共查询到20条相似文献,搜索用时 0 毫秒
1.
Szewczyk MM Davis KA Samson SE Simpson F Rangachari PK Grover AK 《Journal of cellular and molecular medicine》2007,11(1):129-138
Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation. This can be achieved by Ca2+ sequestration by the sarco-/endoplasmic reticulum Ca2+ pumps (SERCA) and Ca2+ extrusion by plasma membrane Ca2+ pumps (PMCA) and Na+-Ca2+-exchangers (NCX). Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots. The activity of NCX is higher in EC than in SMC but those of PMCA and SERCA is lower. Consistently, the protein abundance for NCX protein is higher in EC than in SMC and those of PMCA and SERCA is lower. Based on RT-PCR experiments, the types of RNA present are as follows: EC for PMCA1 while SMC for PMCA4 and PMCA1; EC for SERCA2 and SERCA3 and SMC for SERCA2. Both EC and SMC express NCX1 (mainly NCX1.3). PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation. Based on these observations and the literature, we conclude that the tightly regulated Ca2+ removal systems in SMC are consistent with the cyclical control of contractility of the filaments and those in EC are consistent with Ca2+ regulation of the endothelial nitric oxide synthase near the cell surface. The differences between EC and SMC should be considered in therapeutic interventions of cardiovascular diseases. 相似文献
2.
In a rat model of streptozotocin (STZ)-induced diabetes, we earlier showed that under these conditions the concentration of free cytosolic Ca2+ in input neurons of the nociceptive system increases, Ca2+ signals are prolonged, while Ca2+ release from intracellular calcium stores decreases. The aim of our study was to test the hypothesis that changes in the activities of Ca2+,Mg2+-ATPases of the endoplasmic reticulum (SERCA) and plasmalemma (PMCA) could be responsible for diabetes-induced disorders of calcium homeostasis in nociceptive neurons. We measured the Ca2+,Mg2+-ATPase activities in microsomal fractions obtained from tissues of the dorsal root ganglia (DRG) and spinal dorsal horn (DH) of control rats and rats with experimentally induced diabetes. The integral specific Ca2+,Mg2+-ATPase activity in microsomes from diabetic rats was lower than that in the control group. The activity of SERCA in samples of DRG and DH of diabetic rats was reduced by 50 ± 8 and 48 ± 12%, respectively, as compared with the control (P < 0.01). At the same time, the activity of PMCA decreased by 63 ± 6% in DRG and by 60 ± 9% in DH samples (P < 0.01). We conclude that diabetic polyneuropathy is associated with the reduction of the rate of recovery of the Ca2+ level in the cytosol of DRG and DH neurons due to down-regulation of the SERCA and PMCA activities. 相似文献
3.
John P. Reeves 《Journal of bioenergetics and biomembranes》1998,30(2):151-160
The Na+/Ca2+ exchange system is the primary Ca2+ efflux mechanism in cardiac myocytes, and plays an important role in controlling the force of cardiac contraction. The exchanger protein contains 11 transmembrane segments plus a large hydrophilic domain between the 5th and 6th transmembrane segments; the transmembrane regions are reponsible for mediating ion translocation while the hydrophilic domain is responsible for regulation of activity. Exchange activity is regulated in vitro by interconversions between an active state and either of two inactive states. High concentrations of cytosolic Na+ or the absence of cytosolic Ca2+ promote the formation of the inactive states; phosphatidylinositol-(4,5)bisphosphate (or other negatively charged phospholipids) and cytosolic Ca2+ counteract the inactivation process. The importance of these mechanisms in regulating exchange activity under normal physiological conditions is uncertain. Exchanger function is also dependent upon cytoskeletal interactions, and the exchanger's location with respect to intracellular Ca2+-sequestering organelles. An understanding of the exchanger's function in normal cell physiology will require more detailed information on the proximity of the exchanger and other Ca2+-transporting proteins, their interactions with the cytoskeleton, and local concentrations of anionic phospholipids and transported ions. 相似文献
4.
It is obvious that disruption of functions of the nervous system in diabetes mellitus is to a great extent related to the changes of synthesis or exocytosis of neurotransmitters. Since the mechanisms underlying exocytosis are similar in cells of different types, it may be assumed that studying these mechanisms in secretory cells will allow experimenters to obtain information on ways to control this process in neurons. Based on the supposition that changes in the activity of Ca2+-controlling systems in exocrine cells play an important role in functional disorders in the salivary glands in diabetes mellitus, we demonstrated, using the fura-2/AM dye, that the intracellular calcium concentration ([Ca2+]
i
) in secretory cells of the above glands in rats with streptozotocin-induced diabetes mellitus (being in the resting state) is significantly increased (on average, by 65%). In our study, we showed that Ca2+-ATPases play an important role in the control of calcium homeostasis in secretory cells of salivary glands in diabetes mellitus. In particular, we demonstrated that the kinetic parameters of microsomal Ca2+-ATPases decreased: V
0, by 50 ± 7, and P
max, by 52 ± 6%, on average. In diabetes mellitus, V
max of Ca2+-ATPases also dropped significantly, by 47 ± 8 and 79 ± 9%, on average, for PMCA and SERCA, respectively. The decrease in K
ATP was 71 ± 11% for SERCA and that in K
Ca was 92 ± 3% for PMCA. We concluded that the activity of Ca2+-ATPases of secretory cells in diabetes mellitus is suppressed because of a decrease in the turnover and/or in the specific number of active molecules of the enzyme. 相似文献
5.
Previously it demonstrated that in the absence of Ca2+ entry, evoked secretion occurs neither by membrane depolarization, induction of [Ca2+]
i
rise, nor by both combined (Ashery, U., Weiss, C., Sela, D., Spira, M. E., and Atlas, D. (1993). Receptors Channels
1:217–220.). These studies designate Ca2+ entry as opposed to [Ca2+]
i
rise, essential for exocytosis. It led us to propose that the channel acts as the Ca2+ sensor and modulates secretion through a physical and functional contact with the synaptic proteins. This view was supported by protein–protein interactions reconstituted in the Xenopus oocytes expression system and release experiments in pancreatic cells (Barg, S., Ma, X., Elliasson, L., Galvanovskis, J., Gopel, S. O., Obermuller, S., Platzer, J., Renstrom, E., Trus, M., Atlas, D., Streissnig, G., and Rorsman, P. (2001). Biophys. J.; Wiser, O., Bennett, M. K., and Atlas, D. (1996). EMBO J.
15:4100–4110; Wiser, O., Trus, M., Hernandez, A., Renström, E., Barg, S., Rorsman, P., and Atlas, D. (1999). Proc. Natl. Acad. Sci. U.S.A.
96:248–253). The kinetics of Cav1.2 (Lc-type) and Cav2.2 (N-type) Ca2+ channels were modified in oocytes injected with cRNA encoding syntaxin 1A and SNAP-25. Conserved cysteines (Cys271, Cys272) within the syntaxin 1A transmembrane domain are essential. Synaptotagmin I, a vesicle-associated protein, accelerated the activation kinetics indicating Cav2.2 coupling to the vesicle. The unique modifications of Cav1.2 and Cav2.2 kinetics by syntaxin 1A, SNAP-25, and synaptotagmin combined implied excitosome formation, a primed fusion complex of the channel with synaptic proteins. The Cav1.2 cytosolic domain Lc753–893, acted as a dominant negative modulator, competitively inhibiting insulin release of channel-associated vesicles (CAV), the readily releasable pool of vesicles (RRP) in islet cells. A molecular mechanism is offered to explain fast secretion of vesicles tethered to SNAREs-associated Ca2+ channel. The tight arrangement facilitates the propagation of conformational changes induced during depolarization and Ca2+-binding at the channel, to the SNAREs to trigger secretion. The results imply a rapid Ca2+-dependent CAV (RRP) release, initiated by the binding of Ca2+ to the channel, upstream to intracellular Ca2+ sensor thus establishing the Ca2+ channel as the Ca2+ sensor of neurotransmitter release. 相似文献
6.
NAADP mobilizes Ca2+ from a thapsigargin-sensitive store in the nuclear envelope by activating ryanodine receptors 总被引:3,自引:0,他引:3
Gerasimenko JV Maruyama Y Yano K Dolman NJ Tepikin AV Petersen OH Gerasimenko OV 《The Journal of cell biology》2003,163(2):271-282
Ca2+ release from the envelope of isolated pancreatic acinar nuclei could be activated by nicotinic acid adenine dinucleotide phosphate (NAADP) as well as by inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose (cADPR). Each of these agents reduced the Ca2+ concentration inside the nuclear envelope, and this was associated with a transient rise in the nucleoplasmic Ca2+ concentration. NAADP released Ca2+ from the same thapsigargin-sensitive pool as IP3. The NAADP action was specific because, for example, nicotineamide adenine dinucleotide phosphate was ineffective. The Ca2+ release was unaffected by procedures interfering with acidic organelles (bafilomycin, brefeldin, and nigericin). Ryanodine blocked the Ca2+-releasing effects of NAADP, cADPR, and caffeine, but not IP3. Ruthenium red also blocked the NAADP-elicited Ca2+ release. IP3 receptor blockade did not inhibit the Ca2+ release elicited by NAADP or cADPR. The nuclear envelope contains ryanodine and IP3 receptors that can be activated separately and independently; the ryanodine receptors by either NAADP or cADPR, and the IP3 receptors by IP3. 相似文献
7.
Sujata Misra Kshudiram Naskar Dwijen Sarkar Dilip Kumar Ghosh 《Molecular and cellular biochemistry》1991,102(1):13-18
Summary
Leishmania donovani, the etiological agent for the disease visceral leishmaniasis, attach themselves to the macrophages for initiation of the disease. The attachment process has been found to be regulated by Ca2+ ions. Verapamil, a Ca2+-channel blocker inhibits Leishmania-macrophage attachment. The inhibitory effect is increased with time. Nifedipine, another Ca2+-channel blocker exhibits the same effect. The attachment process is stimulated by Ca2+-ionophore alone. The inhibitory effects of the calcium channel blockers are reversed by the ionophore. 相似文献
8.
K. Narita T. Akita M. Osanai T. Shirasaki H. Kijima K. Kuba 《The Journal of general physiology》1998,112(5):593-609
The extent to which Ca2+-induced Ca2+ release (CICR) affects transmitter release is unknown. Continuous nerve stimulation (20–50 Hz) caused slow transient increases in miniature end-plate potential (MEPP) frequency (MEPP-hump) and intracellular free Ca2+ ([Ca2+]i) in presynaptic terminals (Ca2+-hump) in frog skeletal muscles over a period of minutes in a low Ca2+, high Mg2+ solution. Mn2+ quenched Indo-1 and Fura-2 fluorescence, thus indicating that stimulation was accompanied by opening of voltage-dependent Ca2+ channels. MEPP-hump depended on extracellular Ca2+ (0.05–0.2 mM) and stimulation frequency. Both the Ca2+- and MEPP-humps were blocked by 8-(N,N-diethylamino)octyl3,4,5-trimethoxybenzoate hydrochloride (TMB-8), ryanodine, and thapsigargin, but enhanced by CN−. Thus, Ca2+-hump is generated by the activation of CICR via ryanodine receptors by Ca2+ entry, producing MEPP-hump. A short interruption of tetanus (<1 min) during MEPP-hump quickly reduced MEPP frequency to a level attained under the effect of TMB-8 or thapsigargin, while resuming tetanus swiftly raised MEPP frequency to the previous or higher level. Thus, the steady/equilibrium condition balancing CICR and Ca2+ clearance occurs in nerve terminals with slow changes toward a greater activation of CICR (priming) during the rising phase of MEPP-hump and toward a smaller activation during the decay phase. A short pause applied after the end of MEPP- or Ca2+-hump affected little MEPP frequency or [Ca2+]i, but caused a quick increase (faster than MEPP- or Ca2+-hump) after the pause, whose magnitude increased with an increase in pause duration (<1 min), suggesting that Ca2+ entry-dependent inactivation, but not depriming process, explains the decay of the humps. The depriming process was seen by giving a much longer pause (>1 min). Thus, ryanodine receptors in frog motor nerve terminals are endowed with Ca2+ entry-dependent slow priming and fast inactivation mechanisms, as well as Ca2+ entry-dependent activation, and involved in asynchronous exocytosis. Physiological significance of CICR in presynaptic terminals was discussed. 相似文献
9.
The regulation of insulin secretion from RINm5F cells exposed to high voltage discharge has been investigated. Electron microscopy revealed that the overall structure of the cells was preserved after permeabilization. In this preparation insulin release was stimulated by Ca2+ (EC50=2.4 M). The stable GTP analogue GTPS enhanced secretion both at intermediate (nano- to micromolar) and vanishingly low (<10 pM) Ca2+ concentrations. At optimal Ca2+ (10 M) the effect of GTPS was greatly reduced. We investigated whether the secretory response to GTP analogues was mediated by any of three enzyme systems regulated by GTP-binding proteins, i.e. generation of cyclic AMP by adenylate cyclase, of diacylglycerol by phospholipase C and of arachidonic acid by phospholipase A2. The involvement of these messenger systems could be excluded as (i) cyclic AMP only had minor, Ca2+ dependent effects, (ii) phospholipase C was not activated in the absence of Ca2+ and insulin secretion due to the phorbol ester TPA displayed a different Ca2+ dependency, (iii) arachidonic acid did not elicit Ca2+ independent insulin secretion. These results, taken together with the finding that insulin secretion due to Ca2+ or TPA is attenuated by the inhibitory guanine nucleotide GDPS, suggest the existence of a regulatory site in exocytosis which is sensitive to guanine nucleotides.Abbreviations InsP3
inositol trisphosphate
- Ptd-InsP2
phosphatidylinositol 4,5-bisphosphate
- GTPS
guanosine 5-(3-O-thio)triphosphate
- GDPS
guanosine 5-(2-O-thio)diphosphate
- Gpp(NH)p
guanyl-5-yl imidodiphosphate
- TPA
12-O-tetradecanoylphorbol-13-acetate
- OAG
1-oleoyl-2-acetylglycerol
- Hepes
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- EGTA
(ethylenebis(oxyethylenenitrilo)tetraacetic acid
- DAG
diacylglycerol
- [Ca2+]i
cytosolic free Ca2+ concentration 相似文献
10.
G. Thiel 《Plant biology (Stuttgart, Germany)》1991,104(5):345-354
The effect of the membrane impermeant sulfhydryl group (SH) reagent, p-chloromercuribenzenesulfonic acid (p-CMBS), on electrical membrane transport properties of the giant alga, Chara corallina, was determined. In an external medium with a high K+ concentration (5 mM) cells typically exhibited stable membrane potentials close to the K+equilibrium potential. The steady-state current-voltage (I-V) relation could be dissected into two distinct components: an almost linear ohmic leak current and a voltage-dependent K+ current. Adding 0.5 mM p-CMBS to the external medium resulted in an immediate, short depolarization transient (resembling the time course of an action potential) and was associated with a slow down of the cytoplasmic streaming velocity. The depolarization, as well as the streaming inhibition, could be abolished by pretreating cells with the Ca2+ channel inhibitor, LaCl3. This suggests that the depolarization transient reflected a p-CMBS induced Ca2+ influx, a scenario known to trigger membrane excitation and slow down of cytoplasmic streaming. From the I-V analysis it appeared that p-CMBS also caused a reversible inhibition of two additional transmembrane currents: (1) a reduction of a leak current and (2) a modification of the deactivation kinetics of the voltage-dependent K+ channels. From the I-V difference analysis, the inhibited leak current was identified as a K+ current, because the reversal potential was close to the estimated K+ equilibrium potential. Control experiments have furthermore shown that the mercapto reagent, dithiothreitol, partly reversed the effect of p-CMBS. This strengthens the view that the action of the mercurial is related to a specific and direct modification of SH groups. The p-CMBS-evoked inhibition of K+ currents was not abolished by the LaCl3 pretreatment, which suggests that the effect of the SH reagent is not induced indirectly by p-CMBS-triggered Ca2+ influx. Therefore, it is suggested that the mercurial interacts direcly with the K+ transport protein. 相似文献
11.
Sanyal S Jennings T Dowse H Ramaswami M 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2006,176(3):253-263
To analyze the role of cytosolic calcium in regulating heart beat frequency and rhythm, we studied conditional mutations in
Drosophila Sarco-endoplasmic reticulum Ca2+-ATPase, believed to be predominantly responsible for sequestering free cytosolic calcium. Abnormalities in the amount or
structure of the SERCA protein have been linked to cardiac malfunction in mammals. Drosophila SERCA protein (dSERCA) is highly enriched in Drosophila larval heart with a distinct membrane distribution of SERCA at cardiac Z-lines, suggesting evolutionarily conserved zones
for calcium uptake into the sarcoplasmic reticulum. Heart beat frequency is strikingly reduced in mutant animals following
dSERCA inactivation, (achieved by a brief exposure of these conditional mutants to non-permissive temperature). Cardiac contractions
also show abnormal rhythmicity and electrophysiological recordings from the heart muscle reveal dramatic alterations in electrical
activity. Overall, these studies underscore the utility of the Drosophila heart to model SERCA dysfunction dependent cardiac disorders and constitute an initial step towards developing Drosophila as a viable genetic model system to study conserved molecular determinants of cardiac physiology. 相似文献
12.
The regulation of cytosolic Ca2+ has been investigated in growing root-hair cells of Sinapis alba L. with special emphasis on the role of the plasmamembrane Ca2+-ATPase. For this purpose, erythrosin B was used to inhibit the Ca2+-ATPase, and the Ca2+ ionophore A23187 was applied to manipulate cytosolic free [Ca2+] which was then measured with Ca2+-selective microelectrodes. (i) At 0.01 M, A23187 had no effect on the membrane potential but enhanced the Ca2+ permeability of the plasma membrane. Higher concentrations of this ionophore strongly depolarized the cells, also in the presence of cyanide. (ii) Unexpectedly, A23187 first caused a decrease in cytosolic Ca2+ by 0.2 to 0.3 pCa units and a cytosolic acidification by about 0.5 pH units, (iii) The depletion of cytosolic free Ca2+ spontaneously reversed and became an increase, a process which strongly depended on the external Ca2+ concentration, (iv) Upon removal of A23187, the cytosolic free [Ca2+] returned to its steady-state level, a process which was inhibited by erythrosin B. We suggest that the first reaction to the intruding Ca2+ is an activation of Ca2+ transporters (e.g. ATPases at the endoplasmic reticulum and the plasma membrane) which rapidly remove Ca2+ from the cytosol. The two observations that after the addition of A23187, (i) Ca2+ gradients as steep as-600 mV could be maintained and (ii) the cytosolic pH rapidly and immediately decreased without recovery indicate that the Ca2+-exporting plasma-membrane ATPase is physiologically connected to the electrochemical pH gradient, and probably works as an nH+/Ca2+-ATPase. Based on the finding that the Ca2+-ATPase inhibitor erythrosin B had no effect on cytosolic Ca2+, but caused a strong Ca2+ increase after the addion of A23187 we conclude that these cells, at least in the short term, have enough metabolic energy to balance the loss in transport activity caused by inhibition of the primary Ca2+-pump. We further conclude that this ATPase is a major Ca2+ regulator in stress situations where the cytosolic Ca2+ has been shifted from its steady-state level, as may be the case during processes of signal transduction.Abbreviations and Symbols EB
erythrosin B
- Em
membrane potential
- pCa
negative logarithm of the Ca2+ concentration
This work was supported by the Deutche Forschungsgemeinschaft (H.F.) and the Alexander-von-Humboldt-Foundation (A.T.). 相似文献
13.
Thylakoids and Photosystem II particles prepared from the cyanobacterium Synechococcus PCC 7942 washed with a HEPES/glycerol buffer exhibited low rates of light-induced oxygen evolution. Addition of either Ca2+ or Mg2+ to both thylakoids and Photosystem II particles increased oxygen evolution independently, maximal rates being obtained by addition of both ions. If either preparation was washed with NaCl, light induced O2 evolution was completely inhibited, but re-activated in the same manner by Ca2+ and Mg2+ but to a lower level. In the presence of Mg2+, the reactivation of O2 evolution by Ca2+ allowed sigmoid kinetics, implying co-operative binding. The results are interpreted as indicating that not only Ca2+, but also Mg2+, is essential for light-induced oxygen evolution in thylakoids and Photosystem II particles from Synechococcus PC 7942. The significance of the reactivation kinetics is discussed. Reactivation by Ca2+ was inhibited by antibodies to mammalian calmodulin, indicating that the binding site in Photosystem II may be analogous to that of this protein.Abbreviation HEPES
n-2-Hydroxyethylpiperazine--2-ethane sulphonic acid 相似文献
14.
Supplementation with CaCl22H2O (40 mg l–1) or CuSO45H2O (10 mg l–1) improved vancomycin production by Amycolatopsis orientalis by 12 and 11%, respectively; used in combination, they acted synergistically. Ca2+ decreased the intracellular concentration of vancomycin 36%, whereas Cu2+ increased the intracellular activity of TDP-glucose:aglycosylvancomycin glucosyltransferase 3 times more than control. Ca2+ probably works by altering the permeability of cells to vancomycin, whereas Cu2+ increases the activity of an enzyme responsible for vancomycin biosynthesis. 相似文献
15.
Summary Veratridine opens voltage-dependent Na+ channels in many metazoans. InParamecium, which has voltage-dependent Ca2+ channels and a Ca/K action potential, no such Na+ channels are known. A Ca-inward current is correlated to an intracellular increase in cGMP. The addition of veratridine toParamecium wildtype and to pawn mutant cells, which lack the Ca-inward current, transiently increased intracellular levels of cGMP about sevenfold to 40 pmol/mg protein. A half-maximal effect was obtained with 250 m veratridine. The increase in cGMP was maximal about 15 sec after the addition of veratridine and declined rapidly afterwards. Intracellular cAMP levels were not affected. The effect of veratridine on cGMP was dependent on the presence of extracellular Ca2+. The time dependence and extent of stimulation closely resembled the effects observed after stimulation by Ba2+, which causes the repetitive firing of action potentials, Ca-dependent ciliary reversal, and cGMP formation. The effects of Ba2+ and veratridine were not additive. Wildtype cells and, surprisingly, also pawn mutant cells showed avoiding reactions upon addition of veratridine indicating that it induced a Ca2+ influx into the cilia, which causes ciliary reversal. The potency of veratridine to stimulate cGMP formation was little affected by Na+ in wildtype cells, three pawn mutant strains, and in the cell line fast-2, which is defective in a Ca-dependent Na-inward current. Divalent cations (Ca2+, Mg2+, and Ba2+) inhibited the effects the veratridine similar to metazoan cells. The results indicate that veratridine can open the voltage-operated Ca2+ channels inParamecium wildtype and, most interestingly, in pawn mutant cells. The pawn mutation is suggested to represent a defect in the activation of the Ca2+ channel. This explains the lack of differences in ciliary proteins between wildtype and pawn cells reported earlier. 相似文献
16.
We investigated signal transduction between receptor-operated Ca(2+) influx (ROCI) and Src-related nonreceptor protein tyrosine kinase (PTK) in rat pancreatic acini. CCK and the Ca(2+) ionophore enhanced the Src-related PTK activity, whereas the high-affinity CCK-A receptor agonists, fibroblast growth factor (FGF), and the protein kinase C (PKC) activator had no or little effect. This increase was abolished by eliminating [Ca(2+)](o), loading of the intracellular Ca(2+) chelator, and administering the PTK inhibitor genistein. While genistein inhibited extracellular Ca(2+) or Mn(2+) entry induced by CCK and carbachol, it did not affect intracellular Ca(2+) release and oscillations. CCK dose-dependently increased the Src phosphotransferase activity, which was abolished by inhibitors of G(q) protein, phospholipase C (PLC), and Src, but not by the calmodulin kinase (CaMK) inhibitor. Intensities of the Src band and amounts of tyrosine phosphorylated Src were enhanced by CCK stimulation. Thus, Src cascades appear to be coupled to the low-affinity CCK-A receptor and utilize G(q)-PLC pathways for their activation, independent of PKC and CaMK cascades. The low-affinity CCK-A receptor regulates ROCI via mediation of Src-related PTK and activates Src pathways to cause [Ca(2+)](o)-dependent pancreatic exocytosis. 相似文献
17.
Ca2+ levels in plants are controlled in part by H+/Ca2+ exchangers. Structure/function analysis of the Arabidopsis H+/cation exchanger, CAX1, revealed that a nine amino acid region (87–95) is involved in CAX1-mediated Ca2+ specificity. CAX3 is 77% identical (93% similar) to CAX1, and when expressed in yeast, localizes to the vacuole but does not suppress yeast mutants defective in vacuolar Ca2+ transport. Transgenic tobacco plants expressing CAX3 containing the 9 amino acid Ca2+ domain (Cad) from CAX1 (CAX3-9) displayed altered stress sensitivities similar to CAX1-expressing plants, whereas CAX3-9-expressing plants did not have any altered stress sensitivities. A single leucine-to-isoleucine change at position 87 (CAX3-I) within the Cad of CAX3 allows this protein to weakly transport Ca2+ in yeast (less than 10% of CAX1). Site-directed mutagenesis of the leucine in the CAX3 Cad demonstrated that no amino acid change tested could confer more activity than CAX3-I. Transport studies in yeast demonstrated that the first three amino acids of the CAX1 Cad could confer twice the Ca2+ transport capability compared to CAX3-I. The entire Cad of CAX3 (87–95) inserted into CAX1 abolishes CAX1-mediated Ca2+ transport. However, single, double, or triple amino acid replacements within the native CAX1 Cad did not block CAX1 mediated Ca2+ transport. Together these findings suggest that other domains within CAX1 and CAX3 influence Ca2+ transport. This study has implications for the ability to engineer CAX-mediated transport in plants by manipulating Cad residues. 相似文献
18.
Some features of the Ca2+-transport system in mitochondria of the yeast Endomyces magnusii are considered. The Ca2+ uniporter was shown to be specifically activated by low concentrations of physiological modulators such as ADP, NADH, spermine, and Ca2+ itself. The Na+-independent system responsible for Ca2+ release from Ca2+-preloaded yeast mitochondria was characterized. The rate of the Ca2+ release was proportional to the Ca2+ load, insensitive to cyclosporin A and to Na+, inhibited by La3+, TPP+, Pi, and nigericin, while being activated by spermine. We conclude that Ca2+ release from preloaded E. magnusii yeast mitochondria is mediated by a Na+-independent pathway, very similar to that in mitochondria from nonexcitable mammalian tissues. A scheme describing an arrangement of the Ca2+ transport system of yeast mitochondria is proposed. 相似文献
19.
Yukiharu Hiramatsu Bruce J. Baum Indu S. Ambudkar 《The Journal of membrane biology》1992,129(3):277-286
This study examines the activation of divalent cation entry into rat parotid gland acinar cells by using Mn2+ as a Ca2+ surrogate cation. Following muscarinic-cholinergic stimulation of dispersed parotid acini with carbachol (10 microM), the onset of internal Ca2+ release (cytosolic [Ca2+], [Ca2+]i, increase) and the stimulation of Mn2+ entry (increase in fura2 quenching) are not simultaneously detected. [Ca2+]i elevation, due to intracellular release, is detected almost immediately following carbachol addition and peak [Ca2+]i increase occurs at 6.0 +/- 0.8 sec. However, there is an interval (apparent lag) between carbachol addition and the detection of stimulated Mn2+ entry. This apparent lag is decreased from 26 +/- 3.1 sec to 9.2 +/- 1.5 sec when external Mn2+ ([Mn2+]0) is increased from 12.5 to 500 microM. It is not decreased further with increase in [Mn2+]0 from 500 microM to 1 mM (9.8 +/- 2.1 sec), although both intracellular free Mn2+ and [Mn2+-fura2]/[fura2] increase. Thus, at [Mn2+]0 < 500 microM, the observed lag time is partially due to a limitation in the magnitude of Mn2+ entry. Furthermore, neither peak [Ca2+]i nor the time required to reach peak [Ca2+]i is significantly altered by [Mn2+]0 (12.5 microM to 1 mM). At every [Mn2+]0 tested (i.e., 12.5 microM-1 mM), the apparent lag is significantly greater than the time required to reach peak [Ca2+]i. However, when carbachol stimulation of the [Ca2+]i increase is attenuated by loading the acini with the Ca2+ chelator, 2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA), there is no detectable lag in carbachol stimulation of Mn2+ entry (with 1 mM [Mn2+]0). Importantly, in BAPTA-loaded acini, carbachol stimulates Mn2+ entry via depletion of the internal Ca2+ pool and not via direct activation of other divalent cation entry mechanisms. Based on these results, we suggest that the apparent lag in the detection of carbachol stimulation of Mn2+ entry into parotid acinar cells is due to a retardation of Mn2+ entry by the initial increase in [Ca2+]i, due to internal release, which most likely occurs proximate to the site of divalent cation entry. 相似文献
20.
Shmuel Muallem Timothy Beeker Stephen J. Pandol 《The Journal of membrane biology》1988,102(2):153-162
Summary The relative contributions of the Na+/Ca2+ exchange and the plasma membrane Ca2+ pump to active Ca2+ efflux from stimulated rat pancreatic acini were studied. Na+ gradients across the plasma membrane were manipulated by loading the cells with Na+ or suspending the cells in Na+-free media. The rates of Ca2+ efflux were estimated from measurements of [Ca2+]
i
using the Ca2+-sensitive fluorescent dye Fura 2 and45Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca2+ efflux is described by a single exponential function under control, Na+-loaded, and Na+-depleted conditions. Manipulation of Na+ gradients had no effect on the hormone-induced increase in [Ca2+]
i
. The results indicate that Ca2+ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca2+ pump. The effects of several cations, which were used to substitute for Na+, on cellular activity were also studied. Choline+ and tetramethylammonium+ (TMA+) released Ca2+ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na+, TMA+, choline+, or N-methyl-d-glucamine+ (NMG+) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG+, choline+, and TMA+ inhibited amylase release by 55, 40 and 14%, respectively. NMG+ also increased the Ca2+ permeability of the plasma membrane. Thus, to study Na+ dependency of cellular function, TMA+ is the preferred cation to substitute for Na+. The stimulatory effect of TMA+ can be blocked by atropine. 相似文献