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T Lockwich

Publications and source records attributed to T Lockwich.

12 recordsLinked to original sources
PubMed2001

Stabilization of cortical actin induces internalization of transient receptor potential 3 (Trp3)-associated caveolar Ca2+ signaling complex and loss of Ca2+ influx without disruption of Trp3-inositol trisphosphate receptor association.

Ca(2+) influx via plasma membrane Trp3 channels is proposed to be regulated by a reversible interaction with inositol trisphosphate receptor (IP(3)R) in the endoplasmic reticulum. Condensation of the cortical actin layer has been suggested to physically disrupt this interaction and inhibit Trp3-mediated Ca(2+) influx. This study examines the effect of cytoskeletal reorganization on the localization and function of Trp3 and key Ca(2+) signaling proteins. Calyculin-A treatment resulted in formation of condensed actin layer at the plasma membrane; internalization of Trp3, Galpha(q/11), phospholipase Cbeta, and caveolin-1; and attenuation of 1-oleoyl-2-acetyl-sn-glycerol- and ATP-stimulated Sr(2+) influx. Importantly, Trp3 and IP(3)R-3 remained co-localized inside the cell and were co-immunoprecipitated. Jasplakinolide also induced internalization of Trp3 and caveolin-1. Pretreatment of cells with cytochalasin D or staurosporine did not affect Trp3 but prevented calyculin-A-induced effects. Based on these data, we suggest that Trp3 is assembled in a caveolar Ca(2+) signaling complex with IP(3)R, SERCA, Galpha(q/11), phospholipase Cbeta, caveolin-1, and ezrin. Furthermore, our data demonstrate that conditions which stabilize cortical actin induce loss of Trp3 activity due to internalization of the Trp3-signaling complex, not disruption of IP(3)R-Trp3 interaction. This suggests that localization of the Trp3-associated signaling complex, rather than Trp3-IP(3)R coupling, depends on the status of the actin cytoskeleton.

Actins↗
PubMed2000

Trp1, a candidate protein for the store-operated Ca(2+) influx mechanism in salivary gland cells.

The trp gene family has been proposed to encode the store-operated Ca(2+) influx (SOC) channel(s). This study examines the role of Trp1 in the SOC mechanism of salivary gland cells. htrp1, htrp3, and Trp1 were detected in the human submandibular gland cell line (HSG). HSG cells stably transfected with htrp1alpha cDNA displayed (i) a higher level of Trp1, (ii) a 3-5-fold increase in SOC (thapsigargin-stimulated Ca(2+) influx), determined by [Ca(2+)](i) and Ca(2+)-activated K(+) channel current measurements, and (iii) similar basal Ca(2+) permeability, and inhibition of SOC by Gd(3+) but not by Zn(2+), as compared with control cells. Importantly, (i) transfection of HSG cells with antisense trp1alpha cDNA decreased endogenous Trp1 level and significantly attenuated SOC, and (ii) transfection of HSG cells with htrp3 cDNA did not increase SOC. These data demonstrate an association between Trp1 and SOC and strongly suggest that Trp1 is involved in this mechanism in HSG cells. Consistent with this suggestion, Trp1 was detected in the plasma membrane region, the proposed site of SOC, of acinar and ductal cells in intact rat submandibular glands. Based on these aggregate data, we propose Trp1 as a candidate protein for the SOC mechanism in salivary gland cells.

Animals↗
PubMed1995

Reconstitution of a passive Ca(2+)-transport pathway from the basolateral plasma membrane of rat parotid gland acinar cells.

We have previously reported that rat parotid gland basolateral plasma membrane vesicles (BLMV) have a relatively high affinity Ca2+ transport pathway and an unsaturable Ca2+ flux component (Lockwich et al., 1994. J. Membrane Biol. 141:289-296). In this study, we have solubilized BLMV with octylglucoside (1.5%) and have reconstituted the solubilized proteins into proteoliposomes (PrL) composed of E. coli bulk phospholipids, by using a detergent dilution method. PrL exhibited 3-5-fold higher 45Ca2+ influx than control liposomes (without protein). Ca2+ uptake into PrL was dependent on the [protein] in PrL and steady state [Ca2+] in PrL was in equilibrium with external [Ca2+]. These data demonstrate that a passive, protein-mediated Ca2+ transport has been reconstituted from BLMV into PrL. 45Ca2+ influx into liposomes did not saturate with increasing [Ca2+] in the assay medium. In contrast, PrL displayed saturable 45Ca2+ influx and exhibited a single Ca2+ flux component with an apparent KCa = 242 +/- 50.9 microM and Vmax = 13.5 +/- 1.14 nmoles Ca2+/mg protein/ minute. The KCa of Ca(2+)-transport in PrL was similar to that of the high affinity Ca2+ influx component in BLMV while the Vmax was about 4-fold higher. The unsaturable Ca2+ flux component was not detected in PrL. 45Ca2+ influx in PrL was inhibited by divalent cations in the order of efficacy, Zn2+ > Mn2+ > Co2+ = Ni2+, and appeared to be more sensitive to lower concentrations of Zn2+ than in BLMV. Consistent with our observations with BLMV, the carboxyl group reagent N,N'-dicyclohexylcarbodiimide (DCCD) inhibited the reconstituted Ca2+ transport in PrL. Importantly, in both BLMV and PrL, DCCD induced a 40-50% decrease in Vmax of Ca2+ transport without an alteration in KCa. These data strongly suggest that the high affinity, passive Ca2+ transport pathway present in BLMV has been functionally reconstituted into PrL. We suggest that this approach provides a useful experimental system towards isolation of the protein(s) involved in mediating Ca2+ influx in the rat parotid gland basolateral plasma membrane.

Animals↗
PubMed1993

Involvement of carboxyl groups in the divalent cation permeability of rat parotid gland basolateral plasma membrane.

Divalent cation permeability of rat parotid gland basolateral plasma membranes was examined in dispersed parotid acini (by Ca2+ or Mn2+ entry) and in isolated basolateral plasma membrane vesicles (BLMV, by 45Ca2+ influx). Mn2+ entry (fura2 quenching) was about 1.6 fold higher in internal Ca2+ pool-depleted acini (Ca(2+)-depl acini) than in unstimulated cells. Mn2+ entry into Ca(2+)-depl acini was increased at external pH > 7.4 and decreased at pH < 7.4. Pretreatment of Ca(2+)-depl acini with the relatively hydrophobic carboxylic group reagent, N,N'-dicyclohexylcarbodiimide (DCCD, 50 microM for 30 min) resulted in the inhibition of Mn2+ entry into Ca(2+)-depl acini to unstimulated levels. Another hydrophobic carboxyl group reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and the relatively hydrophilic carboxyl group reagents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMCD) did not affect Mn2+ entry. Similar to the effects in intact acini, Ca2+ influx into BLMV was decreased when the external pH was lowered below 7.4. Also DCCD (5 mM, 30 min), but not EEDQ, decreased (40%) Ca2+ influx in BLMV. However, unlike in acini, the hydrophilic reagents, EDC, EAC, and CMCD decreased Ca2+ permeability in BLMV and the effects were nonadditive with the decrease induced by DCCD. The aggregate effects of carboxyl group reagents on the Ca2+ and Mn2+ permeability in BLMV and intact acini, respectively, suggest that a critical carboxyl group (most likely accessible from the cytoplasmic side of the plasma membrane) is involved in divalent cation flux in rat parotid acinar cells.

Animals↗
PubMed1991

Uncoupling of Ca2+ transport from ATP hydrolysis activity of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.

In reconstituted rabbit skeletal muscle (Ca2+ + Mg2+)-ATPase proteoliposomes, Ca(2+)-uptake is decreased by more than 90% with T2 cleavage (Arg-198). However, no difference in the ATP dependence of hydrolysis activity is seen between SR and trypsin-treated SR. A large decrease in E-P formation and hydrolysis activity of the enzyme appear only at T3 cleavage, which represents the cleavage of A1 fragment to A1a + A1b forms. The disappearance of hydrolysis activity due to digestion is prior to the disappearance of E-P formation. No significant difference is found in the passive Ca2+ efflux between control SR and tryptically digested SR in the absence of Mg2+ + ruthenium red or in the presence of ATP. However, the passive Ca2+ efflux rate for tryptically digested SR is much larger than control SR in the presence of Mg2+ + ruthenium red. These results show that the Ca2+ channel cannot be closed after trypsin digestion of SR membranes by the presence of the Ca2+ channel inhibitors, Mg2+ and ruthenium red. In the reconstituted proteoliposomes, the Ca2+ efflux rates are the same regardless of digestion (T2); also, efflux is not affected by the presence or absence of Mg2+ + ruthenium red. These results indicate that T2 cleavage causes 'uncoupling' of the 'Ca(2+)-pump' from ATP hydrolytic activity. A theoretical model is developed in order to fit the extent of tryptic digestion of the A fragment of the (Ca2+ + Mg2+)-ATPase polypeptide with the loss of Ca(2+)-transport. Fits of the theoretical equations to the data are consistent with that Ca(2+)-transport system appears to require a dimer of the polypeptide (Ca2+ + Mg2+)-ATPase.

Adenosine Triphosphate↗
PubMed1990

Tertiary structure and energy coupling in Ca2(+)-pump system.

Europium luminescence from europium bound to sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase indicates that there are two high affinity calcium binding sites. Furthermore, the two calcium ions at the binding sites are highly coordinated by the protein as the number of H2O molecules surrounding the Ca2+ ions are 3 and 0.5. In the presence of ATP, calcium ions are occluded even further down to 2 and zero H2O molecules, respectively. The Ca2+ - Ca2+ intersite distance is estimated to be 8-9 A and the average distance from the Ca2+ sites to CrATP is about 18 A. Digestion of the (Ca2+ + Mg2+)-ATPase at the T2 site (Arg 198) causes uncoupling of Ca2(+)-transport from ATPase activity while calcium occlusion due to E1-P formation remains unchanged. Further tryptic digestion beyond T2 and in the presence of ATP diminishes Ca2+ occlusion to zero while 50% of the ATPase hydrolytic activity remains. Tryptic digestion beyond T2 and in the absence of ATP diminishes ATPase hydrolytic activity to 50% of normal while Ca2+ occlusion remains intact. These data are consistent with a mechanism in which the functional enzyme must be in the dimeric form for occlusion and calcium uptake to occur, but each monomer can hydrolyze ATP.

Adenosine Triphosphate↗
PubMedDate not supplied

Regulation of cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.

The two high affinity calcium binding sites of the cardiac (Ca2+ + Mg2+)-ATPase have been identified with the use of Eu3+. Eu3+ competes for the two high affinity calcium sites on the enzyme. With the use of laser-pulsed fluorescent spectroscopy, the environment of the two sites appear to be heterogeneous and contain different numbers of H2O molecules coordinated to the ion. The ion appears to be occluded even further in the presence of ATP. Using non-radiative energy transfer studies, we were able to estimate the distance between the two Ca2+ sites to be between 9.4 to 10.2 A in the presence of ATP. Finally, from the assumption that the calcium site must contain four carboxylic side chains to provide the 6-8 ligands needed to coordinate calcium, and based on our recently published data, we predict the peptidic backbone of the two sites.

Adenosine Triphosphate↗
PubMedDate not supplied

Ca2+ permeability of rat parotid gland basolateral plasma membrane vesicles is modulated by membrane potential and extravesicular [Ca2+].

This study examines the Ca2+ permeability of basolateral plasma membrane vesicles (BLMVs) isolated from the rat parotid gland by monitoring the rate of 45Ca2+ efflux from actively-loaded (via the Ca(2+)-ATPase) inside-out BLMVs. Ca2+ efflux from BLMVs into a K(+)-gluconate medium which hyperpolarizes the cytoplasmic side (i.e. outside) of the inside-out BLMVs resulted in a faster rate of Ca2+ efflux compared with a control medium containing N-methyl-D-glucamine (NMDG)-gluconate. Conversely, Ca2+ efflux into a medium which depolarizes the cytoplasmic side of the BLMVs (NMDG-chloride) resulted in slower rates of efflux compared with those observed with the control medium. This increased rate of 45Ca2+ efflux from the hyperpolarized BLMV was inhibited by 1 mM Ni2+, yielding a rate of efflux similar to the rate observed in depolarized BLMVs. The rate of Ca2+ efflux from BLMVs was affected by [Ca2+]o ([Ca2+] on the extravesicular, cytoplasmic side of the vesicle). When [Ca2+]o was kept > 200 nM during efflux, the rate of Ca2+ efflux from both hyper- and depolarized BLMVs was slow and relatively unresponsive to changes in [Ca2+]o, despite sizeable changes in the Ca2+ gradient across the BLMV. However, when [Ca2+]o was lowered < 200 nM, there was an abrupt increase in the rate of Ca2+ efflux from both hyper- and depolarized BLMVs. Additionally, when [Ca2+] was < 200 nM, the rate of Ca2+ efflux appeared to be more sensitive to driving force changes. These data suggest that Ca2+ permeability across the rat parotid gland basolateral plasma membrane is modulated by membrane potential and [Ca2+] on the cytoplasmic side.

Animals↗
PubMedDate not supplied

Uncoupling of occlusion from ATP hydrolysis activity in sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.

The uncoupling of Ca2+ transport from ATP hydrolysis in the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by trypsin digestion was re-investigated by comparing ATPase activity with the ability of the enzyme to occlude Eu3+ (a transport parameter) after various tryptic digests. With this method, re-examination of uncoupling by tryptic digest of the ATPase revealed that TD2 cleavage (Arg-198) had no effect on either occlusion or ATPase activity. Digestion past TD2 in the presence of 5 mM Ca2+ and at 25 degrees C resulted in the loss of about 70% of the ATPase activity, but no loss of occlusion. Digestion past TD2 in the presence of 5 mM Ca2+, 3 mM ATP, and at 25 degrees C resulted in a partially uncoupled enzyme complex which retained about 50% of the ATPase activity, but completely lost the ability to occlude Eu3+. Digest past TD2 in the presence of 5 mM Ca2+ and 3 mM AMP-PNP (a non-hydrolyzable ATP analog) at 25 degrees C resulted in no loss of occlusion, thus revealing the absolute requirement of ATP during the digest to eliminate occlusion. From these findings we conclude that uncoupling of Ca2+ transport from ATPase activity is possible by tryptic digestion of the (Ca2+ + Mg2+)-ATPase. Interestingly, only after phosphorylation of the enzyme do the susceptible bond(s) which lead to the loss of occlusion become exposed to trypsin.

Adenosine Triphosphate↗