A model for receptor-regulated calcium entry

A model is proposed for the mechanism by which activation of surface membrane receptors causes sustained Ca2+ entry into cells from the extracellular space. Reassessment of previously published findings on the behavior of receptor-regulated intracellular Ca2+ pools leads to the conclusion that when such pools are empty, a pathway from the extracellular space to the pool is opened; conversely when the pool is filled, the pathway is closed and it becomes relatively stable to depletion by low Ca2+ media or chelating agents. The biphasic nature of agonist-activated Ca2+-mobilization is thus seen as an initial emptying of the intracellular Ca2+ pool by inositol (1,4,5) trisphosphate, followed by rapid entry of Ca2+ into the pool and, in the continued presence of inositol (1,4,5) trisphosphate, into the cytosol. On withdrawal of agonist, inositol (1,4,5) trisphosphate is then rapidly degraded, the pathway from the pool to the cytosol is closed, and rapid entry from the outside continues until the Ca2+ content of the pool reaches a level that inactivates Ca2+ entry. This capacitative model allows for Ca2+ release and Ca2+ entry to be controlled by a single messenger, inositol (1,4,5) trisphosphate.     

容量性钙内流通道的分子代表

细胞内钙库排空产生一种信号,诱导细胞膜上的钙库操纵的钙通道（SOC）开放,使Ca^2 由细胞外进入细胞内,称为容量性钙内流（CCE）,或钙释放激活的钙通道（CRAC）,可能由果蝇一过性受体电位（trp)和trp样（trpl)基因编码,钙库排空和通道开放之间的偶联机制不清,目前主要提出三种机制：（1）弥散信使;（2）蛋白质－蛋白质之间的相互作用;（3）囊泡分泌。本文综述了CCE的分子代表 ,可能机制及电生理表型。     

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).     

TRPV4 calcium entry channel: a paradigm for gating diversity

The vanilloid receptor-1 (VR1, now TRPV1) was the founding member of a subgroup of cation channels within the TRP family. The TRPV subgroup contains six mammalian members, which all function as Ca2+ entry channels gated by a variety of physical and chemical stimuli. TRPV4, which displays 45% sequence identity with TRPV1, is characterized by a surprising gating promiscuity: it is activated by hypotonic cell swelling, heat, synthetic 4alpha-phorbols, and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids. This review summarizes data on TRPV4 as a paradigm of gating diversity in this subfamily of Ca2+ entry channels.     

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.     

A minimal gating model for the cardiac calcium release channel.

A Markovian model of the cardiac Ca release channel, based on experimental single-channel gating data, was constructed to understand the transient nature of Ca release. The rate constants for a minimal gating scheme with one Ca-free resting state, and with two open and three closed states with one bound Ca2+, were optimized to simulate the following experimental findings. In steady state the channel displays three modes of activity: inactivated 1 mode without openings, low-activity L mode with single openings, and high-activity H mode with bursts of openings. At the onset of a Ca2+ step, the channel first activates in H mode and then slowly relaxes to a mixture of all three modes, the distribution of which depends on the new Ca2+. The corresponding ensemble current shows rapid activation, which is followed by a slow partial inactivation. The transient reactivation of the channel (increment detection) in response to successive additions of Ca2+ is then explained by the model as a gradual recruitment of channels from the extant pool of channels in the resting state. For channels in a living cell, the model predicts a high level of peak activation, a high extent of inactivation, and rapid deactivation, which could underlie the observed characteristics of the elementary release events (calcium sparks).     

Effects of actin cytoskeleton rearrangements on channel activation by calcium entry into A431 cells

Using patch clamp and ion-selective fluorescence dye techniques, we investigated the influence of actin cytoskeleton rearrangements on the activity of calcium entry channels in plasma membrane of human carcinoma A431 cells. It is shown that disruption of actin microfilaments by cytohalasin D has no significant effect on calcium release from the stores and its entry from the extracellular space. It also does not interfere with the activation of inositol 1,4,5-trisphosphate (IP3)-dependent high-selective low-conductance calcium channels Imin. The treatment of cells with calyculin A induces the formation of actin filament layer beneath plasma membrane and also inhibits Imin activation and calcium entry through the plasma membrane, though calcium efflux from the stores was nearly unchanged. Thus, it is concluded that calcium signalling in A431 cells can be modulated by actin cytoskeleton rearrangements, and may be well described in terms of "conformational coupling" model.     

A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL.

Intracellular Ca2+ signalling evoked by Ca2+ mobilizing agonists, like angiotensin II in the adrenal gland, involves the activation of inositol(1,4,5)trisphosphate(InsP3)-mediated Ca2+ release from internal stores followed by activation of a Ca2+ influx termed capacitative calcium entry. Here we report the amino acid sequence of a functional capacitative Ca2+ entry (CCE) channel that supports inward Ca2+ currents in the range of the cell resting potential. The expressed CCE channel opens upon depletion of Ca2+ stores by InsP3 or thapsigargin, suggesting that the newly identified channel supports the CCE coupled to InsP3 signalling.     

On mutations that uncouple sodium channel activation from inactivation

Computations on sodium channel gating were conducted using a closed-open-inactivated coupled kinetic scheme. The time constant of inactivation (tauh) derives a voltage dependency from coupling to voltage-dependent activation even when rate constants between inactivated and other states are strictly voltage independent. The derived voltage dependency does not require any physical, molecular link between the structures responsible for inactivation and the charges producing voltage-dependent activation. The only requirement is that the closed to inactivated rate constant (kCI) differs from the open to inactivated (kOI), consistent with experimental results. A number of mutations and other treatments uncouple sodium channel activation and inactivation in that the voltage dependency of tauh is substantially reduced while voltage-dependent activation persists. However, a clear basis for uncoupling has not been described. A variety of experimental results are accounted for just by changes in the difference between kOI and kCI. In wild type channels, kOI > kCI and inactivation develops with a delay whose time constant is just that for channel opening. Mutations that reduce the kOI - kCI difference reduce the amplitude of the delay process and the derived voltage dependency of tauh. If kOI = kCI, inactivation develops as a single exponential (no matter what the number of closed states), activation and inactivation become independent, parallel processes, and any voltage dependency of tauh is then entirely intrinsic to inactivation. If kOI < kCI, inactivation develops as the sum of exponentials, tauh at negative potentials speeds and then slows with more positive potentials. These predicted kOI < kCI effects have all been seen experimentally (O'Leary, M.E., L.-Q. Chen, R.G. Kallen, and R. Horn. 1995. J. Gen. Physiol. 106: 641-658). An open to closed rate constant of zero also removes the derived voltage dependency of tauh, but activation and inactivation are still coupled and the inactivation delay remains.     

A molecular dynamics study of an L-type calcium channel model

In this work, we propose a molecular model of the L-type calcium channel pore from the human cardiac alpha1 subunit. Four glutamic acid residues, the EEEE locus, located at highly conserved P loops (also called SS1-SS2 segments) of the alpha1 subunit, molecularly express the calcium channel selectivity. The proposed alpha-helix structure for the SS1 segment, analyzed through molecular dynamics simulations in aqueous-phase, was validated by the plotting of Ramachandran diagrams for the averaged structures and by the analysis of i and i + 4 helical hydrogen bonding between the amino acid residues. The results of the simulation of the calcium channel model with one and two Ca2+ ions at the binding site are in accordance with mutation studies which suggest that the EEEE locus in the L-type calcium channel must form a single high-affinity binding site. These results suggest that the Ca2+ permeation through the channel would be derived from competition between two ions for the only high-affinity binding site. Furthermore, the experimentally observed blocking of the Na+ flux at micromolar Ca2+ concentrations, probably due to the occupancy of the single high-affinity binding site for one Ca2+, was also reproduced by our model.     

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   

Hepatitis C virus entry depends on clathrin-mediated endocytosis

Due to difficulties in cell culture propagation, the mechanisms of hepatitis C virus (HCV) entry are poorly understood. Here, postbinding cellular mechanisms of HCV entry were studied using both retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the HCV clone JFH-1 propagated in cell culture (HCVcc). HCVpp entry was measured by quantitative real-time PCR after 3 h of contact with target cells, and HCVcc infection was quantified by immunoblot analysis and immunofluorescence detection of HCV proteins expressed in infected cells. The functional role of clathrin-mediated endocytosis in HCV entry was assessed by small interfering RNA-mediated clathrin heavy chain depletion and with chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. In both conditions, HCVpp entry and HCVcc infection were inhibited. HCVcc infection was also inhibited by pretreating target cells with bafilomycin A1 or chloroquine, two drugs known to interfere with endosome acidification. These data indicate that HCV enters target cells by clathrin-mediated endocytosis, followed by a fusion step from within an acidic endosomal compartment.     

Methods for studying store-operated calcium entry

Activation of surface membrane receptors coupled to phospholipase C results in the generation of cytoplasmic Ca2+ signals comprised of both intracellular Ca2+ release, and enhanced entry of Ca2+ across the plasma membrane. A primary mechanism for this Ca2+ entry process is attributed to store-operated Ca2+ entry, a process that is activated by depletion of Ca2+ ions from an intracellular store by inositol 1,4,5-trisphosphate. Our understanding of the mechanisms underlying both Ca2+ release and store-operated Ca2+ entry have evolved from experimental approaches that include the use of fluorescent Ca2+ indicators and electrophysiological techniques. Pharmacological manipulation of this Ca2+ signaling process has been somewhat limited; but recent identification of key molecular players, STIM and Orai family proteins, has provided new approaches. Here we describe practical methods involving fluorescent Ca2+ indicators and electrophysiological approaches for dissecting the observed intracellular Ca2+ signal to reveal characteristics of store-operated Ca2+ entry, highlighting the advantages, and limitations, of these approaches.     

A random flight chain model for the tether of the Shaker K+ channel inactivation domain.

Rapid inactivation of Shaker K+ channels occurs when a domain in the amino terminal region of the channel protein blocks the pore. Some part of the sequence between the inactivating domain and the first transmembrane segment may form a flexible tether. We consider the possibility that the tether has no secondary structure, but is rather a polypeptide random coil. The local concentration of the tethered inactivation domain and the dependence of the inactivation rate on chain length can then be calculated by using the Jacobson-Stockmayer equation. A chain of 30-100 amino acids is consistent with the sensitivity of the inactivation rate to chain length mutations.     

D1 receptors physically interact with N-type calcium channels to regulate channel distribution and dendritic calcium entry

Dopamine signaling through D1 receptors in the prefrontal cortex (PFC) plays a critical role in the maintenance of higher cognitive functions, such as working memory. At the cellular level, these functions are predicated to involve alterations in neuronal calcium levels. The dendrites of PFC neurons express D1 receptors and N-type calcium channels, yet little information exists regarding their coupling. Here, we show that D1 receptors potently inhibit N-type channels in dendrites of rat PFC neurons. Using coimmunoprecipitation, we demonstrate the existence of a D1 receptor-N-type channel signaling complex in this region, and we provide evidence for a direct receptor-channel interaction. Finally, we demonstrate the importance of this complex to receptor-channel colocalization in heterologous systems and in PFC neurons. Our data indicate that the N-type calcium channel is an important physiological target of D1 receptors and reveal a mechanism for D1 receptor-mediated regulation of cognitive function in the PFC.     

Measurement of calcium channel inactivation is dependent upon the test pulse potential.

We have developed two methods to measure Ca2+ channel inactivation in Lymnaea neurons-one method, based upon the conventional double-pulse protocol, uses currents during a moderately large depolarizing pulse, and the other uses tail currents after a very strong activating pulse. Both methods avoid contamination by proton currents and are unaffected by rundown of Ca2+ current. The magnitude of inactivation measured differs for the two methods; this difference arises because the measurement of inactivation is inherently dependent upon the test pulse voltage used to monitor the Ca2+ channel conductance. We discuss two models that can generate such test pulse dependence of inactivation measurements-a two-channel model and a two-open-state model. The first model accounts for this by assuming the existence of two types of Ca2+ channels, different proportions of which are activated by the different test pulses. The second model assumes only one Ca2+ channel type, with two closed and open states; in this model, the test pulse dependence is due to the differential activation of channels in the two closed states by the test pulses. Test pulse dependence of inactivation measurements of Ca2+ channels may be a general phenomenon that has been overlooked in previous studies.     

A new class of calcium entry blockers defined by 1,3-diphosphonates. Interactions of SR-7037 (belfosdil) with receptors for calcium channel ligands

Tetrabutyl-2(2-phenoxyethyl)-1,3-propylidene diphosphonate (SR-7037) completely displaced dihydropyridine [( 3H]PN200-110), phenylalkylamine [( 3H]D888), and benzothiazepine [( 3H]diltiazem) ligands from brain L-type calcium channels. Half-maximal inhibition of [3H]PN200-110 binding occurred at 19 nM with a Hill coefficient of 0.96. SR-7037 primarily decreased the affinity for [3H]PN200-110 with a small, but significantly, effect on the maximal binding capacity. Kinetic studies showed that this was due to an increased radioligand dissociation rate from 0.04 min-1 to 0.43 min-1 in the presence of the diphosphonate. Displacement of [3H]D888 by SR-7037 was biphasic with respective IC50 of 44 and 8400 nM. Likewise, unlabeled (-)-D888 identified two sites with IC50 values of 0.9 and 27 nM. Both SR-7037 (1000 nM) and D888 (200 nM) accelerated radioligand dissociation about 2-fold. [3H]Diltiazem binding was inhibited by SR-7037 with an IC50 value of 29 nM. The inhibition of dihydropyridine binding by SR-7037 is enhanced by most divalent cations at millimolar concentrations with the following potency: Mn2+ greater than Mg2+ greater than Ca2+ greater than Co2+. Barium has the opposite effect. The half-maximal effect of calcium occurred at 6 microM free ion. Specific binding of [3H]D888 was antagonized in the presence of 1 mM CaCl2. It is concluded that SR-7037 has allosteric interactions with the dihydropyridine receptor of the L-type calcium channel. The differential effect of Ca2+ on the potency of D888 and diltiazem relative to that of SR-7037 indicates that the three drugs may bind to nonequivalent sites. These results support specific calcium channel inhibition, possibly at a novel site, as the primary mechanism of the diphosphonate's pharmacological actions.