Activation of the atrial KACh channel by the βγ subunits of G proteins or intracellular Na+ ions depends on the presence of phosphatidylinositol phosphates

The βγ subunits of GTP-binding proteins (G_βγ) activate the muscarinic K⁺ channel (K_ACh) in heart by direct binding to both of its component subunits. K_ACh channels can also be gated by internal Na⁺ ions. Both activation mechanisms show dependence on hydrolysis of intracellular ATP. We report that phosphatidylinositol 4,5-bisphosphate (PIP₂) mimics the ATP effects and that depletion or block of PIP₂ retards the stimulatory effects of G_βγ subunits or Na⁺ ions on channel activity, effects that can be reversed by restoring PIP₂. Thus, regulation of K_ACh channel activity may be crucially dependent on PIP₂ and phosphatidylinositol signaling. These striking functional results are in agreement with in vitro biochemical studies on the PIP₂ requirement for G_βγ stimulation of G protein receptor kinase activity, thus implicating phosphatidylinositol phospholipids as a potential control point for G_βγ-mediated signal transduction.     

Loss of protein kinase C inhibition in the β-T594M variant of the amiloride-sensitive Na+ channel

We previously reported the presence of a novel variant (β-T594M) of the amiloride-sensitive Na⁺ channel (ASSC) in which the threonine residue at position 594 in the β-subunit has been replaced by a methionine residue. Electrophysiological studies of the ASSC on Epstein–Barr virus (EBV)-transformed lymphocytes carrying this variant showed that the 8-(4-chlorophenylthio) adenosine 3′:5′-cyclic monophosphate (8cpt-cAMP)-induced responses were enhanced when compared to wild-type EBV-transformed lymphocytes. Furthermore, in wild-type EBV-transformed cells, the 8cpt-cAMP-induced response was totally blocked by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This inhibitory effect of PMA was blocked by a protein kinase C inhibitor, chelerythrine. We now have identified individuals who are homozygous for this variant, and showed that PMA had no effect on the 8cpt-cAMP-induced responses in the EBV-transformed lymphocytes from such individuals. Cells heterozygous for this variant showed mixed responses to PMA, with the majority of cells partially inhibited by PMA. Our results demonstrate that an alteration in a single amino acid residue in the β-subunit of the ASSC can lead to a total loss of inhibition to PMA, and establish the β-subunit as having an important role in conferring a regulatory effect on the ASSC of lymphocytes.     

Targeted inactivation of αi2 or αi3 disrupts activation of the cardiac muscarinic K+ channel, IK+Ach, in intact cells

Cardiac muscarinic receptors activate an inwardly rectifying K⁺ channel, I_K^+Ach, via pertussis toxin (PT)-sensitive heterotrimeric G proteins (in heart G_i2, G_i3, or G_o). We have used embryonic stem cell (ES cell)-derived cardiocytes with targeted inactivations of specific PT-sensitive α subunits to determine which G proteins are required for receptor-mediated regulation of I_K^+Ach in intact cells. The muscarinic agonist carbachol increased I_K^+Ach activity in ES cell-derived cardiocytes from wild-type cells, in cells lacking α_o, and in cells lacking the PT-insensitive G protein α_q. In cells with targeted inactivation of α_i2 or α_i3, channel activation by both carbachol and adenosine was blocked. Carbachol-induced channel activation was restored in the α_i2- and α_i3-null cells by reexpressing the previously targeted gene and guanosine 5′-[γ-thio] triphosphate was able to fully activate I_K^+Ach in excised membranes patches from these mutants. In contrast, negative chronotropic responses to both carbachol and adenosine were preserved in cells lacking α_i2 or α_i3. Our results show that expression of two specific PT-sensitive α subunits (α_i2 and α_i3 but not α_o) is required for normal agonist-dependent activation of I_K^+Ach and suggest that both α_i2- and α_i3-containing heterotrimeric G proteins may be involved in the signaling process. Also the generation of negative chronotropic responses to muscarinic or adenosine receptor agonists do not require activation of I_K^+Ach or the expression of α_i2 or α_i3.     

Cotransport of water by the Na+/glucose cotransporter

Water is transported across epithelial membranes in the absence of any hydrostatic or osmotic gradients. A prime example is the small intestine, where 10 liters of water are absorbed each day. Although water absorption is secondary to active solute transport, the coupling mechanism between solute and water flow is not understood. We have tested the hypothesis that water transport is directly linked to solute transport by cotransport proteins such as the brush border Na⁺/glucose cotransporter. The Na⁺/glucose cotransporter was expressed in Xenopus oocytes, and the changes in cell volume were measured under sugar-transporting and nontransporting conditions. We demonstrate that 260 water molecules are directly coupled to each sugar molecule transported and estimate that in the human intestine this accounts for 5 liters of water absorption per day. Other animal and plant cotransporters such as the Na⁺/Cl⁻/γ-aminobutyric acid, Na⁺/iodide and H⁺/amino acid transporters are also able to transport water and this suggests that cotransporters play an important role in water homeostasis.     

Fatty acids suppress voltage-gated Na+ currents in HEK293t cells transfected with the α-subunit of the human cardiac Na+ channel

Studies have shown that fish oils, containing n-3 fatty acids, have protective effects against ischemia-induced, fatal cardiac arrhythmias in animals and perhaps in humans. In this study we used the whole-cell voltage-clamp technique to assess the effects of dietary, free long-chain fatty acids on the Na⁺ current (I_Na,α) in human embryonic kidney (HEK293t) cells transfected with the α-subunit of the human cardiac Na⁺ channel (hH1_α). Extracellular application of 0.01 to 30 μM eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced I_Na,α with an IC₅₀ of 0.51 ± 0.06 μM. The EPA-induced suppression of I_Na,α was concentration- and voltage-dependent. EPA at 5 μM significantly shifted the steady-state inactivation relationship by −27.8 ± 1.2 mV (n = 6, P < 0.0001) at the V_1/2 point. In addition, EPA blocked I_Na,α with a higher “binding affinity” to hH1_α channels in the inactivated state than in the resting state. The transition from the resting state to the inactivated state was markedly accelerated in the presence of 5 μM EPA. The time for 50% recovery from the inactivation state was significantly slower in the presence of 5 μM EPA, from 2.1 ± 0.8 ms for control to 34.8 ± 2.1 ms (n = 5, P < 0.001). The effects of EPA on I_Na,α were reversible. Furthermore, docosahexaenoic acid (C22:6n-3), α-linolenic acid (C18:3n-3), conjugated linoleic acid (C18:2n-7), and oleic acid (C18:1n-9) at 5 μM and all-trans-retinoic acid at 10 μM had similar effects on I_Na,α as EPA. Even 5 μM of stearic acid (C18:0) or palmitic acid (C16:0) also significantly inhibited I_Na,α. In contrast, 5 μM EPA ethyl ester did not alter I_Na,α (8 ± 4%, n = 8, P > 0.05). The present data demonstrate that free fatty acids suppress I_Na,α with high “binding affinity” to hH1_α channels in the inactivated state and prolong the duration of recovery from inactivation.     

Ca2+-induced rebound potentiation of γ-aminobutyric acid-mediated currents requires activation of Ca2+/calmodulin-dependent kinase II

In cerebellar Purkinje neurons, γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission undergoes a long-lasting “rebound potentiation” after the activation of excitatory climbing fiber inputs. Rebound potentiation is triggered by the climbing-fiber-induced transient elevation of intracellular Ca²⁺ concentration and is expressed as a long-lasting increase of postsynaptic GABA_A receptor sensitivity. Herein we show that inhibitors of the Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII) signal transduction pathway effectively block the induction of rebound potentiation. These inhibitors have no effect on the once established rebound potentiation, on voltage-gated Ca²⁺ channel currents, or on the basal inhibitory transmission itself. Futhermore, a protein phosphatase inhibitor and the intracellularly applied CaM-KII markedly enhanced GABA-mediated currents in Purkinje neurons. Our results demonstrate that CaM-KII activation and the following phosphorylation are key steps for rebound potentiation.     

Molecular picture of folding of a small α/β protein

We characterize, at the atomic level, the mechanism and thermodynamics of folding of a small α/β protein. The thermodynamically significant states of segment B1 of streptococcal protein G (GB1) are probed by using the statistical mechanical methods of importance sampling and molecular dynamics. From a thermodynamic standpoint, folding commences with overall collapse, accompanied by formation of ~35% of the native structure. Specific contacts form at the loci experimentally inferred to be structured early in folding kinetics studies. Our study reveals that these initially structured regions are not spatially adjacent. As folding progresses, fluid-like nonlocal native contacts form, with many contacts forming and breaking as the structure searches for the native conformation. Although the α-helix forms early, the β-sheet forms concomitantly with the overall topology. Water is present in the protein core up to a late stage of folding, lubricating conformational transitions during the search process. Once 80% of the native contacts have formed, water is squeezed from the protein interior and the structure descends into the native manifold. Examination of the onset of side-chain mobility within our model indicates side-chain motion is most closely linked to the overall volume of the protein and no sharp order–disorder transition appears to occur. Exploration of models for hydrogen deuterium exchange show qualitative agreement with equilibrium measurement of hydrogen/deuterium protection factors.     

Na+ pump low and high ouabain affinity α subunit isoforms are differently distributed in cells

Three isoforms (α1, α2, and α3) of the catalytic (α) subunit of the plasma membrane (PM) Na⁺ pump have been identified in the tissues of birds and mammals. These isoforms differ in their affinities for ions and for the Na⁺ pump inhibitor, ouabain. In the rat, α1 has an unusually low affinity for ouabain. The PM of most rat cells contains both low (α1) and high (α2 or α3) ouabain affinity isoforms, but precise localization of specific isoforms, and their functional significance, are unknown. We employed high resolution immunocytochemical techniques to localize α subunit isoforms in primary cultured rat astrocytes, neurons, and arterial myocytes. Isoform α1 was ubiquitously distributed over the surfaces of these cells. In contrast, high ouabain affinity isoforms (α2 in astrocytes, α3 in neurons and myocytes) were confined to a reticular distribution within the PM that paralleled underlying endoplasmic or sarcoplasmic reticulum. This distribution is identical to that of the PM Na/Ca exchanger. This raises the possibility that α1 may regulate bulk cytosolic Na⁺, whereas α2 and α3 may regulate Na⁺ and, indirectly, Ca²⁺ in a restricted cytosolic space between the PM and reticulum. The high ouabain affinity Na⁺ pumps may thereby modulate reticulum Ca²⁺ content and Ca²⁺ signaling.     

Effect of β-amyloid block of the fast-inactivating K+ channel on intracellular Ca2+ and excitability in a modeled neuron

β-Amyloid peptide (Aβ), one of the primary protein components of senile plaques found in Alzheimer disease, is believed to be toxic to neurons by a mechanism that may involve loss of intracellular calcium regulation. We have previously shown that Aβ blocks the fast-inactivating potassium (A) current. In this work, we show, through the use of a mathematical model, that the Aβ-mediated block of the A current could result in increased intracellular calcium levels and increased membrane excitability, both of which have been observed in vitro upon acute exposure to Aβ. Simulation results are compared with experimental data from the literature; the simulations quantitatively capture the observed concentration dependence of the neuronal response and the level of increase in intracellular calcium.     

Disruption of the β subunit of the epithelial Na+ channel in mice: Hyperkalemia and neonatal death associated with a pseudohypoaldosteronism phenotype

The epithelial Na⁺ channel (ENaC) is composed of three homologous subunits: α, β and γ. We used gene targeting to disrupt the β subunit gene of ENaC in mice. The βENaC-deficient mice showed normal prenatal development but died within 2 days after birth, most likely of hyperkalemia. In the −/− mice, we found an increased urine Na⁺ concentration despite hyponatremia and a decreased urine K⁺ concentration despite hyperkalemia. Moreover, serum aldosterone levels were increased. In contrast to αENaC-deficient mice, which die because of defective lung liquid clearance, neonatal βENaC deficient mice did not die of respiratory failure and showed only a small increase in wet lung weight that had little, if any, adverse physiologic consequence. The results indicate that, in vivo, the β subunit is required for ENaC function in the renal collecting duct, but, in contrast to the α subunit, the β subunit is not required for the transition from a liquid-filled to an air-filled lung. The phenotype of the βENaC-deficient mice is similar to that of humans with pseudohypoaldosteronism type 1 and may provide a useful model to study the pathogenesis and treatment of this disorder.     

Reciprocal regulation of Gsα by palmitate and the βγ subunit

Hormonal activation of G_s, the stimulatory regulator of adenylyl cyclase, promotes dissociation of α_s from Gβγ, accelerates removal of covalently attached palmitate from the Gα subunit, and triggers release of a fraction of α_s from the plasma membrane into the cytosol. To elucidate relations among these three events, we assessed biochemical effects in vitro of attached palmitate on recombinant α_s prepared from Sf9 cells. In comparison to the unpalmitoylated protein (obtained from cytosol of Sf9 cells, treated with a palmitoyl esterase, or expressed as a mutant protein lacking the site for palmitoylation), palmitoylated α_s (from Sf9 membranes, 50% palmitoylated) was more hydrophobic, as indicated by partitioning into TX-114, and bound βγ with 5-fold higher affinity. βγ protected GDP-bound α_s, but not α_s· GTP[γS], from depalmitoylation by a recombinant esterase. We conclude that βγ binding and palmitoylation reciprocally potentiate each other in promoting membrane attachment of α_s and that dissociation of α_s·GTP from βγ is likely to mediate receptor-induced α_s depalmitoylation and translocation of the protein to cytosol in intact cells.     

CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages

It has been demonstrated that CD8⁺ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4⁺ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8⁺ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8⁺ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8⁺ T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8⁺ T-cell supernatants; and (iii) the CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8⁺ T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8⁺ T-cell-derived HIV-suppressor factors.     

An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus

T cell receptor (TCR) α and δ gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3′ of TCR δ gene segments and 5′ of TCR α joining gene segments within this locus. BEAD-1 blocked the ability of the TCR δ enhancer (E_δ) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR α/δ locus into distinct regulatory domains controlled by E_δ and the TCR α enhancer, and that it prevents E_δ from opening the chromatin of the TCR α joining gene segments for VDJ recombination at an early stage of T cell development.     

RGS proteins reconstitute the rapid gating kinetics of Gβγ-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K⁺ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Gα(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20–40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of “regulators of G protein signaling” (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor → GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m₂ receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent “basal” GIRK current was suppressed by ≈40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of “slow” postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.     

Angiogenesis promoted by vascular endothelial growth factor: Regulation through α1β1 and α2β1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors—the α₁β₁ and α₂β₁ integrins—through induction of mRNAs encoding the α₁ and α₂ subunits. In contrast, VEGF did not induce increased expression of the α₃β₁ integrin, which also has been implicated in collagen binding. Integrin α₁-blocking and α₂-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and α₁-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of α₁β₁ and α₂β₁ expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of α₁-blocking and α₂-blocking Abs. In vivo, a combination of α₁-blocking and α₂-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of α₁β₁ and α₂β₁ expression by EC is an important mechanism by which VEGF promotes angiogenesis and that α₁β₁ and α₂β₁ antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.     

The amino-terminal region of Tyk2 sustains the level of interferon α receptor 1, a component of the interferon α/β receptor

Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-α (IFN-α) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-α. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-α receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficient in vitro IFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-α. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.     

Gαo is necessary for muscarinic regulation of Ca2+ channels in mouse heart

Heterotrimeric G proteins, composed of Gα and Gβγ subunits, transmit signals from cell surface receptors to cellular effector enzymes and ion channels. The Gα_o protein is the most abundant Gα subtype in the nervous system, but it is also found in the heart. Its function is not completely known, although it is required for regulation of N-type Ca²⁺ channels in GH₃ cells and also interacts with GAP43, a major protein in growth cones, suggesting a role in neuronal pathfinding. To analyze the function of Gα_o, we have generated mice lacking both isoforms of Gα_o by homologous recombination. Surprisingly, the nervous system is grossly intact, despite the fact that Gα_o makes up 0.2–0.5% of brain particulate protein and 10% of the growth cone membrane. The Gα_o−/− mice do suffer tremors and occasional seizures, but there is no obvious histologic abnormality in the nervous system. In contrast, Gα_o−/− mice have a clear and specific defect in ion channel regulation in the heart. Normal muscarinic regulation of L-type calcium channels in ventricular myocytes is absent in the mutant mice. The L-type calcium channel responds normally to isoproterenol, but there is no evident muscarinic inhibition. Muscarinic regulation of atrial K⁺ channels is normal, as is the electrocardiogram. The levels of other Gα subunits (Gα_s, Gα_q, and Gα_i) are unchanged in the hearts of Gα_o−/− mice, but the amount of Gβγ is decreased. Whichever subunit, Gα_o or Gβγ, carries the signal forward, these studies show that muscarinic inhibition of L-type Ca²⁺ channels requires coupling of the muscarinic receptor to Gα_o. Other cardiac Gα subunits cannot substitute.     

Distinct functional stoichiometry of potassium channel β subunits

Shaker-type potassium channels play important roles in determining the electrical excitability of cells. The native channel complex is thought to be formed by four pore-forming α subunits that provide four interaction sites for auxiliary modulatory Kvβ subunits. Because Kvβ subunits possess diverse modulatory activities including either up-regulation or down-regulation of potassium currents, differential assembly of the α–β complex could give rise to diverse current properties. However, the detailed physical and functional stoichiometry of the α–β complex remains unknown. Kvβ1 subunits reduce potassium currents through inactivation, whereas Kvβ2 subunits enhance potassium currents by inhibiting the Kvβ1-mediated inactivation and at the same time by promoting the surface expression of certain potassium channels. In this report we show that Kvβ1 and Kvβ2 of the Shaker-type potassium channels display distinct functional stoichiometry to interact with the Kv1 α subunits, a subfamily of Shaker-type potassium channels. The interaction of Kvβ1 subunits with α subunits is consistent with the α₄β_n model, where n equals 0, 1, 2, 3, or 4, depending upon the relative concentration of α and β subunits. The α₄β_n stoichiometry allows for gradual changes of the Kvβ1-mediated inactivation. In contrast, Kvβ2 subunits self-associate to form oligomers and interact with the α subunits via α₄β₄ stoichiometry, which permits effective multivalent associations with α subunits. Such distinct functional stoichiometry of Kvβ1 and Kvβ2 provides a molecular mechanism that is well suited to their contrasting activities of up-regulation or down-regulation of potassium currents.     

Characterization of human cardiac Na+ channel mutations in the congenital long QT syndrome

The congenital long QT syndrome (LQTS) is an inherited disorder characterized by a prolonged cardiac action potential. This delay in cellular repolarization can lead to potentially fatal arrhythmias. One form of LQTS (LQT3) has been linked to the human cardiac voltage-gated sodium channel gene (SCN5A). Three distinct mutations have been identified in the sodium channel gene. The biophysical and functional characteristics of each of these mutant channels were determined by heterologous expression of a recombinant human heart sodium channel in a mammalian cell line. Each mutation caused a sustained, non-inactivating sodium current amounting to a few percent of the peak inward sodium current, observable during long (>50 msec) depolarizations. The voltage dependence and rate of inactivation were altered, and the rate of recovery from inactivation was changed compared with wild-type channels. These mutations in diverse regions of the ion channel protein, all produced a common defect in channel gating that can cause the long QT phenotype. The sustained inward current caused by these mutations will prolong the action potential. Furthermore, they may create conditions that promote arrhythmias due to prolonged depolarization and the altered recovery from inactivation. These results provide insights for successful intervention in the disease.