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1.
P2X2 receptor channel, a homotrimer activated by the binding of extracellular adenosine triphosphate (ATP) to three intersubunit ATP-binding sites (each located ∼50 Å from the ion permeation pore), also shows voltage-dependent activation upon hyperpolarization. Here, we used tandem trimeric constructs (TTCs) harboring critical mutations at the ATP-binding, linker, and pore regions to investigate how the ATP activation signal is transmitted within the trimer and how signals generated by ATP and hyperpolarization converge. Analysis of voltage- and [ATP]-dependent gating in these TTCs showed that: (a) Voltage- and [ATP]-dependent gating of P2X2 requires binding of at least two ATP molecules. (b) D315A mutation in the β-14 strand of the linker region connecting the ATP-binding domains to the pore-forming helices induces two different gating modes; this requires the presence of the D315A mutation in at least two subunits. (c) The T339S mutation in the pore domains of all three subunits abolishes the voltage dependence of P2X2 gating in saturating [ATP], making P2X2 equally active at all membrane potentials. Increasing the number of T339S mutations in the TTC results in gradual changes in the voltage dependence of gating from that of the wild-type channel, suggesting equal and independent contributions of the subunits at the pore level. (d) Voltage- and [ATP]-dependent gating in TTCs differs depending on the location of one D315A relative to one K308A that blocks the ATP binding and downstream signal transmission. (e) Voltage- and [ATP]-dependent gating does not depend on where one T339S is located relative to K308A (or D315A). Our results suggest that each intersubunit ATP-binding signal is directly transmitted on the same subunit to the level of D315 via the domain that contributes K308 to the β-14 strand. The signal subsequently spreads equally to all three subunits at the level of the pore, resulting in symmetric and independent contributions of the three subunits to pore opening.  相似文献   

2.
The voltage sensitivity of voltage-gated cation channels is primarily attributed to conformational changes of a four transmembrane segment voltage-sensing domain, conserved across many levels of biological complexity. We have identified a remarkable point mutation that confers significant voltage dependence to Kir6.2, a ligand-gated channel that lacks any canonical voltage-sensing domain. Similar to voltage-dependent Kv channels, the Kir6.2[L157E] mutant exhibits time-dependent activation upon membrane depolarization, resulting in an outwardly rectifying current-voltage relationship. This voltage dependence is convergent with the intrinsic ligand-dependent gating mechanisms of Kir6.2, since increasing the membrane PIP2 content saturates Po and eliminates voltage dependence, whereas voltage activation is more dramatic when channel Po is reduced by application of ATP or poly-lysine. These experiments thus demonstrate an inherent voltage dependence of gating in a “ligand-gated” K+ channel, and thereby provide a new view of voltage-dependent gating mechanisms in ion channels. Most interestingly, the voltage- and ligand-dependent gating of Kir6.2[L157E] is highly sensitive to intracellular [K+], indicating an interaction between ion permeation and gating. While these two key features of channel function are classically dealt with separately, the results provide a framework for understanding their interaction, which is likely to be a general, if latent, feature of the superfamily of cation channels.  相似文献   

3.
Point mutations in pore-lining S6 segments of CaV1.2 shift the voltage dependence of activation into the hyperpolarizing direction and significantly decelerate current activation and deactivation. Here, we analyze theses changes in channel gating in terms of a circular four-state model accounting for an activation R–A–O and a deactivation O–D–R pathway. Transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements. Voltage-independent pore openings and closures during activation (A–O) and deactivation (D–R) are described by rate constants α and β, and γ and δ, respectively. Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)). Estimated model parameters of 15 CaV1.2 constructs well describe the activation and deactivation processes. Voltage dependence of the “pore-releasing” sensor movement ((x(V)) was much weaker than the voltage dependence of “pore-locking” sensor movement (y(V)). Our data suggest that changes in membrane voltage are more efficient in closing than in opening CaV1.2. The model failed to reproduce current kinetics of mutation A780P that was, however, accurately fitted with individually adjusted x(V) and y(V). We speculate that structural changes induced by a proline substitution in this position may disturb the voltage-sensing domain.  相似文献   

4.
Macrophages are unique innate immune cells that play an integral role in the defense of the host by virtue of their ability to recognize, engulf, and kill pathogens while sending out danger signals via cytokines to recruit and activate inflammatory cells. It is becoming increasingly clear that purinergic signaling events are essential components of the macrophage response to pathogen challenges and disorders such as sepsis may be, at least in part, regulated by these important sensors. The activation of the P2X7 receptor is a powerful event in the regulation of the caspase-1 inflammasome. We provide evidence that the inflammasome activation requires “priming” of macrophages prior to ATP activation of the P2X7R. Inhibition of the inflammasome activation by the tyrosine kinase inhibitor, AG126, suggests regulation by phosphorylation. Finally, the P2X7R may also be activated by other elements of the host response such as the antimicrobial peptide LL-37, which adds a new, physiologically relevant agonist to the P2X7R pathway. Therapeutic approaches to inflammation and sepsis will certainly be enhanced by an increased understanding of how purinergic receptors modulate the inflammasomes.  相似文献   

5.
We have previously reported that Porphyromonas gingivalis infection of gingival epithelial cells (GEC) requires an exogenous danger signal such as ATP to activate an inflammasome and caspase-1, thereby inducing secretion of interleukin (IL)-1β. Stimulation with extracellular ATP also stimulates production of reactive oxygen species (ROS) in GEC. However, the mechanism by which ROS is generated in response to ATP, and the role that different purinergic receptors may play in inflammasome activation, is still unclear. In this study, we revealed that the purinergic receptor P2X4 is assembled with the receptor P2X7 and its associated pore, pannexin-1. ATP induces ROS production through a complex consisting of the P2X4, P2X7, and pannexin-1. P2X7−mediated ROS production can activate the NLRP3 inflammasome and caspase-1. Furthermore, separate depletion or inhibition of P2X4, P2X7, or pannexin-1 complex blocks IL-1β secretion in P. gingivalis-infected GEC following ATP treatment. However, activation via P2X4 alone induces ROS generation but not inflammasome activation. These results suggest that ROS is generated through stimulation of a P2X4/P2X7/pannexin-1 complex, and reveal an unexpected role for P2X4, which acts as a positive regulator of inflammasome activation during microbial infection.  相似文献   

6.
The voltage-dependent gating mechanism of KAT1 inward rectifier potassium channels was studied using single channel current recordings from Xenopus oocytes injected with KAT1 mRNA. The inward rectification properties of KAT1 result from an intrinsic gating mechanism in the KAT1 channel protein, not from pore block by an extrinsic cation species. KAT1 channels activate with hyperpolarizing potentials from −110 through −190 mV with a slow voltage-dependent time course. Transitions before first opening are voltage dependent and account for much of the voltage dependence of activation, while transitions after first opening are only slightly voltage dependent. Using burst analysis, transitions near the open state were analyzed in detail. A kinetic model with multiple closed states before first opening, a single open state, a single closed state after first opening, and a closed-state inactivation pathway accurately describes the single channel and macroscopic data. Two mutations neutralizing charged residues in the S4 region (R177Q and R176L) were introduced, and their effects on single channel gating properties were examined. Both mutations resulted in depolarizing shifts in the steady state conductance–voltage relationship, shortened first latencies to opening, decreased probability of terminating bursts, and increased burst durations. These effects on gating were well described by changes in the rate constants in the kinetic model describing KAT1 channel gating. All transitions before the open state were affected by the mutations, while the transitions after the open state were unaffected, implying that the S4 region contributes to the early steps in gating for KAT1 channels.  相似文献   

7.
ATP is emerging as an ubiquitous extracellular messenger. However, measurement of ATP concentrations in the pericellular space is problematic. To this aim, we have engineered a firefly luciferase-folate receptor chimeric protein that retains the N-terminal leader sequence and the C-terminal GPI anchor of the folate receptor. This chimeric protein, named plasma membrane luciferase (pmeLUC), is targeted and localized to the outer aspect of the plasma membrane. PmeLUC is sensitive to ATP in the low micromolar to millimolar level and is insensitive to all other nucleotides. To identify pathways for nonlytic ATP release, we transfected pmeLUC into cells expressing the recombinant or native P2X7 receptor (P2X7R). Both cell types release large amounts of ATP (100–200 μM) in response to P2X7R activation. This novel approach unveils a hitherto unsuspected nonlytic pathway for the release of large amounts of ATP that might contribute to spreading activation and recruitment of immune cells at inflammatory sites.  相似文献   

8.
We have characterized the effects of prepulse hyperpolarization and extracellular Mg(2+) on the ionic and gating currents of the Drosophila ether-à-go-go K(+) channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg(2+) dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg(2+) modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg(2+) slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg(2+) modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg(2+). We have also identified mutations in the S3-S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg(2+), indicating an important role for this region in the voltage-dependent activation of eag.  相似文献   

9.
Connexin hemichannels are robustly regulated by voltage and divalent cations. The basis of voltage-dependent gating, however, has been questioned with reports that it is not intrinsic to hemichannels, but rather is derived from divalent cations acting as gating particles that block the pore in a voltage-dependent manner. Previously, we showed that connexin hemichannels possess two types of voltage-dependent gating, termed Vj and loop gating, that in Cx46 operate at opposite voltage polarities, positive and negative, respectively. Using recordings of single Cx46 hemichannels, we found both forms of gating persist in solutions containing no added Mg2+ and EGTA to chelate Ca2+. Although loop gating persists, it is significantly modulated by changing levels of extracellular divalent cations. When extracellular divalent cation concentrations are low, large hyperpolarizing voltages, exceeding −100 mV, could still drive Cx46 hemichannels toward closure. However, gating is characterized by continuous flickering of the unitary current interrupted by occasional, brief sojourns to a quiet closed state. Addition of extracellular divalent cations, in this case Mg2+, results in long-lived residence in a quiet closed state, suggesting that hyperpolarization drives the hemichannel to close, perhaps by initiating movements in the extracellular loops, and that divalent cations stabilize the fully closed conformation. Using excised patches, we found that divalent cations are only effective from the extracellular side, indicative that the binding site is not cytoplasmic or in the pore, but rather extracellular. Vj gating remains essentially unaffected by changing levels of extracellular divalent cations. Thus, we demonstrate that both forms of voltage dependence are intrinsic gating mechanisms in Cx46 hemichannels and that the action of external divalent cations is to selectively modulate loop gating.  相似文献   

10.
Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N3-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N3-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1–NBD2 interface. The open state of CFTR has been shown to represent a two-ATP–bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a “partial NBD dimer” state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and “partial” separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR’s NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed.  相似文献   

11.
The voltage-dependent anion channel (VDAC) is the major pathway for ATP, ADP, and other respiratory substrates through the mitochondrial outer membrane, constituting a crucial point of mitochondrial metabolism regulation. VDAC is characterized by its ability to “gate” between an open and several “closed” states under applied voltage. In the early stages of tumorigenesis or during ischemia, partial or total absence of oxygen supply to cells results in cytosolic acidification. Motivated by these facts, we investigated the effects of pH variations on VDAC gating properties. We reconstituted VDAC into planar lipid membranes and found that acidification reversibly increases its voltage-dependent gating. Furthermore, both VDAC anion selectivity and single channel conductance increased with acidification, in agreement with the titration of the negatively charged VDAC residues at low pH values. Analysis of the pH dependences of the gating and open channel parameters yielded similar pKa values close to 4.0. We also found that the response of VDAC gating to acidification was highly asymmetric. The presumably cytosolic (cis) side of the channel was the most sensitive to acidification, whereas the mitochondrial intermembrane space (trans) side barely responded to pH changes. Molecular dynamic simulations suggested that stable salt bridges at the cis side, which are susceptible to disruption upon acidification, contribute to this asymmetry. The pronounced sensitivity of the cis side to pH variations found here in vitro might provide helpful insights into the regulatory role of VDAC in the protective effect of cytosolic acidification during ischemia in vivo.  相似文献   

12.
The TREK subfamily of two-pore domain (K2P) K+ channels exhibit polymodal gating by a wide range of physical and chemical stimuli. Crystal structures now exist for these channels in two main states referred to as the “up” and “down” conformations. However, recent studies have resulted in contradictory and mutually exclusive conclusions about the functional (i.e., conductive) status of these two conformations. To address this problem, we have used the state-dependent TREK-2 inhibitor norfluoxetine that can only bind to the down state, thereby allowing us to distinguish between these two conformations when activated by different stimuli. Our results reconcile these previously contradictory gating models by demonstrating that activation by pressure, temperature, voltage, and pH produce more than one structurally distinct open state and reveal that channel activation does not simply involve switching between the up and down conformations. These results also highlight the diversity of structural mechanisms that K2P channels use to integrate polymodal gating signals.  相似文献   

13.
Ginseng, the root of Panax ginseng C.A. Meyer, is used as a general tonic. Recently, we isolated a novel ginsengderived lysophosphatidic acid (LPA) receptor ligand, gintonin. Gintonin activates G protein-coupled LPA receptors with high affinity in cells endogenously expressing LPA receptors, e.g., Xenopus oocytes. P2X receptors are ligandgated ion channels activated by extracellular ATP, and 7 receptor subtypes (P2X1–P2X7) have been identified. Most of the P2X1 receptors are expressed in the smooth muscles of genitourinary organs involved in reproduction. A main characteristic of the P2X1 receptor is rapid desensitization after repeated ATP treatment of cells or tissues expressing P2X1 receptors. In the present study, we examined the effect of gintonin on P2X1 receptor channel activity. P2X1 receptors were heterologously expressed in Xenopus oocytes. ATP treatment of oocytes expressing P2X1 receptors induced large inward currents (I ATP ), but repetitive ATP treatments induced a rapid desensitization of I ATP . Gintonin treatment after P2X1 receptor desensitization potentiated I ATP in a concentration-dependent manner. We further examined the signaling transduction pathways involved in gintonin-mediated potentiation of I ATP . Gintoninmediated I ATP potentiation was blocked by Ki16425, an LPA1/3 receptor antagonist, a PKC inhibitor, a PLC inhibitor, and a PI4-Kinase inhibitor but not by a calcium chelator. In addition, mutations of the phosphoinositide binding site of the P2X1 receptor greatly attenuated the gintonin-mediated I ATP potentiation. These results indicate that G protein-coupled LPA receptor activation by gintonin is coupled to the potentiation of the desensitized P2X1 receptor through a phosphoinositide-dependent pathway.  相似文献   

14.
Each of the four subunits in a voltage-gated potassium channel has a voltage sensor domain (VSD) that is formed by four transmembrane helical segments (S1–S4). In response to changes in membrane potential, intramembrane displacement of basic residues in S4 produces a gating current. As S4 moves through the membrane, its basic residues also form sequential electrostatic interactions with acidic residues in immobile regions of the S2 and S3 segments. Transition metal cations interact with these same acidic residues and modify channel gating. In human ether-á-go-go–related gene type 1 (hERG1) channels, Cd2+ coordinated by D456 and D460 in S2 and D509 in S3 induces a positive shift in the voltage dependence of activation of ionic currents. Here, we characterize the effects of Cd2+ on hERG1 gating currents in Xenopus oocytes using the cut-open Vaseline gap technique. Cd2+ shifted the half-point (V1/2) for the voltage dependence of the OFF gating charge–voltage (QOFF-V) relationship with an EC50 of 171 µM; at 0.3 mM, V1/2 was shifted by +50 mV. Cd2+ also induced an as of yet unrecognized small outward current (ICd-out) upon repolarization in a concentration- and voltage-dependent manner. We propose that Cd2+ and Arg residues in the S4 segment compete for interaction with acidic residues in S2 and S3 segments, and that the initial inward movement of S4 associated with membrane repolarization displaces Cd2+ in an outward direction to produce ICd-out. Co2+, Zn2+, and La3+ at concentrations that caused ∼+35-mV shifts in the QOFF-V relationship did not induce a current similar to ICd-out, suggesting that the binding site for these cations or their competition with basic residues in S4 differs from Cd2+. New Markov models of hERG1 channels were developed that describe gating currents as a noncooperative two-phase process of the VSD and can account for changes in these currents caused by extracellular Cd2+.  相似文献   

15.
Rat P2X2 receptors open at an undetectably low rate in the absence of ATP. Furthermore, two allosteric modulators, zinc and acidic pH, cannot by themselves open these channels. We describe here the properties of a mutant receptor, K69C, before and after treatment with the thiol-reactive fluorophore Alexa Fluor 546 C5-maleimide (AM546). Xenopus oocytes expressing unmodified K69C were not activated under basal conditions nor by 1,000 µM ATP. AM546 treatment caused a small increase in the inward holding current which persisted on washout and control experiments demonstrated this current was due to ATP independent opening of the channels. Following AM546 treatment, zinc (100 µM) or acidic external solution (pH 6.5) elicited inward currents when applied without any exogenous ATP. In the double mutant K69C/H319K, zinc elicited much larger inward currents, while acidic pH generated outward currents. Suramin, which is an antagonist of wild type receptors, behaved as an agonist at AM546-treated K69C receptors. Several other cysteine-reactive fluorophores tested on K69C did not cause these changes. These modified receptors show promise as a tool for studying the mechanisms of P2X receptor activation.  相似文献   

16.
The Ca2+-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at –40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs2+-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca2+ with Ba2+ slowed the current decay. Increasing the external Ca2+ or Ba2+ concentration increased the amplitude of the inward current and the current–voltage (I–V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na+ in the absence of extracellular Ca2+. Current carried by Na+ (I Na) was almost completely blocked by the dihydropyridine Ca2+ channel antagonist, nifedipine, suggesting that the I Na is through voltage-dependent L-type Ca2+ channels. The other inward current is voltage-independent and its I–V relationship was linear between –100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs+-aspartate and 140 mM Na+-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na+ was replaced with an equimolar amount of K+ or Cs+, or 50 mM Ca2+ or Ba2+. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I–V relationship for the current evoked by the hypotonic solution also showed a linear relationship between –100 mV to 0 mV. Bath application of Gd3+ (10 M) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca2+-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca2+ channel and stretch-activated Ca2+-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.Abbreviations I Ca Ca2+ current - I Na Na+ current - I–V current–voltage - NSCC nonselective cation channel Communicated by G. Heldmaier  相似文献   

17.
Extracellular ATP and 5-hydroxytryptamine (5-HT) are both involved in visceral sensory pathways by interacting with P2X and 5-HT3 receptors, respectively. We have investigated the changes in P2X and 5-HT3-mediated signalling in pelvic afferent neurons in mice deficient in P2X2 and/or P2X3 subunits by whole-cell recording of L6–S2 dorsal root ganglion (DRG) neurons and by multi-unit recording of pelvic afferents of the colorectum. In wildtype DRG neurons, ATP evoked transient, sustained or mixed (biphasic) inward currents. Transient currents were absent in P2X3 −/− neurons, whereas sustained currents were absent in P2X2 −/− DRG neurons. Neither transient nor sustained currents were observed following application of ATP or α,β-methylene ATP (α,β-meATP) in P2X2/P2X3 Dbl−/− DRG neurons. 5-HT was found to induce a fast inward current in 63% of DRG neurons from wildtype mice, which was blocked by tropisetron, a 5-HT3 receptor antagonist. The percentage of DRG neurons responding to 5-HT was significantly increased in P2X 2 −/−, P2X3 −/− and P2X2/P2X3 Dbl−/− mice, and the amplitude of 5-HT response was significantly increased in P2X2/P2X3 Dbl−/− mice. The pelvic afferent response to colorectal distension was attenuated in P2X2/P2X3 Dbl−/− mice, but the response to serosal application of 5-HT was enhanced. Furthermore, tropisetron resulted in a greater reduction in pelvic afferent responses to colorectal distension in the P2X2/P2X3 Dbl−/− preparations. These data suggest that P2X receptors containing the P2X2 and/or P2X3 subunits mediate purinergic activation of colorectal afferents and that 5-HT signalling in pelvic afferent neurons is up-regulated in mice lacking P2X2 or P2X3 receptor genes. This effect is more pronounced when both subunits are absent.  相似文献   

18.
This work shows that ATP activates JNK1, but not JNK2, in rat osteoblasts and ROS-A 17/2.8 osteoblast-like cells. In ROS-A 17/2.8 cells ATP induced JNK1 phosphorylation in a dose- and time-dependent manner. JNK1 phosphorylation also increased after osteoblast stimulation with ATPγS and UTP, but not with ADPβS. RT-PCR studies supported the expression of P2Y2 receptor subtype. ATP-induced JNK1 activation was reduced by PI-PLC, IP3 receptor, PKC and Src inhibitors and by gadolinium, nifedipine and verapamil or a Ca2+-free medium. ERK 1/2 or p38 MAPK inhibitors diminished JNK1 activation by ATP, suggesting a cross-talk between these pathways. ATP stimulated osteoblast-like cell proliferation consistent with the participation of P2Y2 receptors. These results show that P2Y2 receptor stimulation by ATP induces JNK1 phosphorylation in ROS-A 17/2.8 cells in a way dependent on PI-PLC/IP3/intracellular Ca2+ release and Ca2+ influx through stress activated and L-type voltage-dependent calcium channels and involves PKC and Src kinases.  相似文献   

19.
Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K+ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K+ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K+, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current.  相似文献   

20.
Ca(2+)-activated Cl(-) channels play important roles in a variety of physiological processes, including epithelial secretion, maintenance of smooth muscle tone, and repolarization of the cardiac action potential. It remains unclear, however, exactly how these channels are controlled by Ca(2+) and voltage. Excised inside-out patches containing many Ca(2+)-activated Cl(-) channels from Xenopus oocytes were used to study channel regulation. The currents were mediated by a single type of Cl(-) channel that exhibited an anionic selectivity of I(-) > Br(-) > Cl(-) (3.6:1.9:1.0), irrespective of the direction of the current flow or [Ca(2+)]. However, depending on the amplitude of the Ca(2+) signal, this channel exhibited qualitatively different behaviors. At [Ca(2+)] < 1 microM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state current-voltage relationship was strongly outwardly rectifying. At higher [Ca(2+)], the currents did not rectify and were time independent. This difference in behavior at different [Ca(2+)] was explained by an apparent voltage-dependent Ca(2+) sensitivity of the channel. At +120 mV, the EC(50) for channel activation by Ca(2+) was approximately fourfold less than at -120 mV (0.9 vs. 4 microM). Thus, at [Ca(2+)] < 1 microM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca(2+) binding. The voltage-dependent Ca(2+) sensitivity was explained by a kinetic gating scheme in which channel activation was Ca(2+) dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca(2+) concentration jumps were voltage sensitive, but that the activation time constants were Ca(2+) sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca(2+) concentrations adds a new dimension to Ca(2+) signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca(2+) signal.  相似文献   

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