Divalent Cation-free Solution Research Articles

Linoleic acid permeabilizes gastric epithelial cells by increasing connexin 43 levels in the cell membrane via a GPR40- and Akt-dependent mechanism

Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC activity of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintains the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt.

Editorial: Single membrane channels formed by connexins or pannexins: focus on the nervous system

Editorial: Single membrane channels formed by connexins or pannexins: focus on the nervous system

Isoform-specific phosphorylation-dependent regulation of connexin hemichannels.

Connexins form gap junction channels made up of two connexons (hemichannels) from adjacent cells. Unopposed hemichannels may open toward the extracellular space upon stimulation by, e.g., removal of divalent cations from the extracellular solution and allow isoform-specific transmembrane flux of fluorescent dyes and physiologically relevant molecules, such as ATP and ions. Connexin (Cx)43 and Cx30 are the major astrocytic connexins. Protein kinase C (PKC) regulates Cx43 in its cell-cell gap junction configuration and may also act to keep Cx43 hemichannels closed. In contrast, the regulation of Cx30 hemichannels by PKC is unexplored. To determine phosphorylation-dependent regulation of Cx30 and Cx43 hemichannels, these were heterologously expressed in Xenopus laevis oocytes and opened with divalent cation-free solution. Inhibition of PKC activity did not affect hemichannel opening of either connexin. PKC activation had no effect on Cx43-mediated hemichannel activity, whereas both dye uptake and current through Cx30 hemichannels were reduced. We detected no PKC-induced connexin internalization from the plasma membrane, indicating that PKC reduced Cx30 hemichannel activity by channel closure. In an attempt to resolve the PKC phosphorylation site(s) on Cx30, alanine mutations of putative cytoplasmic PKC consensus sites were created to prevent phosphorylation (T5A, T8A, T102A, S222A, S225A, S239A, and S258A). These Cx30 mutants responded to PKC activation, suggesting that Cx30 hemichannels are not regulated by phosphorylation of a single site. In conclusion, Cx30, but not Cx43, hemichannels close upon PKC activation, illustrating that connexin hemichannels display not only isoform-specific permeability profiles but also isoform-specific regulation by PKC.

Enzyme-free passage of human pluripotent stem cells by controlling divalent cations.

Enzymes used for passaging human pluripotent stem cells (hPSCs) digest cell surface proteins, resulting in cell damage. Moreover, cell dissociation using divalent cation-free solutions causes apoptosis. Here we report that Mg2+ and Ca2+ control cell-fibronectin and cell-cell binding of hPSCs, respectively, under feeder- and serum-free culture conditions without enzyme. The hPSCs were detached from fibronectin-, vitronectin- or laminin-coated dishes in low concentrations of Mg2+ and remained as large colonies in high concentrations of Ca2+. Using enzyme-free solutions containing Ca2+ without Mg2+, we successfully passaged hPSCs as large cell clumps that showed less damage than cells passaged using a divalent cation-free solution or dispase. Under the same conditions, the undifferentiated and early-differentiated cells could also be harvested as a cell sheet without being split off. Our enzyme-free passage of hPSCs under a serum- and feeder-free culture condition reduces cell damage and facilitates easier and safer cultures of hPSCs.

Cation permeation through connexin 43 hemichannels is cooperative, competitive and saturable with parameters depending on the permeant species

Kinetics of permeation through connexin 43-EGFP hemichannels (Cx43-EGFP HCs) were evaluated in divalent cation-free solutions, which enhance HC open probability and thus, allow measurements during initial velocity. Three cations that become fluorescent upon binding to intracellular nucleic acids [ethidium (Etd), propidium (Prd) and 4′,6-diamidino-2-phenylindole (DAPI)] and Cx43-EGFP or Cx43 wild type HeLa cell transfectants (Cx43-EGFP- and Cx43-WT-HeLa cells, respectively) were used. Levels of Cx43-EGFP at the cell periphery and rate of dye uptake were directly related. The rate of uptake of each dye reached saturation consistent with a facilitated transport mechanism. Before saturation, the relation between rate of uptake and concentration of each dye was sigmoidal with Hill coefficients >1, indicating positive cooperativity of transport at low concentrations. The maximal rate of Etd uptake was not affected by the presence of DAPI and vice versa, but under each condition the apparent affinity constant of the main permeant molecule increased significantly consistent with competitive inhibition or competition for binding sites within the channel. Moreover, Cx43-EGFP and Cx43-WT HCs had similar permeability properties, indicating that EGFP bound to the C-terminal of Cx43 does not significantly alter the permeability of Cx43 HCs to positively charged molecules. Thus, competitive inhibition of permeation through hemichannels might contribute to cellular retention of essential molecules and/or uptake inhibition of toxic compounds.

Contribution of the S5-Pore-S6 Domain to the Gating Characteristics of the Cation Channels TRPM2 and TRPM8

The closely related cation channels TRPM2 and TRPM8 show completely different requirements for stimulation and are regulated by Ca(2+) in an opposite manner. TRPM8 is basically gated in a voltage-dependent process enhanced by cold temperatures and cooling compounds such as menthol and icilin. The putative S4 voltage sensor of TRPM8 is closely similar to that of TRPM2, which, however, is mostly devoid of voltage sensitivity. To gain insight into principal interactions of critical channel domains during the gating process, we created chimeras in which the entire S5-pore-S6 domains were reciprocally exchanged. The chimera M2-M8P (i.e. TRPM2 with the pore of TRPM8) responded to ADP-ribose and hydrogen peroxide and was regulated by extracellular and intracellular Ca(2+) as was wild-type TRPM2. Single-channel recordings revealed the characteristic pattern of TRPM2 with extremely long open times. Only at far-negative membrane potentials (-120 to -140 mV) did differences become apparent because currents were reduced by hyperpolarization in M2-M8P but not in TRPM2. The reciprocal chimera, M8-M2P, showed currents after stimulation with high concentrations of menthol and icilin, but these currents were only slightly larger than in controls. The transfer of the NUDT9 domain to the C terminus of TRPM8 produced a channel sensitive to cold, menthol, or icilin but insensitive to ADP-ribose or hydrogen peroxide. We conclude that the gating processes in TRPM2 and TRPM8 differ in their requirements for specific structures within the pore. Moreover, the regulation by extracellular and intracellular Ca(2+) and the single-channel properties in TRPM2 are not determined by the S5-pore-S6 region.

Expression of Orai genes and ICRAC activation in the human retinal pigment epithelium

The retinal pigment epithelium (RPE) fulfills a large variety of tasks that are important for visual function. Many of these tasks, such as phagocytosis, growth factor secretion, or transepithelial ion transport, are regulated by increases in intracellular Ca²(+) as second-messenger. Despite the multitude of Ca²(+)-dependently regulated functions, only few Ca²(+) channels have been described so far in the RPE to couple Ca²(+) conductance and Ca²(+) signaling. RT-PCR experiments with mRNA of freshly isolated RPE cells as well as from the RPE cell line ARPE-19 and measurements of the intracellular free Ca²(+) concentration were performed. The RT-PCR experiments revealed the expression of the I(CRAC) channel proteins Orai 1, 2, and 3 and their stimulators Stim-1 and Stim-2. The classic maneuver to stimulate capacitive Ca²(+) entry (depletion of Ca²(+) stores by 1 μM thapsigargin under extracellular Ca²(+)-free conditions and then re-adding extracellular Ca²(+)) led to an increase in intracellular free Ca²(+), which could be blocked by application of a high concentration of 2-APB (75 μM) either before or during induction of capacitive Ca²(+) entry. On the other hand, application of a low concentration of 2-APB (2 μM) led to enhancement of the Ca²(+) increase induced by capacitive Ca²(+) entry. Depletion of cytosolic Ca²(+) stores by administration of an extracellular divalent cation-free solution led to an increase in the whole-cell conductance. With these data we show a new Ca²(+) entry pathway linked to the Ca²(+)/inositolphosphate second-messenger system in RPE cells which help to further understand regulatory pathways of agonists. The expression of Orai channels enables the RPE cells to generate sustained or repetitive Ca²(+) signals as they are known to be induced by different stimuli like ATP, bFGF, and the stimulation with photoreceptor outer segments.

The Antibacterial Cell Division Inhibitor PC190723 Is an FtsZ Polymer-stabilizing Agent That Induces Filament Assembly and Condensation

Cell division protein FtsZ can form single-stranded filaments with a cooperative behavior by self-switching assembly. Subsequent condensation and bending of FtsZ filaments are important for the formation and constriction of the cytokinetic ring. PC190723 is an effective bactericidal cell division inhibitor that targets FtsZ in the pathogen Staphylococcus aureus and Bacillus subtilis and does not affect Escherichia coli cells, which apparently binds to a zone equivalent to the binding site of the antitumor drug taxol in tubulin (Haydon, D. J., Stokes, N. R., Ure, R., Galbraith, G., Bennett, J. M., Brown, D. R., Baker, P. J., Barynin, V. V., Rice, D. W., Sedelnikova, S. E., Heal, J. R., Sheridan, J. M., Aiwale, S. T., Chauhan, P. K., Srivastava, A., Taneja, A., Collins, I., Errington, J., and Czaplewski, L. G. (2008) Science 312, 1673-1675). We have found that the benzamide derivative PC190723 is an FtsZ polymer-stabilizing agent. PC190723 induced nucleated assembly of Bs-FtsZ into single-stranded coiled protofilaments and polymorphic condensates, including bundles, coils, and toroids, whose formation could be modulated with different solution conditions. Under conditions for reversible assembly of Bs-FtsZ, PC190723 binding reduced the GTPase activity and induced the formation of straight bundles and ribbons, which was also observed with Sa-FtsZ but not with nonsusceptible Ec-FtsZ. The fragment 2,6-difluoro-3-methoxybenzamide also induced Bs-FtsZ bundling. We propose that polymer stabilization by PC190723 suppresses in vivo FtsZ polymer dynamics and bacterial division. The biochemical action of PC190723 on FtsZ parallels that of the microtubule-stabilizing agent taxol on the eukaryotic structural homologue tubulin. Both taxol and PC190723 stabilize polymers against disassembly by preferential binding to each assembled protein. It is yet to be investigated whether both ligands target structurally related assembly switches.

Gap junction hemichannel-mediated release of glutathione from cultured rat astrocytes

Release of glutathione (GSH) from astrocytes is essential for the supply of neurons with the GSH precursor cysteine. In order to test whether gap junction hemichannels could contribute to GSH release from astrocytes, we incubated astrocyte-rich primary cultures from neonatal rat brain in the absence of divalent cations, a condition that is known to increase the opening probability of hemichannels. During incubation in divalent cation free incubation solution (DCFS) the cells remained viable and released about 50% of the initial cellular GSH within 15 min. This extracellular GSH accumulation in DCFS was lowered by the presence of Ca 2+ in a concentration dependent manner with a half-maximal inhibition at a Ca 2+ concentration of 107 ± 46 μM. Extracellular GSH accumulation in DCFS was also blocked by the divalent cations Mg 2+, Ba 2+ and Sr 2+ as well as by the known gap junction inhibitors carbenoxolone (CBX), flufenamic acid (FFA) and lanthanum chloride. In contrast, the P2X 7 receptor blocker brilliant blue G (BBG) did not affect GSH release in divalent cation free solution. This pharmacological profile strongly suggests that astrocytes are able to release GSH via open hemichannels. This release of GSH may have severe consequences for the antioxidative defense and for the GSH homeostasis in pathological brain.

Functional Hemichannels in Astrocytes: A Novel Mechanism of Glutamate Release

Little is known about the expression and possible functions of unopposed gap junction hemichannels in the brain. Emerging evidence suggests that gap junction hemichannels can act as stand-alone functional channels in astrocytes. With immunocytochemistry, dye uptake, and HPLC measurements, we show that astrocytes in vitro express functional hemichannels that can mediate robust efflux of glutamate and aspartate. Functional hemichannels were confirmed by passage of extracellular lucifer yellow (LY) into astrocytes in nominal divalent cation-free solution (DCFS) and the ability to block this passage with gap junction blocking agents. Glutamate/aspartate release (or LY loading) in DCFS was blocked by multivalent cations (Ca2+, Ba2+, Sr2+, Mg2+, and La3+) and by gap junction blocking agents (carbenoxolone, octanol, heptanol, flufenamic acid, and 18alpha-glycyrrhetinic acid) with affinities close to those reported for blockade of gap junction intercellular communication. Glutamate efflux via hemichannels was also accompanied by greatly reduced glutamate uptake. Glutamate release in DCFS, however, was not significantly mediated by reversal of the glutamate transporter: release did not saturate and was not blocked by glutamate transporter blockers. Control experiments in DCFS precluded glutamate release by volume-sensitive anion channels, P2X7 purinergic receptor pores, or general purinergic receptor activation. Blocking intracellular Ca2+ mobilization by BAPTA-AM or thapsigargin did not inhibit glutamate release in DCFS. Divalent cation removal also induced glutamate release from intact CNS white matter (acutely isolated optic nerve) that was blocked by carbenoxolone, suggesting the existence of functional hemichannels in situ. Our results indicated that astrocyte hemichannels could influence CNS levels of extracellular glutamate with implications for normal and pathological brain function.

Multiple regulation by external ATP of nifedipine‐insensitive, high voltage‐activated Ca2+ current in guinea‐pig mesenteric terminal arteriole

We investigated the receptor-mediated regulation of nifedipine-insensitive, high voltage-activated Ca(2+) currents in guinea-pig terminal mesenteric arterioles (I(mVDCC)) using the whole-cell clamp technique. Screening of various vasoactive substances revealed that ATP, histamine and substance P exert modulatory effects on I(mVDCC). The effects of ATP on I(mVDCC) after complete P2X receptor desensitization exhibited a complex concentration dependence. With 5 mM Ba(2+), ATP potentiated I(mVDCC) at low concentrations (approximately 1-100 microM), but inhibited it at higher concentrations (>100 microM). The potentiating effects of ATP were abolished by suramin (100 microM) and PPADS (10 microM) and by intracellular application of GDPbetaS (500 microM), whereas a substantial part of I(mVDCC) inhibition by milimolar concentrations of ATP remained unaffected; due probably to its divalent cation chelating actions. In divalent cation-free solution, I(mVDCC) was enlarged and underwent biphasic effects by ATPgammaS and ADP, while 2-methylthio ATP (2MeSATP) exerted only inhibition, and pyrimidines such as UTP and UDP were ineffective. ATP-induced I(mVDCC) potentiation was selectively inhibited by anti-Galpha(s) antibodies or protein kinase A (PKA) inhibitory peptides and mimicked by dibutyryl cAMP. In contrast, ATP-induced inhibition was selectively inhibited by Galpha(q/11) antibodies or protein kinase C (PKC) inhibitory peptides and mimicked by PDBu. Pretreatment with pertussis toxin was ineffective. The apparent efficacy for I(mVDCC) potentiation with PKC inhibitors was: ATPgammaS > ATP>/=ADP and for inhibition with PKA inhibitors was: 2MeSATP > ATPgammaS > ATP > ADP. Neither I(mVDCC) potentiation nor inhibition showed voltage dependence. These results suggest that I(mVDCC) is multi-phasically regulated by external ATP via P2Y(11)-resembling receptor/G(s)/PKA pathway, P2Y(1)-like receptor/G(q/11)/PKC pathway, and metal chelation.

Stable Activation of Single Ca2+ Release-activated Ca2+ Channels in Divalent Cation-free Solutions

The regulation of store-operated, calcium-selective channels in the plasma membrane of rat basophilic leukemia cells (RBL-2H3 m1), an immortalized mucosal mast cell line, was studied at the single-channel level with the patch clamp technique by removing divalent cations from both sides of the membrane. The activity of the single channels in excised patches could be modulated by Ca(2+), Mg(2+), and pH. The maximal activation of these channels by divalent cation-free conditions occurred independently of depletion of intracellular Ca(2+) stores, whether in excised patches or in whole cell mode. Yet, a number of points of evidence establish these single-channel openings as amplified store-operated channel events. Specifically, (i) the single channels are exquisitely sensitive to inhibition by intracellular Ca(2+), and (ii) both the store-operated current and the single-channel openings are completely blocked by the capacitative calcium entry blocker, 2-aminoethoxydiphenyl borane. In addition, in Jurkat T cells single-channel openings with lower open probability have been observed in the whole cell mode with intracellular Mg(2+) present (Kerschbaum, H. H., and Cahalan, M. D. (1999) Science 283, 836-839), and in RBL-2H3 m1 cells a current with similar properties is activated by store depletion.

Membrane currents in cultured human intestinal smooth muscle cells.

Using whole-cell patch-clamp recording techniques, we have examined voltage-gated ion currents in a cultured human intestinal smooth muscle cell line (HISM). Experiments were performed at room temperature on cells after passages 16 and 17. Two major components of the whole-cell current were a tetraethylammonium-sensitive (IC50 = 9 mM), iberiotoxin-resistant, delayed rectifier K+ current and a Na+ current inhibited by tetrodotoxin (IC50 A 100 nM). No measurable inward current via voltage-gated Ca2+ channels could be detected in these cells even with 10 mM Ca2+ or Ba2+ in the external solution. No current attributable to calcium-activated K+ channels was found and no cationic current in response to muscarinic receptor activation was present. In divalent cation-free external solution two additional currents were activated: an inwardly rectifying hyperpolarization-activated current, I(HA), and a depolarization-activated current, I(DA) x I(HA) and I(DA) could be carried by several monovalent cations; the sizes of currents in descending order were: K+ > Cs+ > Na+ for I(HA) and Na+ > K+ >> Cs+ for I(DA). I(HA) was activated and deactivated instantaneously and showed no inactivation whereas I(DA) was activated, inactivated and deactivated within tens of milliseconds. These currents were inhibited by external calcium with an IC50 of 0.3 microM for I(DA) and an IC50 of 20 microM for I(HA). Cyclopiazonic acid (CPA) induced an outward, but not an inward current. SK&F 96365, a blocker of store-operated Ca2+ channels, suppressed I(DA) with a half-maximal inhibitory concentration of 9 microM but was ineffective in inhibiting I(HA) at concentrations up to 100 microM. Gd3+ and La3+ strongly suppressed I(DA) at 1 and 10 microM, respectively and were less effective in blocking I(HA) (complete inhibition required a concentration of 100 microM for both). Carbachol at 10-100 microM evoked about a 3-fold increase in I(HA) amplitude and completely abolished I(DA). We conclude that I(HA) and I(DA) are Ca2+-blockable cationic currents with different ion selectivity profiles that are carried by different channels. I(DA) shows novel voltage-dependent properties for a cationic current.

Na+ currents through Ca2+ channels in human retinal glial (M�ller) cells

To detect the presence of voltage-gated Ca(2+) channels in the plasma membranes of freshly isolated Müller glial cells from the human retina and their modulation by GABA(B) receptor agonists. Whole cell voltage-clamp recordings were made to study Ca( 2+), Ba(2+), and Na(+) currents through voltage-gated Ca(2+) channels. The vast majority of the investigated cells displayed no resolvable currents through Ca(2+) channels when Ca(2+) ions (2 mM) were present in the extracellular solution. Small-amplitude inwardly directed currents ( approximately 0.6 pA/pF) were detected when Ba(2+) ions (20 mM) were used as charge carrier. However, when Na(+) ions were used as charge carrier in divalent cation-free external solution, currents of large amplitudes ( approximately 7.5 pA/pF) through voltage-gated Ca(2+) channels were detected. Human Müller cells displayed currents through both transient, low voltage-activated Ca(2+) channels and long-lasting, high voltage-activated channels. The Na(+) fluxes through low voltage-activated Ca( 2+) channels were inhibited in a voltage-independent manner in the presence of GABA(B) receptor agonists. Human Müller glial cells express different kinds of voltage-gated Ca(2+) channels in their plasma membranes that can be activated only under certain physiological or pathophysiological conditions. The record of Na(+) fluxes in divalent cation-free solutions may be a technique to detect the presence of "hidden" voltage-gated Ca(2+) channels in Müller glial cells.

Na+ currents through Ca2+ channels in human retinal glial (Müller) cells

Purpose. To detect the presence of voltage-gated Ca 2+ channels in the plasma membranes of freshly isolated Müller glial cells from the human retina and their modulation by GABA B receptor agonists. Methods. Whole cell voltage-clamp recordings were made to study Ca 2+, Ba 2+, and Na + currents through voltage-gated Ca 2+ channels. Results. The vast majority of the investigated cells displayed no resolvable currents through Ca 2+ channels when Ca 2+ ions (2 mM) were present in the extracellular solution. Small-amplitude inwardly directed currents (~0.6 pA/pF) were detected when Ba 2+ ions (20 mM) were used as charge carrier. However, when Na + ions were used as charge carrier in divalent cation-free external solution, currents of large amplitudes (~7.5 pA/pF) through voltage-gated Ca 2+ channels were detected. Human Müller cells displayed currents through both transient, low voltage-activated Ca 2+ channels and long-lasting, high voltage-activated channels. The Na + fluxes through low voltage-activated Ca 2+ channels were inhibited in a voltage-independent manner in the presence of GABA B receptor agonists. Conclusions. Human Müller glial cells express different kinds of voltage-gated Ca 2+ channels in their plasma membranes that can be activated only under certain physiological or pathophysiological conditions. The record of Na + fluxes in divalent cation-free solutions may be a technique to detect the presence of “hidden” voltage-gated Ca 2+ channels in Müller glial cells.

Plasmalemmal repair of severed neurites of PC12 cells requires Ca(2+) and synaptotagmin.

Ca(2+) and synaptotagmin (a Ca(2+)-binding protein that regulates axolemmal fusion of synaptic vesicles) play essential roles in the repair of axolemmal damage in invertebrate giant axons. We now report that neurites of a rat pheochromocytoma (PC12) cell line transected and maintained in a serum medium form a dye barrier (exclude an external hydrophilic fluorescent dye) and survive for 24-hr posttransection (based on morphology and retention of another hydrophilic dye internally loaded at 6-hr posttransection). Some (25%) transected neurites that form a dye barrier regrow. Most (83%) neurites transected in a saline solution containing divalent cations (PBS(++)) also exclude entry of an externally placed hydrophilic fluorescent dye at 15-min posttransection. In contrast, only 14 or 17% of neurites maintained in a divalent cation-free solution (PBS(=)) or in PBS(=) + Mg(2+), respectively, form a dye barrier. Neurites that do not form a dye barrier do not survive for 24 hr. When PC12 neurites are loaded with an antibody to squid synaptotagmin, most (81%) antibody-loaded neurites do not form a dye barrier, whereas most (>/=81%) neurites loaded with heat-inactivated antibody or preimmune IgG do form a barrier. These data show that: 1) transected neurites of PC12 cells have mechanism(s) for plasmalemmal repair (dye barrier formation and survival); 2) Ca(2+) is necessary for dye barrier formation, which occurs minutes after transection and is necessary for survival and regrowth; and 3) synaptotagmin is an essential mediator of barrier formation. The similarity in the requirements for plasmalemmal repair in this mammalian cell preparation with those reported previously for invertebrate axons suggests that mechanisms necessary for plasmalemmal repair have been conserved phylogenetically.

Comparison of the effects of divalent cations on the noradrenaline‐evoked cation current in rabbit portal vein smooth muscle cells

1. The facilitatory effects of external Ca2+, Sr2+ and Ba2+ (Ca2+o, Sr2+o and Ba2+o) on the noradrenaline-evoked non-selective cation current (Icat) were compared in rabbit portal vein smooth muscle cells using patch pipette techniques. 2. All divalent cations tested potentiated the amplitude of Icat and the potency sequence was Ca2+o > Sr2+o > Ba2+o. Ca2+o and Sr2+o increased the amplitude of Icat by about eight times whereas Ba2+o produced only a threefold facilitation. 3. The current-voltage relationship of Icat was not changed by Ca2+o, Sr2+o or Ba2+o. 4. From noise analysis the single channel conductance (gamma) was approximately 10 pS in divalent cation-free solution but was about 20 pS with Ca2+o, Sr2+o and Ba2+o. 5. From noise and voltage-jump experiments it was apparent that at least three kinetically resolvable channel states are associated with Icat in divalent cation-free solution. Ca2+o and Sr2+o produced marked changes in the characteristics of the power spectrum and relaxations of Icat in response to voltage steps, consistent with a shift in the equilibrium between the channel states, whereas Ba2+o produced minimal effects. 6. The data show that Ca2+o, Sr2+o and Ba2+o increase the amplitude of Icat, which results in part from an increase in the single channel conductance. In addition the results suggest that Ca2+o and Sr2+o alter the kinetic behaviour of the single channels whereas Ba2+o has little effect on the equilibrium between the channel states.

PVP-containing solutions for analysis of divalent cation-dependent NMDA responses in Xenopus oocytes

The electrophysiological analysis of Ca 2+-conducting ion channels in Xenopus oocytes is difficult due to secondary intracellular effects induced by Ca 2+. In the presence of polyvinylpyrrolidone (PVP) membrane currents can be recorded in nominally divalent cation-free solutions. The Ca 2+-permeable recombinant NMDA receptors of the NR1/NR2A subtype were used as assay system and the results show that PVP has no effect on NMDA receptor-induced currents. Ca 2+ and Ba 2+ depress NMDA-induced currents at submillimolar concentrations probably by interfering with the Na +/K + flux. This block is fully reversible as also observed for Mg 2+ but shows in contrast no pronounced voltage dependence. PVP-containing solutions may be useful for the analysis of divalent cation-dependent ion channels.

Fluorescence imaging of extracellular purinergic receptor sites and putative ecto-ATPase sites on isolated cochlear hair cells

Fluorescence imaging of extracellular adenosine-5'-triphosphate (ATP) binding sites on inner and outer hair cells isolated from the guinea pig organ of Corti was achieved using the fluorescent analog of ATP, 2'-(or-3')-O-(trinitrophenyl)adenosine-5'- triphosphate (TNP-ATP; 30-75 microM). This analog, which fluoresces on binding to these sites, was pressure applied by micropipette while hair cells were viewed by fluorescence microscopy. Fluorescence imaging revealed a widespread distribution of extracellular binding sites, including the stereocilia, cuticular plate, and the basolateral margins of the cells, but particularly in infracuticular and infranuclear regions. In support of extracellular binding, simultaneous electrophysiological recordings demonstrated that rapid washout of TNP-ATP-induced fluorescence was dependent upon cell integrity. Suramin, a nonselective P2 purinoceptor antagonist, coapplied with TNP-ATP, reduced the fluorescence observed on the stereocilia and apical surface of the cuticular plate only. This implies that binding sites on the apical surface of hair cells are P2 receptors, consistent with previous electrophysiological evidence for localization of P2 receptors to the apical surface of cochlear hair cells (Housley et al., 1992). Binding of TNP-ATP to P2 purinoceptors was confirmed by its antagonism of the inward current elicited by ATP (10 microM) in voltage-clamped hair cells. Fluorescence from the basolateral margin was significantly quenched when TNP-ATP was applied in divalent cation-free solution. Because divalent cations are required for ATPase activity, this finding provides evidence for the presence of ecto-ATPases on the basolateral membrane of hair cells. The divalent cation-free condition had no significant effect on the ATP-gated P2 purinoceptor conductance. We propose that there are two classes of ATP binding sites on cochlear hair cells: apically located P2 purinoceptors gating nonselective cation channels and basolaterally located ecto-ATPases that may be involved in purine turnover.

Necessity of divalent cations for recovery from carbachol‐induced nicotinic acetylcholine receptor inactivation at snake twitch fibre endplates

1. Previous studies demonstrated that elevation of the extracellular calcium concentration during a prolonged exposure to a high concentration of carbachol reverses the staurosporine-induced decrease in the extent of endplate resensitization in voltage-clamped snake twitch fibres. The present studies were designed to establish the site, specificity and potential mechanisms by which calcium could reverse the effects of staurosporine on acetylcholine receptor recovery. 2. Pretreatment of potassium-depolarized muscle preparations with 0.5 microM staurosporine, followed by a 10 min incubation with 540 microM carbachol, produced a significant decrease in the recovery of miniature endplate current (m.e.p.c.) amplitudes. Raising the extracellular calcium concentration from 1 mM to 10 mM during the agonist application reversed this inhibition. In addition, a brief (3 min) incubation in an elevated calcium (10 mM) solution in staurosporine-treated preparations previously exposed to 540 microM carbachol also reversed the reduction in m.e.p.c. amplitude. 3. Substitution of calcium with 10 mM strontium had no effect on the staurosporine-induced decrease in m.e.p.c. amplitude, whereas 10 mM magnesium partially substituted for calcium. Inclusion of (+)-tubocurarine (13 microM) in the 10 mM calcium solution to prevent the influx of calcium through agonist-activated channels did not prevent the reversal of the staurosporine-induced decrease in m.e.p.c. amplitudes. This suggested that the site of action of calcium on endplate resensitization was extracellular. 4. Analysis of acetylcholine (ACh)-induced single channel currents demonstrated that a population of small conductance channels seen only in the staurosporine-treated preparations following carbachol exposure, was still present in staurosporine-treated preparations incubated in 540 microM carbachol solution containing 10 mM calcium. Thus the effect of calcium on ACh receptor recovery was not due to the conversion of the small conductance channels to the normal, large conductance channels.5. Removal of calcium from the extracellular solution with the magnesium concentration unchanged,resulted in no change in the extent of m.e.p.c. amplitude recovery, nor did it alter the ability of staurosporine to inhibit recovery. Removal of both calcium and magnesium resulted in a significant decrease in the extent of recovery and staurosporine produced no additional decrease. This decrease in m.e.p.c. recovery in the divalent cation-free solution was not associated with a change in mean channel conductance as determined by noise analysis.6. Based on the results from these experiments, we suggest that, with prolonged exposure to agonist,some ACh receptors at the endplate become irreversibly inactivated. For full recovery of endplate sensitivity to occur, inactivated ACh receptors must be replaced. Part of the replacement ACh receptors appear to be recruited from a readily available but not previously activated pool and this process is dependent on extracellular calcium and/or magnesium. Thus, elevation of extracellular calcium may be able to reverse the apparent decrease in endplate sensitivity in staurosporine-treated fibres following carbachol exposure by increasing the number of functional receptors at the endplate.