SERCA pump optimizes Ca2+ release by a mechanism independent of store filling in smooth muscle cells

Thapsigargin-sensitive sarco/endoplasmic reticulum Ca(2+) pumps (SERCAs) are involved in maintaining and replenishing agonist-sensitive internal stores. Although it has been assumed that release channels act independently of SERCA pumps, there are data suggesting the opposite. Our aim was to study the relationship between SERCA pumps and the release channels in smooth muscle cells. To this end, we have rapidly blocked SERCA pumps with thapsigargin, to avoid depletion of the internal Ca(2+) stores, and induced Ca(2+) release with either caffeine, to open ryanodine receptors, or acetylcholine, to open inositol 1,4,5-trisphosphate receptors. Blocking SERCA pumps produced smaller and slower agonist-induced [Ca(2+)](i) responses. We determined the Ca(2+) level of the internal stores both indirectly, measuring the frequency of spontaneous transient outward currents, and directly, using Mag-Fura-2, and demonstrated that the inhibition of SERCA pumps did not produce a reduction of the sarco/endoplasmic reticulum Ca(2+) levels to explain the decrease in the agonist-induced Ca(2+) responses. It appears that SERCA pumps are involved in sustaining agonist-induced Ca(2+) release by a mechanism that involves the modulation of Ca(2+) availability in the lumen of the internal stores.     

Rabbit sarcoplasmic reticulum Ca(2+)-ATPase replaces yeast PMC1 and PMR1 Ca(2+)-ATPases for cell viability and calcineurin-dependent regulation of calcium tolerance

SERCA1a, the fast-twitch skeletal muscle isoform of sarco(endo)plasmic reticulum Ca(2+)-ATPase, was expressed in yeast using the promoter of the plasma membrane H(+)-ATPase. In the yeast Saccharomyces cerevisiae, the Golgi PMR1 Ca(2+)-ATPase and the vacuole PMC1 Ca(2+)-ATPase function together in Ca2+ sequestration and Ca2+ tolerance. SERCA1a expression restored growth of pmc1 mutants in media containing high Ca2+ concentrations, consistent with increased Ca2+ uptake in an internal compartment. SERCA1a expression also prevented synthetic lethality of pmr1 pmc1 double mutants on standard media. Electron microscopy and subcellular fractionation analysis showed that SERCA1a was localized in intracellular membranes derived from the endoplasmic reticulum. Finally, we found that SERCA1a ATPase activity expressed in yeast was regulated by calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase. This result indicates that calcineurin contributes to calcium homeostasis by modulating the ATPase activity of Ca2+ pumps localized in intra-cellular compartments.     

SERCA pump activity is physiologically regulated by presenilin and regulates amyloid beta production

In addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca(2+) is maintained at low levels by pumping Ca(2+) into stores in the endoplasmic reticulum (ER) via the sarco ER Ca(2+)-ATPase (SERCA) pumps. We show that SERCA activity is diminished in fibroblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca(2+), whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca(2+) clearance and exaggerating inositol 1,4,5-trisphosphate-mediated Ca(2+) liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer's disease is underscored by our findings that modulating SERCA activity alters amyloid beta production. Our results point to a physiological role for the presenilins in Ca(2+) signaling via regulation of the SERCA pump.     

Ca2+-pumps and Na2+-Ca2+-exchangers in coronary artery endothelium versus smooth muscle

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.     

Glucose and endoplasmic reticulum calcium channels regulate HIF-1beta via presenilin in pancreatic beta-cells

Pancreatic beta-cell death is a critical event in type 1 diabetes, type 2 diabetes, and clinical islet transplantation. We have previously shown that prolonged block of ryanodine receptor (RyR)-gated release from intracellular Ca(2+) stores activates calpain-10-dependent apoptosis in beta-cells. In the present study, we further characterized intracellular Ca(2+) channel expression and function in human islets and the MIN6 beta-cell line. All three RyR isoforms were identified in human islets and MIN6 cells, and these endoplasmic reticulum channels were observed in close proximity to mitochondria. Blocking RyR channels, but not sarco/endoplasmic reticulum ATPase (SERCA) pumps, reduced the ATP/ADP ratio. Blocking Ca(2+) flux through RyR or inositol trisphosphate receptor channels, but not SERCA pumps, increased the expression of hypoxia-inducible factor (HIF-1beta). Moreover, inhibition of RyR or inositol trisphosphate receptor channels, but not SERCA pumps, increased the expression of presenilin-1. Both HIF-1beta and presenilin-1 expression were also induced by low glucose. Overexpression of presenilin-1 increased HIF-1beta, suggesting that HIF is downstream of presenilin. Our results provide the first evidence of a presenilin-HIF signaling network in beta-cells. We demonstrate that this pathway is controlled by Ca(2+) flux through intracellular channels, likely via changes in mitochondrial metabolism and ATP. These findings provide a mechanistic understanding of the signaling pathways activated when intracellular Ca(2+) homeostasis and metabolic activity are suppressed in diabetes and islet transplantation.     

Gene knockout studies of Ca2+-transporting ATPases.

The biochemical functions of intracellular and plasma membrane Ca2+-transporting ATPases in the control of cytosolic and organellar Ca2+ levels are well established, but the physiological roles of specific isoforms are less well understood. There appear to be three different types of Ca2+ pumps in mammalian tissues: the sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs), which sequester Ca2+ within the endoplasmic or sarcoplasmic reticulum, the plasma membrane Ca2+-ATPases (PMCAs), which extrude Ca2+ from the cell, and the putative secretory pathway Ca2+-ATPase (SPCA), the function of which is poorly understood. This review describes the results of recent analyses of mouse models with null mutations in the genes encoding SERCA and PMCA isoforms and genetic studies of SERCA and SPCA dysfunction in both humans and model organisms. These studies are yielding important insights regarding the physiological functions of individual Ca2+-transporting ATPases in vivo.     

Phenotypes of SERCA and PMCA knockout mice

P-type Ca2+-ATPases of the sarco(endo)plasmic reticulum (SERCAs) and plasma membrane (PMCAs) are responsible for maintaining the Ca2+ gradients across cellular membranes that are required for regulation of Ca2+-mediated signaling and other biological processes. Gene-targeting studies of SERCA isoforms 1, 2, and 3 and PMCA isoforms 1, 2, and 4 have confirmed some of the general functions proposed for these pumps, such as a major role in excitation-contraction coupling for SERCA1 and SERCA2 and housekeeping functions for PMCA1 and SERCA2, but have also revealed some unexpected phenotypes. These include squamous cell cancer and plasticity in the regulation of Ca2+-mediated exocytosis in SERCA2 heterozygous mutant mice, modulation of Ca2+ signaling in SERCA3-deficient mice, deafness and balance disorders in PMCA2 null mice, and male infertility in PMCA4 null mice. These unique phenotypes provide new information about the cellular functions of these pumps, the requirement of their activities for higher order physiological processes, and the pathophysiological consequences of pump dysfunction.     

Improving cardiac Ca⁺² transport into the sarcoplasmic reticulum in heart failure: lessons from the ubiquitous SERCA2b Ca⁺² pump

As a major Ca2+ pump in the sarcoplasmic reticulum of the cardiomyocyte, SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a) controls the relaxation and contraction of the cardiomyocyte. It is meticulously regulated by adapting its expression levels and affinity for Ca2+ ions to the physiological demand of the heart. Dysregulation of the SERCA2a activity entails poor cardiomyocyte contractility, resulting in heart failure. Conversely, improving cardiac SERCA2a activity, e.g. by boosting its expression level or by increasing its affinity for Ca2+, is a promising strategy to rescue contractile dysfunction of the failing heart. The structures of the related SERCA1a Ca2+ pump and the Na+/K+-ATPase of the plasma membrane exposed the pumping mechanism and conserved domain architecture of these ion pumps. However, how the Ca2+ affinity of SERCA2a is regulated at the molecular level remained unclear. A structural and functional analysis of the closely related SERCA2b Ca2+ pump, i.e. the housekeeping Ca2+ pump found in the endoplasmic reticulum and the only SERCA isoform characterized by a high Ca2+ affinity, aimed to fill this gap. We demonstrated the existence of a novel and highly conserved site on the SERCA2 pump mediating Ca2+ affinity regulation by the unique C-terminus of SERCA2b (2b-tail). It differs from the earlier-described target site of the affinity regulator phospholamban. Targeting this novel site may provide a new approach to improve SERCA2a function in the failing heart. Strikingly, the intramembrane interaction site of the 2b-tail in SERCA2b shares sequence and structural homology with the binding site of the β-subunit on the α Na+/K+-ATPase. Thus P-type ATPases seem to have developed related mechanisms of regulation, and it is a future challenge for us to discover these general principles of P-type regulation.     

Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps.

ATP-dependent calcium pumps that reside in intracellular organelles are encoded by a family of structurally related enzymes, termed the sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPases (SERCA), which each have a distinct pattern of tissue-specific and developmentally regulated expression. A COS-1 cell expression system was used to examine the biochemical properties of the isoforms: SERCA1 (fast-twitch skeletal muscle). SERCA2a (cardiac/slow-twitch skeletal muscle), SERCA2b (ubiquitous smooth- and non-muscle), and SERCA3 (non-muscle). Each isoform was expressed efficiently and appeared to be targeted to the endoplasmic reticulum. All isoforms displayed qualitatively similar enzymatic properties and were activated by calcium in a cooperative manner with a Hill coefficient of 2. The quantitative properties of SERCA1 and SERCA2a (the muscle isoforms) were identical in all respects. SERCA2b, however, appeared to have a lower turnover rate for both calcium transport and ATP hydrolysis. SERCA3 displayed a reduced apparent affinity for calcium, an increased apparent affinity for vanadate, and an altered pH dependence when compared with the other isoforms. These properties are consistent with an enzyme in which the equilibrium between the E1 and E2 conformations is shifted toward the E2 state.     

Complex effects of ryanodine on the sarcoplasmic reticulum Ca2+ levels in smooth muscle cells

We have studied the effects of ryanodine and inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) with thapsigargin, on both [Ca(2+)](i) and the sarcoplasmic reticulum (SR) Ca(2+) level during caffeine-induced Ca(2+) release in single smooth muscle cells. Incubation with 10 microM ryanodine did not inhibit the first caffeine-induced [Ca(2+)](i) response, although it abolished the [Ca(2+)](i) response to a second application of caffeine. To assess whether ryanodine was inducing a permanent depletion of the internal Ca(2+) stores, we measured the SR Ca(2+) level with Mag-Fura-2. The magnitude of the caffeine-induced reduction in the SR Ca(2+) level was not augmented by incubating cells with 1 microM ryanodine. Moreover, on removal of caffeine, the SR Ca(2+) levels partially recovered in 61% of the cells due to the activity of thapsigargin-sensitive SERCA pumps. Unexpectedly, 10 microM ryanodine instead of inducing complete depletion of SR Ca(2+) stores markedly reduced the caffeine-induced SR Ca(2+) response. It was necessary to previously inhibit SERCA pumps with thapsigargin for ryanodine to be able to induce caffeine-triggered permanent depletion of SR Ca(2+) stores. These data suggest that the effect of ryanodine on smooth muscle SR Ca(2+) stores was markedly affected by the activity of SERCA pumps. Our data highlight the importance of directly measuring SR Ca(2+) levels to determine the effect of ryanodine on the internal Ca(2+) stores.     

Sarco/endoplasmic reticulum Ca2+ATPase type 3 isoforms (SERCA3b and SERCA3f): distinct roles in cell adhesion and ER stress

Sarco/endoplasmic reticulum Ca(2+)ATPases (SERCAs) pump free Ca(2+) from the cytosol into the endoplasmic reticulum. The human SERCA3 family counts six members named SERCA3a to 3f. However, the exact role of these different isoforms in cellular physiology remains undetermined. In this study, we compared some physiological consequences of SERCA3b and SERCA3f overexpression in HEK-293 cells. We observed that overexpression of SERCA3b affected cell adhesion capacity associated with a major disorganization of F-actin and a decrease in focal adhesion. Furthermore, we found that SERCA3f overexpression resulted in an increase in endoplasmic reticulum stress markers (including processing of X-box-binding protein-1 (XBP-1) mRNA and expression of chaperone glucose-regulated protein 78 (GRP78)). This was associated with the activation of caspase cascade and a higher spontaneous cell death. In conclusion, these data point for the first time to distinct physiological roles of SERCA3 isoforms in cell functions.     

Calcium pumps of plasma membrane and cell interior

Calcium entering the cell from the outside or from intracellular organelles eventually must be returned to the extracellular milieu or to intracellular storage organelles. The two major systems capable of pumping Ca2+ against its large concentration gradient out of the cell or into the sarco/endoplasmatic reticulum are the plasma membrane Ca2+ ATPases (PMCAs) and the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), respectively. In mammals, multigene families code for these Ca2+ pumps and additional isoform subtypes are generated via alternative splicing. PMCA and SERCA isoforms show developmental-, tissue- and cell type-specific patterns of expression. Different PMCA and SERCA isoforms are characterized by different regulatory and kinetic properties that likely are optimized for the distinct functional tasks fulfilled by each pump in setting resting cytosolic or intra-organellar Ca2+ levels, and in shaping intracellular Ca2+ signals with spatial and temporal resolution. The loss or malfunction of specific Ca2+ pump isoforms is associated with defects such as deafness, ataxia or heart failure. Understanding the involvement of different Ca2+ pump isoforms in the pathogenesis of disease allows their identification as therapeutic targets for the development of selective strategies to prevent or combat the progression of these disorders.     

Three novel sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 3 isoforms. Expression, regulation, and function of the membranes of the SERCA3 family

Sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) pump Ca2+ into the endoplasmic reticulum. Recently, three human SERCA3 (h3a-c) proteins and a previously unknown rat SERCA3 (r3b/c) mRNA have been described. Here, we (i) document two novel human SERCA3 splice variants h3d and h3e, (ii) provide data for the expression and mechanisms regulating the expression of all known SERCA3 variants (r3a, r3b/c, and h3a-e), and (iii) show functional characteristics of the SERCA3 isoforms. h3d and h3e are issued from the insertion of an additional penultimate exon 22 resulting in different carboxyl termini for these variants. Distinct distribution patterns of the SERCA3 gene products were observed in a series of cell lines of hematopoietic, epithelial, embryonic origin, and several cancerous types, as well as in panels of rat and human tissues. Hypertension and protein kinase C, calcineurin, or retinoic acid receptor signaling pathways were found to differently control rat and human splice variant expression, respectively. Stable overexpression of each variant was performed in human embryonic kidney 293 cells, and the SERCA3 isoforms were fully characterized. All SERCA3 isoforms were found to pump Ca2+ with similar affinities. However, they modulated the cytosolic Ca2+ concentration ([Ca2+]c) and the endoplasmic reticulum Ca2+ content ([Ca2+]er) in different manners. A newly generated polyclonal antibody and a pan-SERCA3 antibody proved the endogenous expression of the three novel SERCA3 proteins, h3d, h3e, and r3b/c. All these data suggest that the SERCA3 gene products have a more widespread role in cellular Ca2+ signaling than previously appreciated.     

Molecular physiology of the SERCA and SPCA pumps

Intracellular Ca(2+)-transport ATPases exert a pivotal role in the endoplasmic reticulum and in the compartments of the cellular secretory pathway by maintaining a sufficiently high lumenal Ca(2+) (and Mn(2+)) concentration in these compartments required for an impressive number of vastly different cell functions. At the same time this lumenal Ca(2+) represents a store of releasable activator Ca(2+) controlling an equally impressive number of cytosolic functions. This review mainly focuses on the different Ca(2+)-transport ATPases found in the intracellular compartments of mainly animal non-muscle cells: the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pumps. Although it is not our intention to treat the ATPases of the specialized sarcoplasmic reticulum in depth, we can hardly ignore the SERCA1 pump of fast-twitch skeletal muscle since its structure and function is by far the best understood and it can serve as a guide to understand the other members of the family. In a second part of this review we describe the relatively novel family of secretory pathway Ca(2+)/Mn(2+) ATPases (SPCA), which in eukaryotic cells are primarily found in the Golgi compartment.     

Myeloperoxidase-derived oxidants inhibit sarco/endoplasmic reticulum Ca2+-ATPase activity and perturb Ca2+ homeostasis in human coronary artery endothelial cells

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) plays a critical role in Ca(2+) homeostasis via sequestration of this ion in the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in aging tissues and cardiovascular disease. We have shown previously that the myeloperoxidase (MPO)-derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca(2+) levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to preformed or enzymatically generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrent with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca(2+), to HOSCN or HOCl resulted in a time- and concentration-dependent increase in intracellular Ca(2+) under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca(2+) pumps/channels, completely attenuated the increase in intracellular Ca(2+) consistent with a critical role for SERCA in maintaining endothelial cell Ca(2+) homeostasis. Angiotensin II pretreatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca(2+) levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers because of their higher SCN(-) levels.     

Control of protein expression through mRNA stability in calcium signalling

Specific sequences (cis-acting elements) in the 3'-untranslated region (UTR) of RNA, together with stabilizing and destabilizing proteins (trans-acting factors), determine the mRNA stability, and consequently, the level of expression of several proteins. Such interactions were discovered initially for short-lived mRNAs encoding cytokines and early genes like c-jun and c-myc. However, they may also determine the fate of more stable mRNAs in a tissue and disease-dependent manner. The interactions between the cis-acting elements and the trans-acting factors may also be modulated by Ca(2+) either directly or via a control of the phosphorylation status of the trans-acting factors. We focus initially on the basic concepts in mRNA stability with the trans-acting factors AUF1 (destabilizing) and HuR (stabilizing). Sarco/endoplasmic reticulum Ca(2+) pumps, SERCA2a (cardiac and slow twitch muscles) and SERCA2b (most cells including smooth muscle cells), are pivotal in Ca(2+) mobilization during signal transduction. SERCA2a and SERCA2b proteins are encoded by relatively stable mRNAs that contain cis-acting stability determinants in their 3'-regions. We present several pathways where 3'-UTR mediated mRNA decay is key to Ca(2+) signalling: SERCA2a and beta-adrenergic receptors in heart failure, renin-angiotensin system, and parathyroid hormones. Other examples discussed include cytokines vascular endothelial growth factor, endothelin and endothelial nitric oxide synthase. Roles of Ca(2+) and Ca(2+)-binding proteins in mRNA stability are also discussed. We anticipate that these novel modes of control of protein expression will form an emerging area of research that may explore the central role of Ca(2+) in cell function during development and in disease.     

Effects of PMCA and SERCA pump overexpression on the kinetics of cell Ca(2+) signalling

The dynamic interactions of the main pathways for active Ca(2+) transport have been analysed in living cells by altering the expression of their components. The plasma membrane (PMCA) and the endoplasmic reticulum (ER) (SERCA) Ca(2+) pumps were transiently overexpressed in CHO cells, and the Ca(2+) homeostasis in the subcellular compartments was investigated using specifically targeted chimaeras of the Ca(2+)- sensitive photoprotein aequorin. In resting cells, overexpression of the PMCA and SERCA pumps caused a reduction and an increase in ER [Ca(2+)] levels, respectively, while no significant differences were detected in cytosolic and mitochondrial [Ca(2+)]. Upon stimulation with an inositol 1,4, 5-trisphosphate (IP(3))-generating agonist, the amplitude of the mitochondrial and cytosolic Ca(2+) rises correlated with the ER [Ca(2+)] only up to a threshold value, above which the feedback inhibition of the IP(3) channel by Ca(2+) appeared to be limiting.     

Thyroid hormones differentially regulate the distribution of rabbit skeletal muscle Ca(2+)-ATPase (SERCA) isoforms in light and heavy sarcoplasmic reticulum

The sarcoplasmic reticulum (SR) is composed of two fractions, the heavy fraction that contains proteins involved in Ca2+ release, and the light fraction enriched in Ca(2+)-ATPase (SERCA), an enzyme responsible for Ca2+ transport from the cytosol to the lumen of SR. It is known that in red muscle thyroid hormones regulate the expression of SERCA 1 and SERCA 2 isoforms. Here we show the effects of thyroid hormone on SERCA expression and distribution in light and heavy SR fractions from rabbit white and red muscles. In hyperthyroid red muscle there is an increase of SERCA 1 and a decrease of SERCA 2 expression. This is far more pronounced in the heavy than in the light SR fraction. As a result, the rates of Ca(2+)- ATPase activity and Ca(2+)-uptake by the heavy vesicles are increased. In hypothyroidism we observed a decrease in SERCA 1 and no changes in the amount of SERCA 2 expressed. This promoted a decrease of both Ca(2+)-uptake and Ca(2+)-ATPase activity. While the major differences in hyperthyroidism were found in the heavy SR fraction, the effects of hypothyroidism were restricted to light SR fraction. In white muscle we did not observe any significant changes in either hypo- or hyperthyroidism in both SR fractions. Thus, the regulation of SERCA isoforms by thyroid hormones is not only muscle specific but also varies depending on the subcellular compartment analyzed. These changes might correspond to the molecular basis of the altered contraction and relaxation rates detected in thyroid dysfunction.