Sodium channel gating in clonal pituitary cells. The inactivation step is not voltage dependent

We have determined the time course of Na channel inactivation in clonal pituitary (GH3) cells by comparing records before and after the enzymatic removal of inactivation. The cells were subjected to whole-cell patch clamp, with papain included in the internal medium. Inactivation was slowly removed over the course of 10 min, making it possible to obtain control records before the enzyme acted. Papain caused a large (4-100x) increase in current magnitude for small depolarizations (near -40 mV), and a much smaller increase for large ones (approximately 1.5x at +40 mV). For technical reasons it was sometimes convenient to study outward INa recorded with no Na+ outside. The instantaneous I-V (IIV) curve in this condition was nonlinear before papain, and more nearly linear afterwards. The gNa-V curve after papain, obtained by dividing the INa-V curve by the IIV curve, was left-shifted by at least 20 mV and steepened. A spontaneous 5-10 mV left shift occurred in the absence of papain. The rate of the inactivation step was found to vary only slightly from -100 mV to +60 mV, based on the following evidence. (a) Before papain, inactivation rate saturated with voltage and was constant from +20 to +60 mV. (b) We activated the channels with a brief pulse, and studied the time course of the current on changing the voltage to a second, usually more negative level (Na+ present internally and externally). The time course of inactivation at each voltage was obtained by comparing control traces with those after inactivation was removed. When the 5-10-mV spontaneous shift was taken into account, inactivation rate changed by less than 10% from -100 to +60 mV. The data are considered in terms of existing models of the Na channel.     

State-dependent inactivation of the alpha1G T-type calcium channel.

We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the alpha1G channel, with symmetrical Na(+)(i) and Na(+)(o) and 2 mM Ca(2+)(o). After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential -100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from -120 to -70 mV (e-fold for 31 mV; tau = 2.5 ms at -100 mV), but tau = 12-17 ms from-40 to +60 mV. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100-300 ms, inactivation was strong but incomplete (approximately 98%). Inactivation was also produced by long, weak depolarizations (tau = 220 ms at -80 mV; V(1/2) = -82 mV), which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast (tau = 85 ms at -100 mV), but weakly voltage dependent. Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at -100 mV during recovery from inactivation, consistent with 相似文献   

Activation of the calcium release channel (ryanodine receptor) by heparin and other polyanions is calcium dependent.

Heparin has been used as a potent competitive inhibitor of inositol 1,4,5-trisphosphate (IP3)-binding to IP3 receptors and to block IP3-gated calcium channels in bilayer experiments. In contrast to the effect on the IP3-gated channel, heparin (0.1-1 micrograms/ml) opened the Ca release channel (ryanodine receptor). Other polyanions such as pentosan polysulfate and polyvinyl sulfate also activated the Ca release channel. The effect of polyanions on the Ca release channel was Ca dependent. Polyanion addition activated the Ca release channel when free Ca was > 80 nM, but was ineffective when free Ca was < 20 nM. The level of channel activation could be altered by manipulating the free Ca concentration. These results suggest that the polyanions act by increasing the local concentration of Ca near regulatory sites on the channel complex. As most cells have both types of intracellular channels, the opposite effects of the polyanions on the two channel types suggests that addition of polyanions to intact cells may produce multiple effects.     

Islet hormone pulse intervals are dependent upon sampling frequency.

Pulsatile insulin secretion has been reported from a variety of in vivo and in vitro systems. While it is agreed that insulin pulses exist, there is little agreement concerning the basal frequency or interpulse interval either within the same species in vivo, as both long and short term pulses have been reported, or between in vivo and in vitro preparations. We propose that the frequency of sampling may have profound effects upon the calculated pulse interval. Three systems were used to test this hypothesis: 1) artificial test data were designed to produce regular pulses with an exact 11 min period, 2) perfusate insulin concentration from isolated canine pancreata sampled at 1 min intervals and 3) peripheral blood insulin concentrations from human volunteers sampled every 2 and 5 min. Pulse parameters were determined at 1, 2, 5, 20, 15, 30 and 60 min sampling intervals for each data set by the use of the computer algorithms Pulsar and Cycle Detector. The results indicate that for insulin secretory pulses, sampling frequencies longer than 2 min may result in the production of spurious pulse trains with multiple longer term pulse periods. It is concluded that islet hormone secretory pulse period calculations are dependent upon the sampling frequency.     

N-type calcium channel inactivation probed by gating-current analysis

N-type calcium channels inactivate most rapidly in response to moderate, not extreme depolarization. This behavior reflects an inactivation rate that bears a U-shaped dependence on voltage. Despite this apparent similarity to calcium-dependent inactivation, N-type channel inactivation is insensitive to the identity of divalent charge carrier and, in some reports, to the level of internal buffering of divalent cations. Hence, the inactivation of N-type channels fits poorly with the "classic" profile for either voltage-dependent or calcium-dependent inactivation. To investigate this unusual inactivation behavior, we expressed recombinant N-type calcium channels in mammalian HEK 293 cells, permitting in-depth correlation of ionic current inactivation with potential alterations of gating current properties. Such correlative measurements have been particularly useful in distinguishing among various inactivation mechanisms in other voltage-gated channels. Our main results are the following: 1) The degree of gating charge immobilization was unchanged by the block of ionic current and precisely matched by the extent of ionic current inactivation. These results argue for a purely voltage-dependent mechanism of inactivation. 2) The inactivation rate was fastest at a voltage where only approximately (1)/(3) of the total gating charge had moved. This unusual experimental finding implies that inactivation occurs most rapidly from intermediate closed conformations along the activation pathway, as we demonstrate with novel analytic arguments applied to coupled-inactivation schemes. These results provide strong, complementary support for a "preferential closed-state" inactivation mechanism, recently proposed on the basis of ionic current measurements of recombinant N-type channels (Patil et al., . Neuron. 20:1027-1038).     

Lung protection against paraquat is calcium dependent.

Isolated, perfused, and ventilated rat lungs were challenged by paraquat (0.01 M) in the presence of 2.5 mM Ca2+, 2.5 mM Ca2+ with trifluoperazine (100 microM), 0.025 mM Ca2+, or 0.025 mM Ca2+ with sodium metavanadate (10 microM) to establish the effect of varying calcium concentration or calcium-dependent enzyme activities on injury induced by paraquat. Segmental vascular resistances, microvascular permeability (as assessed by the capillary filtration coefficient), lung tissue oxidized glutathione, and lung paraquat accumulation were measured. Exposure to paraquat for 2.5 h did not increase microvascular permeability or pulmonary vascular resistance in the presence of either normal extracellular calcium or low extracellular calcium and sodium metavanadate. Lungs exposed to paraquat were injured (as assessed by increased filtration coefficient) only in the presence of low extracellular calcium or after trifluoperazine was added. This injury was associated with decreased levels of oxidized glutathione and increased paraquat accumulation, suggesting that calcium's protective effect was both by inhibition of paraquat accumulation and maintenance of NADPH. Pulmonary vascular resistance was not increased with paraquat challenge.     

K+ channel gating: C-type inactivation is enhanced by calcium or lanthanum outside

Many voltage-gated K⁺ channels exhibit C-type inactivation. This typically slow process has been hypothesized to result from dilation of the outer-most ring of the carbonyls in the selectivity filter, destroying this ring’s ability to bind K⁺ with high affinity. We report here strong enhancement of C-type inactivation upon extracellular addition of 10–40 mM Ca²⁺ or 5–50 µM La³⁺. These multivalent cations mildly increase the rate of C-type inactivation during depolarization and markedly promote inactivation and/or suppress recovery when membrane voltage (V_m) is at resting levels (−80 to −100 mV). At −80 mV with 40 mM Ca²⁺ and 0 mM K⁺ externally, ShBΔN channels with the mutation T449A inactivate almost completely within 2 min or less with no pulsing. This behavior is observed only in those mutants that show C-type inactivation on depolarization and is distinct from the effects of Ca²⁺ and La³⁺ on activation (opening and closing of the V_m-controlled gate), i.e., slower activation of K⁺ channels and a positive shift of the mid-voltage of activation. The Ca²⁺/La³⁺ effects on C-type inactivation are antagonized by extracellular K⁺ in the low millimolar range. This, together with the known ability of Ca²⁺ and La³⁺ to block inward current through K⁺ channels at negative voltage, strongly suggests that Ca²⁺/La³⁺ acts at the outer mouth of the selectivity filter. We propose that at −80 mV, Ca²⁺ or La³⁺ ions compete effectively with K⁺ at the channel’s outer mouth and prevent K⁺ from stabilizing the filter’s outer carbonyl ring.     

Model of calcium channel inactivation: a qualitative analysis

A simple model of calcium channel inactivation has been developed, based on the accumulation of calcium ions at the inner mouth of the channel and on their binding to a receptor which inactivates the channel. A qualitative analysis has shown that upon an appropriate choice of parameters corresponding to the cell structure and to kinetic properties of its components, the calcium dependent inactivation and that assumed to be voltage dependent can both be emulated. The model suggests that the supposed variety of calcium channels might be explained by quantitative differences in nonlinear interactions of the channels with other cell components.     

Vanadate-activated calcium influx in A431 cells is dependent on the plasma membrane potential

Vanadate can activate the uptake of Ca in A431 epidermal carcinoma cells by two- to fivefold with no detectable lag period. Preincubation with epidermal growth factor (EGF) to down-regulate the EGF receptor prevents subsequent stimulation by EGF but not that by vanadate. Ca uptake is sodium-independent and is not activated by depolarization in high KCl. On the contrary, vanadate-stimulated uptake is completely inhibited by decreasing the plasma membrane potential from about -65 to -30 mV. These results demonstrate that the EGF receptor is not itself functioning as a Ca channel, that vanadate is not acting at the level of EGF receptor, and that the Ca transport system exhibits an unusual potential sensitivity in that it is inhibited by depolarization of the plasma membrane.     

Retrieval of context-associated memory is dependent on the Ca(v)3.2 T-type calcium channel

Among all voltage-gated calcium channels, the T-type Ca²⁺ channels encoded by the Ca_v3.2 genes are highly expressed in the hippocampus, which is associated with contextual, temporal and spatial learning and memory. However, the specific involvement of the Ca_v3.2 T-type Ca²⁺ channel in these hippocampus-dependent types of learning and memory remains unclear. To investigate the functional role of this channel in learning and memory, we subjected Ca_v3.2 homozygous and heterozygous knockout mice and their wild-type littermates to hippocampus-dependent behavioral tasks, including trace fear conditioning, the Morris water-maze and passive avoidance. The Ca_v3.2 ^−/− mice performed normally in the Morris water-maze and auditory trace fear conditioning tasks but were impaired in the context-cued trace fear conditioning, step-down and step-through passive avoidance tasks. Furthermore, long-term potentiation (LTP) could be induced for 180 minutes in hippocampal slices of WTs and Ca_v3.2 ^+/− mice, whereas LTP persisted for only 120 minutes in Ca_v3.2 ^−/− mice. To determine whether the hippocampal formation is responsible for the impaired behavioral phenotypes, we next performed experiments to knock down local function of the Ca_v3.2 T-type Ca²⁺ channel in the hippocampus. Wild-type mice infused with mibefradil, a T-type channel blocker, exhibited similar behaviors as homozygous knockouts. Taken together, our results demonstrate that retrieval of context-associated memory is dependent on the Ca_v3.2 T-type Ca²⁺ channel.     

Transient receptor potential channel A1 is directly gated by calcium ions

Members of the superfamily of transient receptor potential (TRP) channels are proposed to play important roles in sensory physiology. As an excitatory ion channel TRPA1 is robustly activated by pungent irritants in mustard and garlic and is suggested to mediate the inflammatory actions of environmental irritants and proalgesic agents. Here, we demonstrate that, in addition to pungent natural compounds, Ca(2+) directly gates heterologously expressed TRPA1 in whole-cell and excised-patch recordings with an apparent EC(50) of 905 nm. Pharmacological experiments and site-directed mutagenesis indicate that the N-terminal EF-hand calcium-binding domain of the channel is involved in Ca(2+)-dependent activation. Furthermore, we determine Ca(2+) as prerequisite for icilin activity on TRPA1.     

Voltage dependent calcium channel expression in isolated osteoclasts

In this study the expression of voltage-dependent calcium channels on osteoclast plasma membrane has been investigated. We found that osteoclasts were sensitive to KCl-induced depolarization. In this circumstance a 4 fold transient cytosolic calcium concentration ([Ca2+]i) increase was observed. This increase was dose-dependent. Its half maximal effect was achieved at 30 mM KCl. Voltage sensitive calcium channels in osteoclasts were inhibited by specific antagonists. Nicardipine, a dihydropyridine derivative, was the most effective, inducing complete block of the channels at 10(-6) M. Verapamil (phenylalkylamine) and diltiazem (benzodiazepine) were less effective. These results are consistent with the presence, on the osteoclast membrane, of L-type voltage-sensitive calcium channels.     

A binding-site model for calcium channel inactivation that depends on calcium entry

We describe a model for the mechanism by which Ca channel inactivation might depend on calcium entry. Ca is assumed to bind to a site at the internal membrane surface to cause inactivation of Ca channels. We assume that Ca that enters through the membrane accumulates in a submembrane compartment and also make simplifying assumptions about Ca buffering and removal. Our model predicts the results of single- and double-pulse voltage-clamp experiments well. The predicted turn-off of Ca current is non-exponential. The model also predicts that procedures that slow inactivation will increase peak Ca current and suggests that both two-phase turn-off of currents and failure of normalized current to recover to 1.0 in two-pulse experiments may be explained without assuming a voltage-dependent component of inactivation or two populations of Ca channels.     

Calcium-dependent inactivation of the calcium current activated upon hyperpolarization of Paramecium tetraurelia

The Ca2+ current activated upon hyperpolarization of Paramecium tetraurelia decays over a period of 150-200 ms during sustained steps under voltage clamp. At membrane potentials between -70 and approximately -100 mV, the time course of this inactivation is described by a single exponential function. Steps negative to approximately -100 mV elicit currents that decay biexponentially, however. Three lines of evidence suggest that this current's inactivation is a function of intracellular Ca2+ concentration rather than membrane potential: (a) Comparing currents with similar amplitudes but elicited at widely differing membrane potentials suggests that their time course of decay is a sole function of inward current magnitude. (b) The extent of current inactivation is correlated with the amount of Ca2+ entering the cell during hyperpolarization. (c) The onset and time course of recovery from inactivation can be hastened significantly by injecting cells with EGTA. We suggest that the decay of this current during hyperpolarization involves a Ca(2+)-dependent pathway.     

Glucagon-stimulated calcium efflux in the isolated perfused rat liver is dependent on cellular redox potential

In the absence of any exogenous substrates, glucagon (1 X 10(-9) M) stimulated 45Ca2+ efflux from perfused livers derived from fed rats but not in livers of 24-h-fasted animals. In livers of 24-h-fasted animals perfused under conditions which would decrease cellular NAD(P)H/NAD(P)+ ratio (pyruvate (2.0 mM) or acetoacetate (10.0 mM], glucagon (1 X 10(-9) M) did not stimulate 45Ca2+ efflux. Similarly, in livers of 24-h-fasted animals perfused with substrates which increase cellular NAD(P)H content (lactate (2.0 mM) or beta-hydroxybutyrate (10.0 mM], glucagon (1 X 10(-9) M) did not increase 45Ca2+ efflux. Glucagon (1 X 10(-9) M) elicited an increase in 45Ca2+ efflux from livers of 24-h-fasted animals, only when the livers were perfused with [lactate]/[pyruvate] and [beta-hydroxybutyrate]/[acetoacetate] ratios similar to those reported for livers of fed rats. Stimulation of 45Ca2+ efflux elicited by either 8-CPT-cAMP, a cAMP analog, or high glucagon concentrations (1 X 10(-8) M) was not affected whether livers were perfused with pyruvate (2.0 mM) or lactate (2.0 mM). Administration of isobutylmethylxanthine (50 microM) alone, or glucagon (1 X 10(-9) M) in the presence of isobutylmethylxanthine (50 microM) stimulated 45Ca2+ efflux from livers of 24-h-fasted animals perfused with pyruvate (2.0 mM) but not from livers perfused with lactate (2.0 mM). The ability of glucagon (1 X 10(-9) M) to elevate tissue cAMP levels was also regulated by the oxidation-reduction state of the livers. The data indicate that glucagon-stimulated 45Ca2+ efflux from perfused livers is mediated via cAMP and is dependent on the oxidation-reduction state of the livers.     

Effect of calcium upon sodium inactivation in the giant axon ofLoligo pealei

Summary Giant axons ofLoligo pealei were voltage clamped in artificial seawater solutions containing varying concentrations of calcium from 10 to 100mm, and the sodium conductance inactivation was measured with a series of two-pulse experiments. Theh vs. voltage curve showed a shift of about 10 mV in the depolarizing direction on the voltage axis for a tenfold increase in external calcium without substantial alteration in the slope of the voltage dependence. The kinetics of the inactivation process were found to be exponential for hyperpolarizing prepulses, but showed some indication of a sigmoidal decay for depolarizing prepulses in all calcium concentrations employed. Increasing calcium increased the delay in the sigmoidal response. The inactivation time constant _h increased as a function of calcium concentration over the potential range studied, –10 to –90 mV. The values of the rate constants _h and _h are decreased with an increase in calcium and these effects are not consistent with parallel shifts of the rate constant vs. voltage curves along the voltage axis for changes in calcium concentration.Magnesium does not behave as an equimolar substitute for calcium. The effect of a solution containing 10mm calcium and 50mm magnesium is intermediate to that of solutions containing 10 and 30mm calcium alone.Predictions of a recent model for the sodium conductance (Moore, J.W., Cox, E.B., 1976Biophys. J. 16:171) which employs calcium binding were compared with the experimental data.     

Induction of cytosolic calcium flux by CD20 is dependent upon B Cell antigen receptor signaling

The anti-CD20 monoclonal antibody (mAb) rituximab is now routinely used for the treatment of non-Hodgkins lymphoma and is being examined in a wide range of other B-cell disorders, such as rheumatoid arthritis. Despite intensive study, the mechanism of action still remains uncertain. In the current study, anti-CD20 mAb-induced calcium signaling was investigated. Previously, we grouped anti-CD20 mAbs into Type I (rituximab-like) and Type II (B1-like) based upon various characteristics such as their ability to induce complement activation and redistribute CD20 into detergent-insoluble membrane domains. Here we show that only Type I mAbs are capable of inducing a calcium flux in B cells and that this is tightly correlated with the expression of the B-cell antigen receptor (BCR). Inhibitor analysis revealed that the signaling cascade employed by CD20 was strikingly similar to that utilized by the BCR, with inhibitors of Syk, Src, and PI3K, but not EGTA, p38, or ERK1/2, completely ablating calcium flux. Furthermore, binding of Type I but not Type II mAbs caused direct association of CD20 with the BCR as measured by FRET and resulted in the phosphorylation of BCR-specific adaptor proteins BLNK and SLP-76. Crucially, variant Ramos cells lacking BCR expression but with unchanged CD20 expression were completely unable to induce calcium flux following ligation of CD20. Collectively, these data indicate that CD20 induces cytosolic calcium flux through its ability to associate with and "hijack" the signaling potential of the BCR.     

LPS-induced apoptosis is dependent upon mitochondrial dysfunction

Bacterial infection induces apoptotic cell death in human monoblastic U937 cells that have been pretreated with interferon gamma (U937IFN). Apoptosis occurs in a manner that is independent of bacterial virulence proteins. In the present study, we show that lipopolysaccharide (LPS), a membrane constituent of gram-negative bacteria, also induces apoptosis in U937IFN cells. LPS treatment led to the appearance of characteristic markers of apoptosis such as nuclear fragmentation and activation of caspases. While the caspase inhibitor Z-VAD-fmk prevented LPS-induced apoptosis as judged by its inhibition of nuclear fragmentation, it failed to inhibit cytochrome c release and loss of mitochondrial membrane potential. Transfection of peptides containing the BH4 (Bcl-2 homology 4) domain derived from the anti-apoptotic protein Bcl-XL blocked LPS-induced nuclear fragmentation and the limited digestion of PARP. These results suggest that LPS does not require caspase activation to induce mitochondrial dysfunction and that mitochondria play a crucial role in the regulation of LPS-mediated apoptosis in U937IFN cells.