–15 (rodent neuroblastoma × glioma hybrid) cells
Authors: Lukyanetz E. A. , Piper T. P. , Sihra T. S. 
Published: September 2004

We examined the relationship between calcineurin (protein phosphatase 2B (PP2B)) and voltage‐operated Ca2+ channels (VOCCs) in NG108–15 cells. PP2B expression in NG108–15 cells was altered by transfection with plasmid constructs containing a full length cDNA of human PP2Bβ3 in sense (CN‐15) and antisense (CN‐21) orientation.
Confocal immunocytochemical localization showed that in wild‐type cells, PP2B immunoreactivity is uniformly distributed in undifferentiated cells and located at the inner surface of soma membrane and neurites in differentiated cells.
To test the Ca2+ dependence of the VOCC, we used high‐frequency stimulation (HFS). The L‐ and N‐type VOCCs decreased by 37 and 52 %, respectively, whereas the T‐type current was only marginally sensitive to this procedure. FK‐506 (2 μM), a specific blocker of PP2B, reduced the inhibition of L‐ and N‐type VOCCs induced by HFS by 30 and 33 %, respectively.
In CN‐15‐transfected cells overexpressing PP2B, total high‐voltage‐activated (HVA) VOCCs were suppressed by about 60 % at a test potential of +20 mV. Intracellular addition of EGTA or FK‐506 into CN‐15‐transfected cells induced an up to 5‐fold increase of HVA VOCCs.
These findings indicate that PP2B activity does not influence the expression of HVA Ca2+ channels, but modulates their function by Ca2+‐dependent dephosphorylation. Thus HVA VOCCs, in a phosphorylated state under control conditions, are downregulated by PP2B upon stimulation, with the major effect being on N‐type VOCCs.
Inhibition of N‐ and P/Q‐type calcium channels by postsynaptic GABAB receptor activation in rat supraoptic neurones
Authors: Harayama Nobuya , Shibuya Izumi , Tanaka Keiko , Kabashima Narutoshi , Ueta Yoichi , Yamashita Hiroshi 
Published: September 2004

Voltage‐dependent Ca2+ currents of dissociated rat supraoptic nucleus (SON) neurones were measured using the whole‐cell configuration of the patch‐clamp technique to examine direct postsynaptic effects of GABAB receptor activation on SON magnocellular neurones.
The selective GABAB agonist baclofen reversibly inhibited voltage‐dependent Ca2+ currents elicited by voltage steps from a holding potential of −80 mV to depolarized potentials in a dose‐dependent manner. The ED50 of baclofen for inhibiting Ca2+ currents was 1.4 × 10−6 M. Baclofen did not inhibit low threshold Ca2+ currents elicited by voltage steps from −120 to −40 mV.
Inhibition of high threshold Ca2+ currents by baclofen was rapidly and completely reversed by the selective GABAB antagonists, CGP 35348 and CGP 55845A, when the antagonists were added at the molar ratio vs. baclofen of 10 : 1 and 0.01 : 1, respectively. It was also reversed by a prepulse to +150 mV lasting for 100 ms.
The inhibition of Ca2+ currents was abolished when the cells were pretreated with pertussis toxin for longer than 20 h or with N‐ethylmaleimide for 2 min. It was also abolished when GDPβS was included in the patch pipette. When GTPγS was included in the patch pipette, baclofen produced irreversible inhibition of Ca2+ currents and this inhibition was again reversed by the prepulse procedure.
The inhibition of N‐, P/Q‐, L‐ and R‐type Ca2+ channels by baclofen (10−5 M) was 24.1, 10.5, 3.1 and 3.6 %, respectively, of the total Ca2+ currents. Only the inhibition of N‐ and P/Q‐types was significant.
These results suggest that GABAB receptors exist in the postsynaptic sites of the SON magnocellular neurones and mediate selective inhibitory actions on voltage‐dependent Ca2+ channels of N‐ and P/Q‐types via pertussis toxin‐sensitive G proteins, and that such inhibitory mechanisms may play a role in the regulation of SON neurones by the GABA neurones.