Lipids as cofactors in protein folding: stereo-specific lipid-protein interactions are required to form HAMLET (human alpha-lactalbumin made lethal to tumor cells)

Proteins can adjust their structure and function in response to shifting environments. Functional diversity is created not only by the sequence but by changes in tertiary structure. Here we present evidence that lipid cofactors may enable otherwise unstable protein folding variants to maintain their conformation and to form novel, biologically active complexes. We have identified unsaturated C18 fatty acids in the cis conformation as the cofactors that bind apo alpha-lactalbumin and form HAMLET (human alpha-lactalbumin made lethal to tumor cells). The complexes were formed on an ion exchange column, were stable in a molten globule-like conformation, and had attained the novel biological activity. The protein-fatty acid interaction was specific, as saturated C18 fatty acids, or unsaturated C18:1trans conformers were unable to form complexes with apo alpha-lactalbumin, as were fatty acids with shorter or longer carbon chains. Unsaturated cis fatty acids other than C18:1:9cis were able to form stable complexes, but these were not active in the apoptosis assay. The results demonstrate that stereo-specific lipid-protein interactions can stabilize partially unfolded conformations and form molecular complexes with novel biological activity. The results offer a new mechanism for the functional diversity of proteins, by exploiting lipids as essential, tissue-specific cofactors in this process.     

Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis

A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human alpha-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-alpha-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the beta-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the beta-domain of alpha-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.     

Slo1 tail domains,but not the Ca2+ bowl,are required for the beta 1 subunit to increase the apparent Ca2+ sensitivity of BK channels

Functional large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels can be assembled from four alpha subunits (Slo1) alone, or together with four auxiliary beta1 subunits to greatly increase the apparent Ca(2+) sensitivity of the channel. We examined the structural features involved in this modulation with two types of experiments. In the first, the tail domain of the alpha subunit, which includes the RCK2 (regulator of K(+) conductance) domain and Ca(2+) bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca(2+) bowl and high affinity Ca(2+) sensitivity. In the second, the Ca(2+) bowl was disrupted by mutations that greatly reduce the apparent Ca(2+) sensitivity. We found that the beta1 subunit increased the apparent Ca(2+) sensitivity of Slo1 channels, independently of whether the alpha subunits were expressed as separate cores (S0-S8) and tails (S9-S10) or full length, and this increase was still observed after the Ca(2+) bowl was mutated. In contrast, beta1 subunits no longer increased Ca(2+) sensitivity when Slo1 tails were replaced by Slo3 tails. The beta1 subunits were still functionally coupled to channels with Slo3 tails, as DHS-I and 17 beta-estradiol activated these channels in the presence of beta1 subunits, but not in their absence. These findings indicate that the increase in apparent Ca(2+) sensitivity induced by the beta1 subunit does not require either the Ca(2+) bowl or the linker between the RCK1 and RCK2 domains, and that Slo3 tails cannot substitute for Slo1 tails. The beta1 subunit also induced a decrease in voltage sensitivity that occurred with either Slo1 or Slo3 tails. In contrast, the beta1 subunit-induced increase in apparent Ca(2+) sensitivity required Slo1 tails. This suggests that the allosteric activation pathways for these two types of actions of the beta1 subunit may be different.     

Expression of calcium-binding protein regucalcin mRNA in the cloned rat hepatoma cells (Hr-II-E) is stimulated through Ca2+ signaling factors: Involvement of protein kinase C

The expression of hepatic Ca²⁺-binding protein regucalcin in the cloned rat hepatoma cells (H4-II-E) was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open reading frame). Regucalcin mRNA was expressed in H4-II-E hepatoma cells. This expression was clearly stimulated in the presence of serum (10% fetal bovine serum). Bay K 8644 (2. 5 × 10^-6 M), a Ca²⁺ channel agonist, significantly stimulated regucalcin mRNA expression in the absence or presence of 10% serum. Dibutyryl cyclic AMP (10^-3 M) did not have a stimulatory effect on the regucalcin mRNA expression. The presence of phorbol 12-myristate 13-acetate (PMA; 10^-6 M) or estrogen (10^-8 M) caused a significant increase in regucalcin mRNA levels in the hepatoma cells cultured in serum-free medium, while insulin (5 × 10^-9 M) or dexamethasone (10^-6 M) had no effect. Bay K 8644-stimulated regucalcin mRNA expression in the hepatoma cells was completely blocked in the presence of trifluoperazine (10^-5 M), an antagonist of calmodulin, or staurosporine (10^-7 M), an inhibitor of protein kinase C. The stimulatory effect of PMA was clearly inhibited in the presence of stauroporine. The present study demonstrates that regucalcin mRNA is expressed in the transformed H4-II-E hepatoma cells, and that the expression is stimulated through Ca²⁺-dependent signaling factors.     

A Ca2+ signal is found upstream of cytochrome c release during apoptosis in HeLa cells

We showed previously that a cytosolic Ca(2+) signal is involved in regulating UV-induced apoptosis in HeLa cells. In this study, we found evidence that this Ca(2+) signal occurs upstream of the release of cytochrome c from mitochondria. First, when we abolished [Ca(2+)](i) increases by injecting BAPTA or heparin into UV-treated HeLa cells, cytochrome c release was either blocked or severely delayed. Second, using a living cell imaging technique, we observed a series of transient [Ca(2+)](i) increases (typically lasting about 40-60s) in many apoptotic cells induced by either UV- or TNFalpha-treatment. Third, using GFP-tagged cytochrome c, we found that the Ca(2+) spikes appear in a time window before cytochrome c was released. Finally, by fixing the TNFalpha-treated cell at the time when it started to display Ca(2+) spikes, we examined the distribution of its endogenous cytochrome c using immunostaining. We found that cytochrome c was not yet released from mitochondria. These findings suggest the existence of certain apoptotic pathways, in which an early Ca(2+) signal is activated upstream of cytochrome c release.     

Reversed Actrocytic GLT-1 during Ischemia is Crucial to Excitotoxic Death of Neurons, but Contributes to the Survival of Astrocytes themselves

During ischemia, the operation of astrocytic/neuronal glutamate transporters is reversed and glutamate and Na⁺ are co-transported to the extracellular space. This study aims to investigate whether this reversed operation of glutamate transporters has any functional meanings for astrocytes themselves. Oxygen/glucose deprivation (OGD) of neuron/astrocyte co-cultures resulted in the massive death of neurons, and the cell death was significantly reduced by treatment with either AP5 or DHK. In cultured astrocytes with little GLT-1 expression, OGD produced Na⁺ overload, resulting in the reversal of astrocytic Na⁺/Ca²⁺-exchanger (NCX). The reversed NCX then caused Ca²⁺ overload leading to the damage of astrocytes. In contrast, the OGD-induced Na⁺ overload and astrocytic damage were significantly attenuated in PACAP-treated astrocytes with increased GLT-1 expression, and the attenuation was antagonized by treatment with DHK. These results suggested that the OGD-induced reversal of GLT-1 contributed to the survival of astrocytes themselves by releasing Na⁺ with glutamate via reversed GLT-1.     

Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex

The four integral delta subunits of the phosphorylase kinase (PhK) complex are identical to calmodulin (CaM) and confer Ca(2+) sensitivity to the enzyme, but bind independently of Ca(2+). In addition to binding Ca(2+), an obligatory activator of PhK's phosphoryltransferase activity, the delta subunits transmit allosteric signals to PhK's remaining alpha, beta, and gamma subunits in activating the enzyme. Under mild conditions about 10% of the delta subunits can be exchanged for exogenous CaM. In this study, a CaM double-mutant derivatized with a fluorescent donor-acceptor pair (CaM-DA) was exchanged for delta to assess the conformational substates of PhKdelta by single molecule fluorescence resonance energy transfer (FRET) +/-Ca(2+). The exchanged subunits were determined to occupy distinct conformations, depending on the absence or presence of Ca(2+), as observed by alterations of the compact, mid-length, and extended populations of their FRET distance distributions. Specifically, the combined predominant mid-length and less common compact conformations of PhKdelta became less abundant in the presence of Ca(2+), with the delta subunits assuming more extended conformations. This behavior is in contrast to the compact forms commonly observed for many of CaM's Ca(2+)-dependent interactions with other proteins. In addition, the conformational distributions of the exchanged PhKdelta subunits were distinct from those of CaM-DA free in solution, +/-Ca(2+), as well as from exogenous CaM bound to the PhK complex as delta'. The distinction between delta and delta' is that the latter binds only in the presence of Ca(2+), but stoichiometrically and at a different location in the complex than delta.     

Bcl-2 acts subsequent to and independent of Ca fluxes to inhibit apoptosis in thapsigargin- and glucocorticoidmtreated mouse lymphoma cells

The mechanism by which Bcl-2 inhibits apoptosis is unknown. One proposal is that Bcl-2 regulates intracellular Ca²⁺ fluxes thought to mediate apoptosis. In the present study, we investigated Bcl-2's mechanism of action by determining the effect of Bcl-2 on intracellular Ca²⁺ fluxes in the WEH17.2 mouse lymphoma cell line, which does not express Bcl-2, and its stable transfectant, which expresses a high level of Bcl-2. Treatment with the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin produced marked alterations in intracellular Ca²⁺ homeostasis in both WEH17.2 and W.Hb12 cells, including elevation of free cytosolic Ca²⁺, endoplasmic reticulum Ca²⁺ pool depletion, capacitative entry of extracellular Ca²⁺, and increased loading of Ca²⁺ into mitochondria. Similar changes in intracellular Ca²⁺ occurred spontaneously in both cell lines following exponential growth. In both situations, W.Hb12 cells maintained optimal viability despite marked alterations in intracellular Ca ²⁺' whereas WEH17.2 cells underwent apoptosis. Treatment with the glucocorticoid hormone, dexamethasone, induced apoptosis in WEH17.2 cells, but not in W.HB12 cells, even though dexamethasone treatment did not alter intracellular Ca²⁺ homeostasis in either cell line. These findings indicate that Bcl-2 acts downstream from intracellular Ca ²⁺ fluxes in a pathway where Ca²⁺-dependent and Ca²⁺-independent death signals converge.     

Phospholipase A(2) of peroxiredoxin 6 has a critical role in tumor necrosis factor-induced apoptosis

Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with peroxidase and phospholipase A(2) (PLA(2)) activities. Although the cellular function of the peroxidase of Prdx6 has been well elucidated, the function of the PLA(2) of Prdx6 is largely unknown. Here, we report a novel function for the PLA(2) in regulating TNF-induced apoptosis through arachidonic acid (AA) release and interleukin-1β (IL-1β) production. Prdx6 knockdown (Prdx6(KD)) in human bronchial epithelial cells (BEAS2B) shows severe decreases of peroxidase and PLA(2) activities. Surprisingly, Prdx6(KD) cells are markedly resistant to apoptosis induced by TNF-α in the presence of cycloheximide, but are highly sensitive to hydrogen peroxide-induced apoptosis. Furthermore, the release of AA and the production of IL-1β induced by proinflammatory stimuli, such as TNF-α, LPS, and poly I/C, are severely decreased in Prdx6(KD) cells. More interestingly, the restoration of Prdx6 expression with wild-type Prdx6, but not PLA(2)-mutant Prdx6 (S32A), in Prdx6(KD) cells dramatically induces the recovery of TNF-induced apoptosis, AA release, and IL-1β production, indicating specific roles for the PLA(2) activity of Prdx6. Our results provide new insights into the distinct roles of bifunctional Prdx6 with peroxidase and PLA(2) activities in oxidative stress-induced and TNF-induced apoptosis, respectively.     

Immunolocalization of synexin (annexin VII) in adrenal chromaffin granules and chromaffin cells: evidence for a dynamic role in the secretory process

Summary Synexin (annexin VII) is a Ca²⁺- and phospholid-binding protein which has been proposed to play a role in Ca²⁺-dependent membrane fusion processes. Using a monoclonal antibody against synexin, Mab 10E7, and immunogold, we carried out a semiquantitative localization study of synexin in bovine adrenal medullary chromaffin granules, and in resting and nicotine-stimulated adrenal chromaffin cells. Isolated chromaffin granules contained very little synexin, whereas chromaffin granules aggregated with synexin (24 g/mg) and Ca²⁺ (1 mM) clearly showed synexin-associated immunogold particles in the vicinity of the granule membrane (1.88 gold particles per granule profile). In isolated, cultured adrenal chromaffin cells, synexin was present in the nucleus (5.5 particles/m²) and in the cytosol (5.3 particles/m²), but mainly around the granule membrane in the granular cell area (11.7 particles/m²). During the active phase of cholinergically stimulated catecholamine secretion, the amount of synexin label was reduced by 33% in the nucleus, by 23% in the cytosol, and by 51% in the granule area. The plasma membrane contained a small amount of synexin, which did not significantly change upon stimulation of the cells. We conclude that synexin is involved in the secretory process in chromaffin cells.     

NF-kappaB, but not p38 MAP kinase, is required for TNF-alpha-induced expression of cell adhesion molecules in endothelial cells

In response to inflammation stimuli, tumor necrosis factor-alpha (TNF-alpha) induces expression of cell adhesion molecules (CAMs) in endothelial cells (ECs). Studies have suggested that the nuclear factor-kappaB (NF-kappaB) and the p38 MAP kinase (p38) signaling pathways play central roles in this process, but conflicting results have been reported. The objective of this study is to determine the relative contributions of the two pathways to the effect of TNF-alpha. Our initial data indicated that blockade of p38 activity by chemical inhibitor SB203580 (SB) at 10 microM moderately inhibited TNF-alpha-induced expression of three types of CAMs; ICAM-1, VCAM-1 and E-selectin, indicating that p38 may be involved in the process. However, subsequent analysis revealed that neither 1 microM SB that could completely inhibit p38 nor specific knockdown of p38alpha and p38beta with small interference RNA (siRNA) had an apparent effect, indicating that p38 activity is not essential for TNF-alpha-induced CAMs. The most definitive evidence to support this conclusion was from the experiments using cells differentiated from p38alpha knockout embryonic stem cells. We could show that deletion of p38alpha gene did not affect TNF-alpha-induced ICAM-1 and VCAM-1 expression when compared with wild-type cells. We further demonstrated that inhibition of NF-kappaB completely blocked TNF-alpha-induced expression of ICAM-1, VCAM-1 and E-selectin. Taken together, our results clearly demonstrate that NF-kappaB, but not p38, is critical for TNF-alpha-induced CAM expression. The inhibition of SB at 10 microM on TNF-alpha-induced ICAM-1, VCAM-1 and E-selectin is likely due to the nonspecific effect of SB.     

The epsilon isoform of protein kinase C is involved in regulation of the LTD(4)-induced calcium signal in human intestinal epithelial cells

We investigated the potential roles of specific isoforms of protein kinase C (PKC) in the regulation of leukotriene D(4)-induced Ca(2+) signaling in the intestinal epithelial cell line Int 407. RT-PCR and Western blot analysis revealed that these cells express the PKC isoforms alpha, betaII, delta, epsilon, zeta, and mu, but not betaI, gamma, eta, or theta;. The inflammatory mediator leukotriene D(4) (LTD(4)) caused the TPA-sensitive PKC isoforms alpha, delta, and epsilon, but not betaII, to rapidly translocate to a membrane-enriched fraction. The PKC inhibitor GF109203X at 30 microM but not 2 microM significantly impaired the LTD(4)-induced Ca(2+) signal, indicating that the response involves a novel PKC isoform, such as delta or epsilon, but not alpha. LTD(4)-induced Ca(2+) signaling was significantly suppressed in cells pretreated with TPA for 15 min and was abolished when the pretreatment was prolonged to 2 h. Immunoblot analysis revealed that the reduction in the LTD(4)-induced calcium signal coincided with a reduction in the cellular content of PKCepsilon and, to a limited extent, PKCdelta. LTD(4)-induced Ca(2+) signaling was also markedly suppressed by microinjection of antibodies against PKCepsilon but not PKCdelta. These data suggest that PKCepsilon plays a unique role in regulation of the LTD(4)-dependent Ca(2+) signal in intestinal epithelial cells.     

Identification of a calmodulin-regulated autoinhibited Ca2+-ATPase (ACA11) that is localized to vacuole membranes in Arabidopsis

In plant cells, the vacuole functions as a major calcium store. Although a calmodulin-regulated Ca2+-ATPase (ACA4) is known to be present in prevacuolar compartments, the presence of an ACA-type Ca2+-ATPase in the mature vacuole of a plant cell has not been verified. Here we provide evidence that ACA11 localizes to the vacuole membrane. ACA11 tagged with GFP was expressed in stable transgenic plants, and visualized in root cells and protoplasts by confocal microscopy. A Ca2+-ATPase function for ACA11 was confirmed by complementation of yeast mutants. A calmodulin binding domain was identified within the first 37 residues of the N-terminal autoinhibitory region.     

RGD targeted poly(L-glutamic acid)-cystamine-(Gd-DO3A) conjugate for detecting angiogenesis biomarker alpha(v) beta3 integrin with MRT, mapping

Cyclic Arg-Gly-Asp-D-Phe-Lys [c(RGDfK)] targeted poly(L-glutamic acid) (PGA)-(Gd-DO3A) conjugate with a biodegradable cystamine spacer was prepared and evaluated for in vivo detection of an angiogenesis biomarker, alpha(v)beta3 integrin, in neoplastic tissues with T1 mapping, a quantitative magnetic resonance imaging (MRI) technique. The binding activity of the c(RGDfK) containing conjugate was investigated using in vitro vitronectin assay with human prostate carcinoma DU145 cell line and Kaposi's sarcoma SLK cell line. The peptide c(RGDfK) and PGA-cystamine-(Gd-DO3A) conjugate were used as controls. The binding affinity of polymer bound c(RGDfK) was slightly lower than free c(RGDfK) peptide. The RGD targeted conjugate had higher binding affinity to the DU145 cells than the SLK cells, which was consistent to free c(RGDfK). The imaging of alpha(v)beta3 integrin with targeted PGA-cystamine-(Gd-DO3A) was evaluated in nude mice bearing DU145 and SLK xenografts at a dose of 5 micromol-Gd/kg. The targeted conjugate demonstrated higher in vivo binding affinity to the DU145 xenografts than the SLK xenografts, resulting in a significant decrease of T1 values of water protons in the periphery of the DU145 tumors as shown in the MR T1 maps. No significant decrease of T1 values was observed in the SLK tumor with the targeted conjugate and in both tumors with the non-targeted conjugate. The targeted polymeric Gd(III) chelate conjugate with a degradable spacer has the potential to be a new paradigm for safe and effective probes in molecular imaging with quantitative MR T1 mapping.     

ORAI-mediated calcium influx in T cell proliferation, apoptosis and tolerance

Ca²⁺ homeostasis controls a diversity of cellular processes including proliferation and apoptosis. A very important aspect of Ca²⁺ signaling is how different Ca²⁺ signals are translated into specific cell functions. In T cells, Ca²⁺ signals are induced following the recognition of antigen by the T cell receptor and depend mainly on Ca²⁺ influx through store-operated CRAC channels, which are mediated by ORAI proteins following their activation by STIM proteins. The complete absence of Ca²⁺ influx caused by mutations in Stim1 and Orai1 leads to severe immunodeficiency. Here we summarize how Ca²⁺ signals are tuned to regulate important T cell functions as proliferation, apoptosis and tolerance, the latter one being a special state of immune cells in which they can no longer respond properly to an otherwise activating stimulus. Perturbations of Ca²⁺ signaling may be linked to immune suppressive diseases and autoimmune diseases.     

The endoplasmic reticulum (ER)-target protein Bik induces Hep3B cells apoptosis by the depletion of the ER Ca2+ stores

Bik, a BH3-only protein, was identified to induce cells apoptosis. In this study, we reported that Bik exclusively localized to endoplasmic reticulum rather than mitochondria. The apoptosis induced by Bik was inhibited in Hep3B cells, when TM domain of Bik was truncated. The ectopic overexpression of Bik protein caused the rapid and sustained elevation of the intracellular cytosolic Ca²⁺, which originated from the ER Ca²⁺ stores releasing. The Hep3B cells apoptosis induced by Bik was not prevented by establishing the clamped cytosolic Ca²⁺ condition, or by buffering of the extracellular Ca²⁺ with EGTA, suggesting that the depletion of ER Ca²⁺ stores rather than the elevation of cytosolic Ca²⁺ or the extracellular Ca²⁺ entry contributed to Bik-induced Hep3B cells apoptosis. The authors Xiaoping Zhao and Li Wang contributed equally to this work.     

Solution structure of the N-terminal A domain of the human voltage-gated Ca2+channel beta4a subunit

Ca2+ channel beta subunits regulate trafficking and gating (opening and closing) of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, they are thought to be modular structures composed of five domains (A-E) that are related to the large family of membrane associated guanylate-kinase (MAGUK) proteins. The crystal structures of the beta subunit core, B-D, domains have recently been reported; however, very little is known about the structures of the A and E domains. The N-terminal A domain is a hypervariable region that differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4). Furthermore, this domain undergoes alternative splicing to create multiple N-terminal structures within a given gene class that have distinct effects on gating. We have solved the solution structure of the A domain of the human beta4a subunit, a splice variant that we have shown previously to have alpha1 subunit subtype-specific effects on Ca2+ channel trafficking and gating.     

(Questions) on phloem biology. 1. Electropotential waves, Ca fluxes and cellular cascades along the propagation pathway

This review explores the relationships between electrical long-distance signalling, Ca²⁺ influx coincident with propagation of electropotential waves, and cellular responses to Ca²⁺ influx including the consequences for sieve-tube conductivity and mass flow. Ca²⁺ influx is inherent to electropotential waves and appears to constitute the key link between rapid physical signals and resultant chemical cascades in sieve tubes and adjacent cells. Members of several channel groups are likely involved the regulation of Ca²⁺ levels in sieve elements. Among them are hyperpolarization-activated, depolarization-activated, and mechanosensitive Ca²⁺ channels located in the plasma membrane and Ca²⁺ dependent Ca²⁺ channels that reside in ER-membranes of sieve elements. These channels collectively determine intracellular Ca²⁺ levels in sieve elements and their neighbour cells. The latter cells react to Ca²⁺ elevation by inducing diverse functional responses dependent on the cell type. If the Ca²⁺ concentration in sieve elements surpasses a threshold level, dual sieve-plate occlusion by proteins and callose deposition is triggered. Occlusion is reversed when Ca²⁺ levels subside. Electrical messages may regulate the degree of sieve plate hydraulic conductivity in intact plants by partial sieve-plate occlusion that has a major impact on volume flow through sieve tubes. Furthermore, complete but temporary occlusion of sieve tubes may modify mass flow patterns in intact plants.     

Responsiveness of a human parotid epithelial cell line (HSY) to autonomic stimulation: Muscarinic control of K+ transport

Summary Salivary electrolyte secretion is under the control of the autonomic nervous system. In this paper we report that HSY, an epithelial cell line derived from the acinar-intercalated duct region of the human parotid gland, responds to muscarinic-cholinergic (generation of Ca²⁺ signal) andβ-adrenergic (generation of cAMP signal), but not toα-adrenergic (lack of Ca²⁺ signal), receptor stimulation. The muscarinic response was studied in detail. Carbachol (10⁻⁴ M, muscarinic agonist) or A23187 (5 μM, calcium ionophore) stimulation of HSY cells increases both⁸⁶Rb (K⁺) influx and efflux, resulting in no change in net equilibrium⁸⁶Rb content. Atropine (10⁻⁵ M, muscarinic antagonist) blocks both the carbachol-generated Ca²⁺ signal and carbachol-stimulated⁸⁶Rb fluxes, but has no effect on either the A23187-generated Ca²⁺ signal or A23187-stimulated⁸⁶Rb fluxes. Carbachol- and A23187-stimulated⁸⁶Rb fluxes are substantially inhibited by two K⁺ channel blockers, quinine (0.3 mM) and scorpion venom containing charybdotoxin (33 μg/ml). The inhibition of these stimulated fluxes by another K⁺ channel blocker, tetraethylammonium chloride (5 mM), is less pronounced. Protein kinase C (PKC) seems to be involved in the regulation of the⁸⁶Rb fluxes as 10⁻⁷ M PMA (phorbol ester, phorbol-12-myristate-13-acetate) substantially inhibits the muscarinic-stimulated⁸⁶Rb efflux and influx. Because this concentration of PMA totally inhibits the carbachol-generated Ca²⁺ signal and only 80% of the muscarinic-stimulated⁸⁶Rb influx, it seems that a portion of the carbachol-stimulated⁸⁶Rb flux (i.e. that portion not inhibited by PMA) might occur independently of the Ca²⁺ signal. PMA fails to inhibit the A23187-stimulated⁸⁶Rb fluxes, however, suggesting that PKC regulates Ca²⁺-sensitive K⁺ channel activity by regulating the Ca²⁺ signal, and not steps distal to this event. 4-α-Phorbol-12,13-didecanoate, a phorbol ester which fails to activate PKC, fails to inhibit either the carbachol-stimulated increase in intracellular free Ca²⁺, or carbachol-stimulated⁸⁶Rb fluxes.     

The plasma membrane (Mg2+)-dependent adenosine triphosphatase from the human erythrocyte is not an ion pump

Summary The plasma membrane (Mg²⁺)-dependent adenosine triphosphatase ((Mg²⁺)-ATPase) from human erythrocytes has been tested for its ability to transport ions. Using a preparation of inside-out vesicles loaded with the pH-sensitive fluorescence probe 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), we have demonstrated the absence of proton movement during (Mg²⁺)-ATPase activity. From the rate of ATP hydrolysis and the passive proton permeability of these vesicles, an upper limit of 0.03 H⁺ transported per ATP hydrolyzed was calculated. To verify that proton pumping could be detected in this system, the intravesicular pH was monitored during (Ca²⁺)-dependent adenosine triphosphatase ((Ca²⁺)-ATPase) activity. Proton efflux associated with (Ca²⁺)-ATPase activity was observed (in agreement with a recent report of proton pumping by a reconstituted erythrocyte (Ca²⁺)-ATPase (Niggli, V., Sigel, E., Carafoli, E. (1982)J. Biol. Chem. 257:2350–2356)) and was shown to be stimulated by calmodulin. The ability of the (Mg²⁺)-ATPase to pump²⁸Mg²⁺,³⁵SO ₄ ^2– and⁸⁶Rb⁺ was also tested, with the results leading to the conclusion that the human erythrocyte enzyme does not function as an ion transport system.