Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the β-subunit of the amiloride-sensitive epithelial sodium channel

The amiloride-sensitive epithelial sodium channel (ENaC) is a heteromultimer of three homologous subunits (α-, β-, and γ-subunits). To study the role of the β-subunit in vivo, we analyzed mice in which the βENaC gene locus was disrupted. These mice showed low levels of βENaC mRNA expression in kidney (≈1%), lung (≈1%), and colon (≈4%). In homozygous mutant βENaC mice, no βENaC protein could be detected with immunofluorescent staining. At birth, there was a small delay in lung-liquid clearance that paralleled diminished amiloride-sensitive Na⁺ absorption in tracheal explants. With normal salt intake, these mice showed a normal growth rate. However, in vivo, adult βENaC m/m mice exhibited a significantly reduced ENaC activity in colon and elevated plasma aldosterone levels, suggesting hypovolemia and pseudohypoaldosteronism type 1. This phenotype was clinically silent, as βENaC m/m mice showed no weight loss, normal plasma Na⁺ and K⁺ concentrations, normal blood pressure, and a compensated metabolic acidosis. On low-salt diets, βENaC-mutant mice developed clinical symptoms of an acute pseudohypoaldosteronism type 1 (weight loss, hyperkalemia, and decreased blood pressure), indicating that βENaC is required for Na⁺ conservation during salt deprivation.     

A mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism

Aldosterone-dependent epithelial sodium transport in the distal nephron is mediated by the absorption of sodium through the highly selective, amiloride-sensitive epithelial sodium channel (ENaC) made of three homologous subunits (α, β, and γ). In human, autosomal recessive mutations of α, β, or γENaC subunits cause pseudohypoaldosteronism type 1 (PHA-1), a renal salt-wasting syndrome characterized by severe hypovolemia, high plasma aldosterone, hyponatremia, life-threatening hyperkaliemia, and metabolic acidosis. In the mouse, inactivation of αENaC results in failure to clear fetal lung liquid at birth and in early neonatal death, preventing the observation of a PHA-1 renal phenotype. Transgenic expression of αENaC driven by a cytomegalovirus promoter in αENaC(−/−) knockout mice [αENaC(−/−)Tg] rescued the perinatal lethal pulmonary phenotype and partially restored Na⁺ transport in renal, colonic, and pulmonary epithelia. At days 5–9, however, αENaC(−/−)Tg mice showed clinical features of severe PHA-1 with metabolic acidosis, urinary salt-wasting, growth retardation, and 50% mortality. Adult αENaC(−/−)Tg survivors exhibited a compensated PHA-1 with normal acid/base and electrolyte values but 6-fold elevation of plasma aldosterone compared with wild-type littermate controls. We conclude that partial restoration of ENaC-mediated Na⁺ absorption in this transgenic mouse results in a mouse model for PHA-1.     

Targeted disruption of the Ca2+ channel β3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltagedependent activation of P/Q-type Ca2+ channels in neurons

In comparison to the well characterized role of the principal subunit of voltage-gated Ca²⁺ channels, the pore-forming, antagonist-binding α₁ subunit, considerably less is understood about how β subunits contribute to neuronal Ca²⁺ channel function. We studied the role of the Ca²⁺ channel β₃ subunit, the major Ca²⁺ channel β subunit in neurons, by using a gene-targeting strategy. The β₃ deficient (β₃−/−) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic β₃−/− neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca²⁺ channels was described by two Boltzmann components with different voltage dependence, analogous to the “reluctant” and “willing” states reported for N-type channels. The absence of the β₃ subunit was associated with a hyperpolarizing shift of the “reluctant” component of activation. Norepinephrine inhibited wt and β₃−/− neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca²⁺ channels. The reduction in the expression of N-type Ca²⁺ channels in the β₃−/− mice may be expected to impair Ca²⁺ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.     

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.     

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.     

Streaming potential measurements in αβγ-rat epithelial Na+ channel in planar lipid bilayers

Streaming potentials across cloned epithelial Na⁺ channels (ENaC) incorporated into planar lipid bilayers were measured. We found that the establishment of an osmotic pressure gradient (Δπ) across a channel-containing membrane mimicked the activation effects of a hydrostatic pressure differential (ΔP) on αβγ-rENaC, although with a quantitative difference in the magnitude of the driving forces. Moreover, the imposition of a Δπ negates channel activation by ΔP when the Δπ was directed against ΔP. A streaming potential of 2.0 ± 0.7 mV was measured across αβγ-rat ENaC (rENaC)-containing bilayers at 100 mM symmetrical [Na⁺] in the presence of a 2 Osmol/kg sucrose gradient. Assuming single file movement of ions and water within the conduction pathway, we conclude that between two and three water molecules are translocated together with a single Na⁺ ion. A minimal effective pore diameter of 3 Å that could accommodate two water molecules even in single file is in contrast with the 2-Å diameter predicted from the selectivity properties of αβγ-rENaC. The fact that activation of αβγ-rENaC by ΔP can be reproduced by the imposition of Δπ suggests that water movement through the channel is also an important determinant of channel activity.     

Absence of the β subunit (cchb1) of the skeletal muscle dihydropyridine receptor alters expression of the α1 subunit and eliminates excitation-contraction coupling

The multisubunit (α_1S, α₂/δ, β₁, and γ) skeletal muscle dihydropyridine receptor transduces transverse tubule membrane depolarization into release of Ca²⁺ from the sarcoplasmic reticulum, and also acts as an L-type Ca²⁺ channel. The α_1S subunit contains the voltage sensor and channel pore, the kinetics of which are modified by the other subunits. To determine the role of the β₁ subunit in channel activity and excitation-contraction coupling we have used gene targeting to inactivate the β₁ gene. β₁-null mice die at birth from asphyxia. Electrical stimulation of β₁-null muscle fails to induce twitches, however, contractures are induced by caffeine. In isolated β₁-null myotubes, action potentials are normal, but fail to elicit a Ca²⁺ transient. L-type Ca²⁺ current is decreased 10- to 20-fold in the β₁-null cells compared with littermate controls. Immunohistochemistry of cultured myotubes shows that not only is the β₁ subunit absent, but the amount of α_1S in the membrane also is undetectable. In contrast, the β₁ subunit is localized appropriately in dysgenic, mdg/mdg, (α_1S-null) cells. Therefore, the β₁ subunit may not only play an important role in the transport/insertion of the α_1S subunit into the membrane, but may be vital for the targeting of the muscle dihydropyridine receptor complex to the transverse tubule/sarcoplasmic reticulum junction.     

Kinetics of T cell receptor β, γ, and δ rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44+CD25+ Pro-T thymocytes

We performed a comprehensive analysis of T cell receptor (TCR) γ rearrangements in T cell precursors of the mouse adult thymus. Using a sensitive quantitative PCR method, we show that TCRγ rearrangements are present in CD44⁺CD25⁺ Pro-T thymocytes much earlier than expected. TCRγ rearrangements increase significantly from the Pro-T to the CD44⁻CD25⁺ Pre-T cell transition, and follow different patterns depending on each Vγ gene segment, suggesting that ordered waves of TCRγ rearrangement exist in the adult mouse thymus as has been described in the fetal mouse thymus. Recombinations of TCRγ genes occur concurrently with TCRδ and D-Jβ rearrangements, but before Vβ gene assembly. Productive TCRγ rearrangements do not increase significantly before the Pre-T cell stage and are depleted in CD4⁺CD8⁺ double-positive cells from normal mice. In contrast, double-positive thymocytes from TCRδ−/− mice display random proportions of TCRγ rearranged alleles, supporting a role for functional TCRγ/δ rearrangements in the γδ divergence process.     

Inhibition of interferon γ induced interleukin 12 production: A potential mechanism for the anti-inflammatory activities of tumor necrosis factor

Inflammation is associated with production of cytokines and chemokines that recruit and activate inflammatory cells. Interleukin (IL) 12 produced by macrophages in response to various stimuli is a potent inducer of interferon (IFN) γ production. IFN-γ, in turn, markedly enhances IL-12 production. Although the immune response is typically self-limiting, the mechanisms involved are unclear. We demonstrate that IFN-γ inhibits production of chemokines (macrophage inflammatory proteins MIP-1α and MIP-1β). Furthermore, pre-exposure to tumor necrosis factor (TNF) inhibited IFN-γ priming for production of high levels of IL-12 by macrophages in vitro. Inhibition of IL-12 by TNF can be mediated by both IL-10-dependent and IL-10-independent mechanisms. To determine whether TNF inhibition of IFN-γ-induced IL-12 production contributed to the resolution of an inflammatory response in vivo, the response of TNF^+/+ and TNF^−/− mice injected with Corynebacterium parvum were compared. TNF^−/− mice developed a delayed, but vigorous, inflammatory response leading to death, whereas TNF^+/+ mice exhibited a prompt response that resolved. Serum IL-12 levels were elevated 3-fold in C. parvum-treated TNF^−/− mice compared with TNF^+/+ mice. Treatment with a neutralizing anti-IL-12 antibody led to resolution of the response to C. parvum in TNF^−/− mice. We conclude that the role of TNF in limiting the extent and duration of inflammatory responses in vivo involves its capacity to regulate macrophage IL-12 production. IFN-γ inhibition of chemokine production and inhibition of IFN-γ-induced IL-12 production by TNF provide potential mechanisms by which these cytokines can exert anti-inflammatory/repair function(s).     

Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: A quantitative approach

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na⁺ movement in Na⁺-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of ¹²⁵I-labeled M₂ anti-FLAG monoclonal antibody (M₂Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the α, β, and γ ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (K_d = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (I_Na) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of α, β, and γ ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (β R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.     

An interleukin 4 (IL-4)-independent pathway for CD4+ T cell IL-4 production is revealed in IL-4 receptor-deficient mice

IL-4 receptor α chain (IL-4Rα)-deficient mice were generated by gene-targeting in BALB/c embryonic stem cells. Mutant mice showed a loss of IL-4 signal transduction and functional activity. The lack of IL-4Rα resulted in markedly diminished, but not absent, TH2 responses after infection with the helminthic parasite Nippostrongylus brasiliensis. CD4⁺, CD62L-high, and CD62L-low T cell populations from uninfected IL-4Rα^−/− mice were isolated by cell sorting. Upon primary stimulation by T cell receptor cross-linkage, the CD62L-low, but not the CD62L-high, cells secreted considerable amounts of IL-4, which was strikingly enhanced upon 4-day culture with anti-CD3 in the presence or absence of IL-4. CD62L-low cells isolated from IL-4Rα^−/−, β₂-microglobulin^−/− double homozygous mice produced less IL-4 than did either IL-4Rα^−/− or wild-type mice. These results indicate that an IL-4-independent, β₂-microglobulin-dependent pathway exists through which the CD62L-low CD4⁺ population has acquired IL-4-producing capacity in vivo, strongly suggesting that these cells are NK T cells.     

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca2+ channel activity by cAMP-dependent protein kinase

Preferential phosphorylation of specific proteins by cAMP-dependent protein kinase (PKA) may be mediated in part by the anchoring of PKA to a family of A-kinase anchor proteins (AKAPs) positioned in close proximity to target proteins. This interaction is thought to depend on binding of the type II regulatory (RII) subunits to AKAPs and is essential for PKA-dependent modulation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor, the L-type Ca²⁺ channel, and the K_Ca channel. We hypothesized that the targeted disruption of the gene for the ubiquitously expressed RIIα subunit would reveal those tissues and signaling events that require anchored PKA. RIIα knockout mice appear normal and healthy. In adult skeletal muscle, RIα protein levels increased to partially compensate for the loss of RIIα. Nonetheless, a reduction in both catalytic (C) subunit protein levels and total kinase activity was observed. Surprisingly, the anchored PKA-dependent potentiation of the L-type Ca²⁺ channel in RIIα knockout skeletal muscle was unchanged compared with wild type although it was more sensitive to inhibitors of PKA–AKAP interactions. The C subunit colocalized with the L-type Ca²⁺ channel in transverse tubules in wild-type skeletal muscle and retained this localization in knockout muscle. The RIα subunit was shown to bind AKAPs, although with a 500-fold lower affinity than the RIIα subunit. The potentiation of the L-type Ca²⁺ channel in RIIα knockout mouse skeletal muscle suggests that, despite a lower affinity for AKAP binding, RIα is capable of physiologically relevant anchoring interactions.     

T cell functions in granulocyte/macrophage colony-stimulating factor deficient mice

Immunological functions were analyzed in mice lacking granulocyte/macrophage colony-stimulating factor (GM-CSF). The response of splenic T cells to allo-antigens, anti-mouse CD3 mAb, interleukin 2 (IL-2), or concanavalin A was comparable in GM-CSF +/+ and GM-CSF −/− mice. To investigate the responses of CD8⁺ and CD4⁺ T cells against exogenous antigens, mice were immunized with ovalbumin peptide or with keyhole limpet hemocyanin (KLH). Cytotoxic CD8⁺ T cells with specificity for ovalbumin peptide could not be induced in GM-CSF −/− mice. After immunization with KLH, there was a delay in IgG generation, particularly IgG2a, in GM-CSF −/− mice. Purified CD4⁺ T cells from GM-CSF −/− mice immunized with KLH showed impaired proliferative responses and produced low amounts of interferon-γ (IFN-γ) and IL-4 when KLH-pulsed B cells or spleen cells were used as antigen presenting cells (APC). When enriched dendritic cells (DC) were used as APC, CD4⁺ T cells from GM-CSF −/− mice proliferated as well as those from GM-CSF +/+ mice and produced high amounts of IFN-γ and IL-4. To analyze the rescue effect of DC on CD4⁺ T cells, supernatants from (i) CD4⁺ T cells cultured with KLH-pulsed DC or (ii) DC cultured with recombinant GM-CSF were transferred to cultures of CD4⁺ T cells and KLH-pulsed spleen cells from GM-CSF −/− mice. Supernatants from both DC sources contained a factor or factors that restored proliferative responses and IFN-γ production of CD4⁺ T cells from GM-CSF −/− mice.     

staggerer phenotype in retinoid-related orphan receptor α-deficient mice

Retinoid-related orphan receptor α (RORα) is a member of the nuclear receptor superfamily. To study its physiological role we generated null-mutant mice by targeted insertion of a lacZ reporter gene encoding the enzyme β-galactosidase. In heterozygous RORα^+/− mice we found β-galactosidase activity, indicative of RORα protein expression, confined to the central nervous system, skin and testis. In the central nervous system, the RORα gene is expressed in cerebellar Purkinje cells, the thalamus, the suprachiasmatic nuclei, and retinal ganglion cells. In skin, RORα is strongly expressed in the hair follicle, the epidermis, and the sebaceous gland. Finally, the peritubular cells of the testis and the epithelial cells of the epididymis also strongly express RORα. Recently, it was reported that the ataxic mouse mutant staggerer (sg/sg) is caused by a deletion in the RORα gene. The analysis of the cerebellar and the behavioral phenotype of homozygous RORα^−/− mice proves identity to sg/sg mice. Although the absence of RORα causes dramatic developmental effects in the cerebellum, it has no apparent morphological effect on thalamus, hypothalamus, and retina. Similarly, testis and skin of RORα^−/− mice display a normal phenotype. However, the pelage hair of both sg/sg and RORα^−/− is significantly less dense and when shaved shows reluctance to regrow.     

Normal cerebellar development but susceptibility to seizures in mice lacking G protein-coupled, inwardly rectifying K+ channel GIRK2

G protein-gated, inwardly rectifying K⁺ channels (GIRK) are effectors of G protein-coupled receptors for neurotransmitters and hormones and may play an important role in the regulation of neuronal excitability. GIRK channels may be important in neurodevelopment, as suggested by the recent finding that a point mutation in the pore region of GIRK2 (G156S) is responsible for the weaver (wv) phenotype. The GIRK2 G156S gene gives rise to channels that exhibit a loss of K⁺ selectivity and may also exert dominant-negative effects on G_βγ-activated K⁺ currents. To investigate the physiological role of GIRK2, we generated mutant mice lacking GIRK2. Unlike wv/wv mutant mice, GIRK2 −/− mice are morphologically indistinguishable from wild-type mice, suggesting that the wv phenotype is likely due to abnormal GIRK2 function. Like wv/wv mice, GIRK2 −/− mice have much reduced GIRK1 expression in the brain. They also develop spontaneous seizures and are more susceptible to pharmacologically induced seizures using a γ-aminobutyric acid antagonist. Moreover, wv/− mice exhibit much milder cerebellar abnormalities than wv/wv mice, indicating a dosage effect of the GIRK2 G156S mutation. Our results indicate that the weaver phenotypes arise from a gain-of-function mutation of GIRK2 and that GIRK1 and GIRK2 are important mediators of neuronal excitability in vivo.     

Nonmuscle myosin II-B is required for normal development of the mouse heart

We used targeted gene disruption in mice to ablate nonmuscle myosin heavy chain B (NMHC-B), one of the two isoforms of nonmuscle myosin II present in all vertebrate cells. Approximately 65% of the NMHC-B^−/− embryos died prior to birth, and those that were born suffered from congestive heart failure and died during the first day. No abnormalities were detected in NMHC-B^+/− mice. The absence of NMHC-B resulted in a significant increase in the transverse diameters of the cardiac myocytes from 7.8 ± 1.8 μm (right ventricle) and 7.8 ± 1.3 μm (left ventricle) in NMHC-B^+/+ and B^+/− mice to 14.7 ± 1.1 μm and 13.8 ± 2.3 μm, respectively, in NMHC-B^−/− mice (in both cases, P < 0.001). The increase in size of the cardiac myocytes was seen as early as embryonic day 12.5 (4.5 ± 0.2 μm for NMHC-B^+/+ and B^+/− vs. 7.2 ± 0.6 μm for NMHC-B^−/− mice (P < 0.01)). Six of seven NMHC-B^−/− newborn mice analyzed by serial sectioning also showed structural cardiac defects, including a ventricular septal defect, an aortic root that either straddled the defect or originated from the right ventricle, and muscular obstruction to right ventricular outflow. Some of the hearts of NMHC-B^−/− mice showed evidence for up-regulation of NMHC-A protein. These studies suggest that nonmuscle myosin II-B is required for normal cardiac myocyte development and that its absence results in structural defects resembling, in part, two common human congenital heart diseases, tetralogy of Fallot and double outlet right ventricle.     

Predominance of the α1D subunit in L-type voltage-gated Ca2+ channels of hair cells in the chicken’s cochlea

The voltage-gated Ca²⁺ channels that effect tonic release of neurotransmitter from hair cells have unusual pharmacological properties: unlike most presynaptic Ca²⁺ channels, they are sensitive to dihydropyridines and therefore are L-type. To characterize these Ca²⁺ channels, we investigated the expression of L-type α₁ subunits in hair cells of the chicken’s cochlea. In PCRs with five different pairs of degenerate primers, we always obtained α_1D products, but only once an α_1C product and never an α_1S product. A full-length α_1D mRNA sequence was assembled from overlapping PCR products; the predicted amino acid sequence of the α_1D subunit was about 90% identical to those of the mammalian α_1D subunits. In situ hybridization confirmed that the α_1D mRNA is present in hair cells. By using a quantitative PCR assay, we determined that the α_1D mRNA is 100–500 times more abundant than the α_1C mRNA. We conclude that most, if not all, voltage-gated Ca²⁺ channels in hair cells contain an α_1D subunit. Furthermore, we propose that the α_1D subunit plays a hitherto undocumented role at tonic synapses.     

Association of calcium channel α1S and β1a subunits is required for the targeting of β1a but not of α1S into skeletal muscle triads

The skeletal muscle L-type Ca²⁺ channel is a complex of five subunits that is specifically localized in the triad. Its primary function is the rapid activation of Ca²⁺ release from cytoplasmic stores in a process called excitation-contraction coupling. To study the role of α_1S–β_1a interactions in the incorporation of the functional channel complex into the triad, α_1S and β_1a [or a β_1a-green fluorescent protein (GFP) fusion protein] were expressed alone and in combination in myotubes of the dysgenic cell line GLT. βGFP expressed in dysgenic myotubes that lack the skeletal muscle α_1S subunit was diffusely distributed in the cytoplasm. On coexpression with the α_1S subunit βGFP distribution became clustered and colocalized with α_1S immunofluorescence. Based on the colocalization of βGFP and α_1S with the ryanodine receptor the clusters were identified as T-tubule/sarcoplasmic reticulum junctions. Expression of α_1S with and without β_1a restored Ca²⁺ currents and depolarization-induced Ca²⁺ release. The translocation of βGFP from the cytoplasm into the junctions failed when βGFP was coexpressed with α_1S mutants in which the β interaction domain had been altered (α_1S-Y366S) or deleted (α_1S-Δ351–380). Although α_1S-Y366S did not associate with βGFP it was incorporated into the junctions, and it restored Ca²⁺ currents and depolarization-induced Ca²⁺ release. Thus, β_1a requires the association with the β interaction domain in the I–II cytoplasmic loop of α_1S for its own incorporation into triad junctions, but stable α_1S–β_1a association is not necessary for the targeting of α_1S into the triads or for its normal function in Ca²⁺ conductance and excitation-contraction coupling.     

Independent signals regulate development of primary and secondary follicle structure in spleen and mesenteric lymph node

Lymphotoxin-α-deficient (LT-α^−/−) mice manifest congenital absence of lymph nodes (LNs) and Peyer’s patches and disturbed spleen follicle structure. The splenic white pulp areas show loss of discrete T and B lymphocyte zones, of follicular dendritic cell (FDC) clusters, and of germinal centers (GCs). Tumor necrosis factor receptor I-deficient (TNFR-I^−/−) mice show similar absence of FDC clusters and GCs but retain segregation of T and B cell zones. Rarely are mesenteric LNs found in LT-α^−/− mice. These mesenteric LNs show segregation of T and B cell zones similar to wild-type mice. In contrast, mesenteric LNs in TNFR-I^−/− mice manifest grossly disturbed organization of T and B cells. Both LT-α^−/− and TNFR-I^−/− mice lacked FDC clusters in LNs and spleen. Interestingly, although both LT-α^−/− and TNFR-I^−/− mice that had been immunized with sheep red blood cells failed to form GCs in the spleen, they both developed GC-like clusters of peanut agglutinin-positive (PNA⁺) cells in their LNs. Furthermore, when lethally irradiated recombination activating gene (RAG)-1-deficient (RAG-1^−/−) mice that had received spleen cells from LT-α^−/− mice were immunized with sheep red blood cells, they failed to generate PNA⁺ clusters in the reconstituted spleen but showed robust PNA⁺ clusters in the reconstituted LNs. These data demonstrate that the signals that regulate the development of distinct T and B cell zones as well as the signals that regulate B cell activation to produce clusters of PNA⁺ cells differ between the spleen and LNs.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Ca2+-sensitive inactivation of L-type Ca2+ channels depends on multiple cytoplasmic amino acid sequences of the α1C subunit

Ca²⁺-dependent inactivation of Ca²⁺ currents is a physiological phenomenon widely associated with L-type Ca²⁺ channels. Although the pore-forming α_1C subunit of the channel is the target for Ca²⁺ binding, the amino acid sequences involved in the binding and/or in the coordination of Ca²⁺-dependent inactivation are still unclear. Based on previous experiments, we have prepared truncation mutants of a human α_1C subunit by systematically deleting an EF-hand motif and sequences in a segment of 80 amino acids in the carboxyl-terminal tail. We found that the rate as well as the Ca²⁺ dependence of inactivation of currents through these mutated channels were very different. We have identified three amino acid sequences, the presence of which is important for Ca²⁺-dependent inactivation: (i) a putative Ca²⁺-binding EF-hand motif, (ii) two hydrophilic residues (asparagine and glutamic acid) 77–78 amino acids downstream of the EF-hand motif, and (iii) a putative IQ calmodulin binding motif. We suggest that Ca²⁺-dependent inactivation is a cooperative process involving several amino acid sequences in cytoplasmic segments of the α_1C subunit.