Calcium channel current of vascular smooth muscle cells: extracellular protons modulate gating and single channel conductance

Modulation of L-type Ca2+ channel current by extracellular pH (pHo) was studied in vascular smooth muscle cells from bovine pial and porcine coronary arteries. Relative to pH 7.4, alkaline pH reversibly increased and acidic pH reduced ICa. The efficacy of pHo in modulating ICa was reduced when the concentration of the charge carrier was elevated ([Ca2+]o or [Ba2+]o varied between 2 and 110 mM). Analysis of whole cell and single Ca2+ channel currents suggested that more acidic pHo values shift the voltage-dependent gating (approximately 15 mV per pH- unit) and reduce the single Ca2+ channel conductance gCa due to screening of negative surface charges. pHo effects on gCa depended on the pipette [Ba2+] ([Ba2+]p), pK*, the pH providing 50% of saturating conductance, increased with [Ba2+]p according to pK* = 2.7-2.log ([Ba2+]p) suggesting that protons and Ba2+ ions complete for a binding site that modulates gCa. The above mechanisms are discussed in respect to their importance for Ca2+ influx and vasotonus.     

High external Ca2+ levels trigger membrane potential oscillations in mouse pancreatic beta-cells during blockade of K(ATP) channels.

Glucose depolarizes the pancreatic beta-cell and induces membrane potential oscillations, but the nature of the underlying oscillatory conductance remains unknown. We have now investigated the effects of the Ca2+ ionophore ionomycin and high external Ca2+ concentration ([Ca2+]o) on glucose-induced electrical activity and whole islet intracellular free Ca2+ concentration ([Ca2+]i), under conditions where the K(ATP) channel was blocked (100 microM tolbutamide or 4 microM glibenclamide). Raising [Ca2+]o to 10.2 or 12.8 mM, but not to 5.1 or 7.7 mM, turned continuous electrical activity into bursting activity. High [Ca2+]o (12.8 mM) regenerated a pattern of fast [Ca2+]i oscillations overshooting the levels recorded in tolbutamide. Ionomycin (10 microM) raised the [Ca2+]i and synergized with 5.1 mM Ca2+ to hyperpolarize the beta-cell membrane. The data indicate that a [Ca2+]i-sensitive and sulphonylurea-insensitive oscillatory conductance underlies the beta-cell bursting activity.     

Protons resolve dual effects of calcium on miniature end-plate potential frequency at frog neuromuscular junctions

Inhibition of transmitter release by protons (H+) was studied at the frog neuromuscular junction at various extracellular concentrations of calcium ([Ca++]o) and potassium ([K+]o) by recording miniature end-plate potential (MEPP) frequency with the intracellular microelectrode. H+ decreased K+ -stimulated MEPP frequency. A double logarithmic graph of MEPP frequency at 7.5 mM K+ vs. [H+]o yielded a straight line with negative slope. At 10 mM K+, there was a parallel shift to the right of the graph. According to the surface charge model, K+ acts solely to depolarize the prejunctional membrane in accordance with the Nernst equation. By decreasing the prejunctional negative surface charge, H+ decreases K+ -stimulated MEPP frequency by decreasing [Ca++]o at the Ca++ channel. An estimated pKa of 4.20 may represent an acidic site at the Ca++ channel associated with Ca++ influx. As [Ca++]o increased above 1 mM for pH 7.40 and 10 mM K+, MEPP frequency decreased, i.e., the inhibitory component of dual effects of Ca++ occurred. At pH 6.40, the inhibitory component was abolished, unmasking the stimulatory effect of Ca++ on MEPP frequency. Reversal of Ca++ action by H+ could not be explained by surface charge theory alone. A double logarithmic graph of MEPP frequency vs. [K+]o at 8.5-10.5 mM was linear with a slope of 4. There were parallel shifts to the right of this graph for changes in pH from 7.40 to 6.90 and in [Ca++]o from 1 to 2.5 mM. These results are explained on the hypothesis that K+ also acts at an acidic prejunctional site to increase Ca++ -dependent quantal transmitter release. This action of K+ was inhibited by H+ and raised Ca++. Based on kinetic theory, the estimated pKa of the acidic prejunctional K+ site was 6.31. Based on free energy calculations, its cation preference was H+ greater than K+ greater than Ca++.     

Extracellular K+ elevates outward currents through Kir2.1 channels by increasing single-channel conductance

Outward currents through inward rectifier K+ channels (Kir) play a pivotal role in determining resting membrane potential and in controlling excitability in many cell types. Thus, the regulation of outward Kir current (IK1) is important for appropriate physiological functions. It is known that outward IK1 increases with increasing extracellular K+ concentration ([K+]o), but the underlying mechanism is not fully understood. A "K+-activation of K+-channel" hypothesis and a "blocking-particle" model have been proposed to explain the [K+]o-dependence of outward IK1. Yet, these mechanisms have not been examined at the single-channel level. In the present study, we explored the mechanisms that determine the amplitudes of outward IK1 at constant driving forces [membrane potential (Vm) minus reversal potential (EK)]. We found that increases in [K+]o elevated the single-channel current to the same extent as macroscopic IK1 but did not affect the channel open probability at a constant driving force. In addition, spermine-binding kinetics remained unchanged when [K+]o ranged from 1 to 150 mM at a constant driving force. We suggest the regulation of K+ permeation by [K+]o as a new mechanism for the [K+]o-dependence of outward IK1.     

Simultaneous measurement of changes in the membrane potential and the intracellular Ca2+ concentration caused by somatostatin in human GH-producing pituitary tumor cells.

Changes in the membrane potential and the intracellular Ca2+ concentration ([Ca2+]i) caused by somatostatin (SRIF) were simultaneously measured in human GH-producing pituitary tumor cells, by means of the nystatin-perforated whole cell clamp technique and Fura-2 AM. An application of 10(-8) M SRIF hyperpolarized the membrane and arrested Ca(2+)-dependent spontaneous action potentials. [Ca2+]i concurrently decreased during membrane hyperpolarization. When the membrane potential was clamped below the threshold for voltage-gated Ca2+ channels, [Ca2+]i decreased and SRIF did not further reduce [Ca2+]i. In cells which did not show spontaneous action potentials, SRIF hyperpolarized the membrane but it affected [Ca2+]i little. From these results it was concluded that the reduction in [Ca2+]i caused by SRIF was ascribed to the decrease in Ca2+ influx through voltage-gated channels during membrane hyperpolarization. The effect of SRIF on the voltage-gated Ca2+ channel current was also examined under the perforated whole cell clamp. SRIF (10(-8) M) inhibited the Ca2+ channel current to 80.8 +/- 15.4% (n = 5) of the control. Because SRIF-induced inhibition of the voltage-gated Ca2+ channel current was not prominent, it was considered that membrane hyperpolarization is the major cause of the reduction in [Ca2+]i in human GH-producing cells.     

Determinants of Deoxyglucose Uptake in Cultured Astrocytes: The Role of the Sodium Pump

Glucose utilization in primary cell cultures of mouse cerebral astrocytes was studied by measuring uptake of tracer concentrations of [3H]2-deoxyglucose ([3H]2-DG). The resting rate of glucose utilization, estimated at an extracellular K+ concentration ([K+]o) of 5.4 mM, was high (7.5 nmol glucose/mg protein/min) and was similar in morphologically undifferentiated and "differentiated" (dibutyryl cyclic AMP-pretreated) cultures. Resting uptake of [3H]2-DG was depressed by ouabain, by reducing [K+]o, and by cooling. These observations suggest that resting glucose utilization in astrocytes was dependent on sodium pump activity. Sodium pump-dependent uptake in 2-3-week-old cultures was about 50% of total [3H]2-DG uptake but this fraction declined with culture age from 1 to 5 weeks. Uptake was not affected by changes in extracellular bicarbonate concentration ([HCO3-]o) in the range of 5-50 mM but was significantly reduced in bicarbonate-free solution. At high [HCO3-]o (50 mM) uptake was insensitive to pH (pH 6-8), whereas at low [HCO3-]o (less than 5 mM) uptake was markedly pH-dependent. Elevation of [K+]o from 2.3 mM to 14.2-20 mM (corresponding to extremes of the physiological range of [K+]o) resulted in a 35-43% increase in [3H]2-DG uptake that was not affected by culture age or by morphological differentiation. Our results indicate a high apparent rate of glucose utilization in astrocytes. This rate is dynamically responsive to changes in extracellular K+ concentration in the physiological range and is partially dependent on sodium pump activity.     

Calcium entry in squid axons during voltage clamp pulses

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca2+ concentration ([Ca2+]o) containing 150mM each of Na+, K+ and an inert cation such as Li+, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca2+ level, [Ca2+]i. The effect of membrane voltage on [Ca2+]i was found to depend on the concentration of internal Na+ ([Na+]i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca2+]i. For depolarizing pulses a relationship between [Ca2+]i gain and [Na+]i indicates that Ca2+ entry is sigmoid with a half maximal response at 22 mM Na+. This Ca2+ entry is a steep function of [Na+]i suggesting that 4 Na+ ions are required to promote the influx of 1 Ca2+. There was little change in Ca2+ entry with depolarizing pulses when [Ca2+]o is varied from 1 to 10mM, while at 50mM [Ca2+]o calcium entry clearly increases suggesting an alternate pathway from that of Na+/Ca2+ exchange. This entry of Ca2+ at high [Ca2+]o, however, was not blocked by Cs+o. The results obtained lend further support to the notion that Na+/Ca2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one.     

Mutations in the pore regions of the yeast K+ channel YKC1 affect gating by extracellular K+.

The product of the Saccharomyces cerevisiae K+-channel gene YKC1 includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarization and is regulated by extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Analysis of the wild-type current now shows that: (i) [K+]o suppresses a very slowly relaxing component, accelerating activation; (ii) [K+]o slows deactivation in a dose-dependent fashion; and (iii) Rb+, Cs+ and, to a lesser extent, Na+ substitute for K+ in its action on gating. We have identified single residues, L293 and A428, at equivalent positions within the two pore loops that affect the [K+]o sensitivity. Substitution of these residues gave channels with reduced sensitivity to [K+]o in macroscopic current kinetics and voltage dependence, but had only minor effects on selectivity among alkali cations in gating and on single-channel conductance. In some mutants, activation was slowed sufficiently to confer a sigmoidicity to current rise at low [K+]o. The results indicate that these residues are involved in [K+]o sensing. Their situation close to the permeation pathway points to an interaction between gating and permeation.     

Ca(2+)-activated anion channels and membrane depolarizations induced by blue light and cold in Arabidopsis seedlings.

The activation of an anion channel in the plasma membrane of Arabidopsis thaliana hypocotyls by blue light (BL) is believed to be a signal-transducing event leading to growth inhibition. Here we report that the open probability of this particular anion channel depends on cytoplasmic Ca2+ ([Ca2+]cyt) within the concentration range of 1 to 10 microM, raising the possibility that BL activates the anion channel by increasing [Ca2+]cyt. Arabidopsis seedlings cytoplasmically expressing aequorin were generated to test this possibility. Aequorin luminescence did not increase during or after BL, providing evidence that Ca2+ does not play a second-messenger role in the activation of anion channels. However, cold shock simultaneously triggered a large increase in [Ca2+]cyt and a 110-mV transient depolarization of the plasma membrane. A blocker of the anion channel, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, blocked 61% of the cold-induced depolarization without affecting the increase in [Ca2+]cyt. These data led us to propose that cold shock opens Ca2+ channels at the plasma membrane, allowing an inward, depolarizing Ca2+ current. The resulting large increase in [Ca2+]cyt activates the anion channel, which further depolarizes the membrane. Although an increase in [Ca2+]cyt may activate anion channels in response to cold, it appears that BL does so via a Ca(2+)-independent pathway.     

Calcium fluxes in T lymphocytes.

Mechanisms controlling Ca2+ fluxes through the plasma membrane of lymphocytes have been characterized in a human T-cell clone and in the Jurkat T-cell line. Due to endogenous buffers, about 1/125 of the Ca2+ ions that enter the cell are free. Ca2+ fluxes were estimated from the variations in intracellular Ca2+ concentration ([Ca2+]i) elicited by concentration jumps in extracellular Ca2+ ([Ca2+]o). Thapsigargin was used to inhibit Ca2+ uptake into intracellular stores and to stimulate Ca2+ entry. Ca2+ extrusion was strictly due to the activity of plasma membrane Ca(2+)-ATPases since there was no detectable Na+/Ca2+ exchange activity in these cells. The rate of Ca2+ extrusion was mainly influenced by [Ca2+]i and less by [Ca2+]o but was insensitive to cell depolarization. In depolarized cells, thapsigargin-induced Ca2+ influx was reduced to 10% of the value measured in normally polarized cells, suggesting that depolarization not only reduces the electrochemical gradient for Ca2+ ions, but also inhibits Ca2+ permeation. When Ca2+ ions enter the cell, they bind to a site inside the channel, with a Kd of 3.3 mM. Stimulation of clonal T-cells with low concentrations of either anti-CD3 antibodies or thapsigargin elicited Ca2+ oscillations. Both the amplitude and the frequency of CD3-induced Ca2+ oscillations were sensitive to [Ca2+]o. These oscillations were immediately interrupted when extracellular Ca2+ was removed. The properties of Ca2+ oscillations in T lymphocytes suggest that they are mainly due to variations of Ca2+ influx, modulated by variations in [Ca2+]i.     

Permeability properties and intracellular ion concentrations of epithelial cells in rat duodenum.

Effects of the K+ concentration in the bathing fluid ([K+]l) on the intracellular K+, Na+ and Cl- concentrations ([K+]i [Na+]i and [Cl-]i) as well as on the electrical potential were studied in rat duodenum. Changes in the mucosal K+ concentration ([K+]m), bringing the sum of Na+ and K+ concentrations to 147.2 mM constant, had little effect on the transmural potential difference (PDt), but did induce marked changes in the mucosal membrane potential (Vm). As [K+]m increased, Vm was depolarized gradually and obeyed the Nernst equation for a potassium electrode in the range of [K+]m greater than approx. 60 mM. Experiments of ion analyses were carried out on strips of duodenum to determine the effect of changing the external K+ concentrations on [K+] i, [Na+]i and [Cl-]i. An increase in [K+]o resulted in increases in [K+]i and [Cl-]i and a decrease in [Na+]i, [K+]i approaching its maximum at [K+]o greater than 70 mM. Such changes in [K+]i and [Na+]i seem to correlate quantitatively with the changes in [K+]o and [Na+]o. The values of the ratio of permeability coefficients, Pna+/PK+ were estimated using the Vm values and intracellular ion concentrations measured in these experiments. The results suggested that there appeared a rather abrupt increase in the PNa+/PK+ ratio from 0 to approx. 0.1, as [K+]m decreased.     

Inhibition of endothelium-dependent vasorelaxation by extracellular K(+): a novel controlling signal for vascular contractility

The effects of extracellular K+ on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) were examined in mouse aorta, mouse aorta endothelial cells (MAEC), and human umbilical vein endothelial cells (HUVEC). In mouse aortic rings precontracted with prostaglandin F2alpha or norepinephrine, an increase in extracellular K+ concentration ([K+]o) from 6 to 12 mM inhibited EDR concentration dependently. In endothelial cells, an increase in [K+]o inhibited the agonist-induced [Ca2+]i increase concentration dependently. Similar to K+, Cs+ also inhibited EDR and the increase in [Ca2+]i concentration dependently. In current-clamped HUVEC, increasing [K+]o from 6 to 12 mM depolarized membrane potential from -32.8 +/- 2.7 to -8.6 +/- 4.9 mV (n = 8). In voltage-clamped HUVEC, depolarizing the holding potential from -50 to -25 mV decreased [Ca2+]i significantly from 0.95 +/- 0.03 to 0.88 +/- 0.03 microM (n = 11, P < 0.01) and further decreased [Ca2+]i to 0.47 +/- 0.04 microM by depolarizing the holding potential from -25 to 0 mV (n = 11, P < 0.001). Tetraethylammonium (1 mM) inhibited EDR and the ATP-induced [Ca2+]i increase in voltage-clamped MAEC. The intermediate-conductance Ca2+-activated K+ channel openers 1-ethyl-2-benzimidazolinone, chlorozoxazone, and zoxazolamine reversed the K+-induced inhibition of EDR and increase in [Ca2+]i. The K+-induced inhibition of EDR and increase in [Ca2+]i was abolished by the Na+-K+ pump inhibitor ouabain (10 microM). These results indicate that an increase of [K+]o in the physiological range (6-12 mM) inhibits [Ca2+]i increase in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K+ efflux, and activating the Na+-K+ pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone.     

A comparative study of the effects of tetrodotoxin and the removal of external Na+ on the resting potential: Evidence of separate pathways for the resting and excitable Na currents in squid axon

To investigate whether the Na permeability of the resting membrane is determined predominantly by the excitable Na channel, we examined the effects of tetrodotoxin (TTX) and the complete removal of external Na+ on the resting potential. In the intact squid axon bathed in K-free artificial seawater, both TTX and the removal of Na+ produced small hyperpolarizations. The effect of Na removal, however, was larger than that of TTX. In the perfused squid axon, the hyperpolarization produced by the removal of external Na+ was greatly enhanced when the internal K concentration ([K+]i) was reduced. The effect of TTX, on the other hand, was not sensitive to the [K+]i or to the membrane potential. For [K+]i = 50 mM and [K+]o = 0, the average hyperpolarization produced by TTX was 1.2 mV, while the hyperpolarization produced by Na removal was approximately 21 mV. The difference between these two effects suggests that the majority of the resting Na current passes through pathways other than the excitable Na channel.     

P2X2 receptors are essential for [Ca2+]i increases in response to ATP in cultured rat myenteric neurons

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP > or = adenosine 5'-O-3-triphosphate > or = CTP > or = 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism.     

Extracellular potassium rapidly inhibits axonal transport of particles in cultured mouse dorsal root ganglion neurites

Changes in extracellular potassium concentration ([K+]o) modulate a variety of neuronal functions. However, whether axonal transport, which conveys materials to the appropriate destination for morphogenesis and other neuronal functions, depends on the extracellular K+ environment remains unclear. We therefore examined the effects of changes in [K+]o on axonal transport of particles visualized by video-enhanced microscopy in cultured mouse dorsal root gan-glion neurites. Increases in [K+]o (delta[K+]o > or = 2.5 mM) from control concentration (5 mM) inhibited both anterograde and retrograde axonal transport within a few minutes in a concentration-dependent manner. Conversely, removal of extracellular K+ induced the rapid facilitation of transport in both directions. These inhibitory and facilitatory responses were completely blocked by the K+ channel blocker tetraethylammonium (TEA), suggesting that the effect of changes in [K+]o involves the TEA-sensitive K+ channels. Increases in [K+]o provoked membrane depolarization in the absence and presence of TEA. Another depolarizing agent, veratridine, did not produce an effect on axonal transport. These results suggest that the extracellular K+-mediated inhibition of axonal transport does not depend on membrane depolarization. The inhibitory effect of increasing [K+]o on axonal transport was retained in calcium (Ca2+)-free extracellular medium, indicating that the inhibitory effect of extracellular K+ does not result from Ca2+ influx through voltage-dependent Ca2+ channels. In chloride (CI-)-free medium, increasing [K+]o failed to inhibit axonal transport, implying that the extracellular K+-mediated inhibition of axonal transport may be due to an increase in intracellular Cl- concentration associated with increases in the net inward movement of K+ and CI- across the membrane. Our results suggest that the extracellular K+ environment is involved in the rapid modulation of axonal transport of particles in dorsal root ganglion neurites.     

Mutations in the yeast two pore K+ channel YKC1 identify functional differences between the pore domains

The K+ channel of Saccharomyces cerevisiae encoded by the YKC1 gene includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarisation and is regulated by the extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Our previous work showed that substitutions of equivalent residues L293 and A428 within the pore-loops had qualitatively similar effects on both the [K+]o-sensitivity of channel gating and its voltage-dependence. Here, we report that mutations of equivalent residues N275 and N410, N-terminal from the K+ channel signature sequences of the two pores, have very different actions on channel gating and, in this case, are without effect on its voltage-sensitivity. The mutation N410D slowed current activation in a [K+]o-dependent manner and it accelerated deactivation, but without significant effect on the apparent affinity for K+. The N275D mutant, by contrast, had little effect on the [K+]o-sensitivity for activation and it greatly altered the. [K+]o-dependence of current deactivation. Neither mutant affected the voltage-dependence of the steady-state current nor the ability for other alkali cations to substitute for K+ in regulating gating. The double mutant N410D-N275D showed characteristics of N410D in the [K+]o-sensitivity of current activation and of N275D in the [K+]o-sensitivity of deactivation, suggesting that little interaction occurs between pore domains with mutations at these sites. The results indicate that the two pore domains are not functionally equivalent and they suggest that the regulation of gating by external K+ is mediated by K+ binding at two physically distinct sites with different actions.     

Intra and extracellular surface charges near Ca2+ channels in neurons and neuroblastoma cells.

The properties of low (LVA) and high (HVA) voltage-activated calcium currents were investigated in rat sensory neurons and a murine neuroblastoma cell line exposed to various concentrations of intra- or extracellular monovalent ([c+]i/o) and trivalent ([c3+]i/o) cations. In neurons, when [c+]i was changed from 150 to 20 mM, positive shifts of 18-28 mV were observed in activation curves of both LVA and HVA currents, as well as in LVA inactivation curves. Extracellularly, in divalent-free solutions, [c+]o of 20-50 mM produced medium (12-22 mV) negative shifts of the LVA channel properties. These data were used to estimate, by a "screening" model, a negative surface charge density around neuron's calcium channels of 1/1,000 and 1/1,325 eA-2 at the outside or inside face, respectively. In the presence of physiological concentrations of divalent cations, [c+]o of 20-60 mM caused smaller (4-11 mV) negative shifts of the activation and inactivation curves, which can be explained by assuming a partial neutralization of negative charges by divalent cations. By applying the above procedure to LVA channels of neuroblastoma cells, the ratio of extra- to intracellular surface charge density turned out to be more than tenfold higher than in neurons. Effects produced by [c3+]i/o were not in agreement with expectations based on screening or binding models.     

Temperature-dependence and conformational basis of inositol 1,4,5-trisphosphate receptor regulated by Ca2+

The inositol 1,4,5-trisphosphate (InsP3) receptor was purified from bovine cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (1:1) successfully.No effect of Ca2+ concentration on [3H]-InsP3 binding to unreconstituted InsP3 receptor could be observed either at 4℃ or at 25℃,whereas the effect of [Ca2+] on reconstituted InsP3 receptor depended on the temperature.The Ca2+ concentration outside the proteolipsome ([Ca2+]o) had no detectable effect on InsP3 binding to InsP3 receptor at 4℃.In contrast,with increase of [Ca2+]o from 0 to 100 nmol/L at 25℃,the InsP3 binding activity increased gradually.Then the InsP3 binding activity was decreased drastically at higher [Ca2+]o and inhibited entirely at 50 mol/L [Ca2+]o.Conformational studies on intrinsic fluorescence of the reconstituted InsP3 receptor and its quenching by KI and HB indicated that the global conformation of reconstituted InsP3 receptor could not be affected by [Ca2+]o at 4℃.While at 25℃,the effects of 10 m mol/L [Ca2+]o on global,membrane and cytoplasmic conformation of the reconstituted InsP3 receptor were different significantly from that of 100 nmol/L [Ca2+]o.     

Calcium-activated potassium channels in human platelets

The cationic fluorescent probe, DiSC3(5) was used to measure the membrane potential in human platelets. Hyperpolarization was induced by the addition of Ca2+ to the medium and also by the addition of the Ca2+ ionophore, A23187. In the absence of extracellular Ca2+ ([Ca2+]o) there was no response to A23187. The threshold concentration for [Ca2+]o was 20 microM and for A23187 was 12 nM. The increase polarity induced by [Ca2+]o was not affected by various K+ channel blockers. However, the effect of A23187 was inhibited by quinine and charybdotoxin, while apamin, tetraethylammonium, and the calmodulin inhibitors trifluoperazine and compound R24571 were ineffective. The resting membrane potential was -66 +/- 0.9 mV and was decreased by quinine. There are three conclusions from this study: (i) Ca2+-activated K+ channels exist in human platelets; (ii) they are the type that are apamin insensitive, charybdotoxin sensitive; and (iii) they may contribute to the resting membrane potential.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.