Increased CaVβ1a expression with aging contributes to skeletal muscle weakness

Ca²⁺ release from the sarcoplasmic reticulum (SR) into the cytosol is a crucial part of excitation–contraction (E‐C) coupling. Excitation–contraction uncoupling, a deficit in Ca²⁺ release from the SR, is thought to be responsible for at least some of the loss in specific force observed in aging skeletal muscle. Excitation–contraction uncoupling may be caused by alterations in expression of the voltage‐dependent calcium channel α_1s (Ca_V1.1) and β_1a (Ca_Vβ_1a) subunits, both of which are necessary for E‐C coupling to occur. While previous studies have found Ca_V1.1 expression declines in old rodents, Ca_Vβ_1a expression has not been previously examined in aging models. Western blot analysis shows a substantial increase of Ca_Vβ_1a expression over the full lifespan of Friend Virus B (FVB) mice. To examine the specific effects of Ca_Vβ_1a overexpression, a Ca_Vβ_1a‐YFP plasmid was electroporated in vivo into young animals. The resulting increase in expression of Ca_Vβ_1a corresponded to decline of Ca_V1.1 over the same time period. YFP fluorescence, used as a measure of Ca_Vβ_1a‐YFP expression in individual fibers, also showed an inverse relationship with charge movement, measured using the whole‐cell patch‐clamp technique. Specific force was significantly reduced in young Ca_Vβ_1a‐YFP electroporated muscle fibers compared with sham‐electroporated, age‐matched controls. siRNA interference of Ca_Vβ_1a in young muscles reduced charge movement, while charge movement in old was restored to young control levels. These studies imply Ca_Vβ_1a serves as both a positive and negative regulator Ca_V1.1 expression, and that endogenous overexpression of Ca_Vβ_1a during old age may play a role in the loss of specific force.     

Functional Coupling of the γ-Aminobutyric AcidB Receptor with Calcium Ion Channel and GTP-Binding Protein and Its Alteration Following Solubilization of the γ-Aminobutyric AcidB Receptor

The coupling mechanism of the gamma-aminobutyric acid (GABA)B receptor, one of the subtypes of GABA receptors, with calcium ion channel and GTP-binding protein was examined using a crude synaptic membrane (P2) fraction from the bovine cerebral cortex and a fraction solubilized with sodium deoxycholate. In the P2 fraction, [3H]GABA binding to the GABAB receptor was increased significantly by the addition of calcium ion, and this enhancement was accentuated further by calcium ion channel blockers such as nicardipine and diltiazem. In contrast, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, did not affect on the calcium ion-induced enhancement of GABAB receptor binding. These results suggest that the GABAB receptor may be functionally coupled with the calcium ion channel, which exhibits an inhibitory modulation against the receptor. On the other hand, GABAB receptor binding, which was noncompetitively inhibited by guanine nucleotides such as GTP, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), guanosine 5'-(beta, gamma-imido)triphosphate [Gpp(NH)p], and GDP, was competitively inhibited by (-)-baclofen. Although the affinity of (-)-baclofen for the GABAB receptor was decreased in the presence of GTP, pretreatment of the P2 fraction with islet-activating protein (IAP) eliminated the effect of GTP. In addition, GABA and (-)-baclofen induced an increase of GTPase activity in the P2 fraction, and this increase was also eliminated by treatment with IAP. These results suggest that the GABAB receptor may also be functionally coupled with IAP-sensitive GTP-binding protein. Treatment of the P2 fraction with sodium deoxycholate resulted in the highest solubilization of GABAB receptor among various detergents examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

α2-Macroglobulin Complexes with and Mediates the Endocytosis of β-Amyloid Peptide via Cell Surface Low-Density Lipoprotein Receptor-Related Protein

Abstract: A primary histopathological feature of Alzheimer's disease is the accumulation of β-amyloid (Aβ) in the brain of afflicted individuals. However, Aβ is produced continuously as a soluble protein in healthy individuals where it is detected in serum and CSF, suggesting the existence of cellular clearance mechanisms that normally prevent its accumulation and aggregation. Here, we demonstrate that Aβ forms stable complexes with activated α₂-macroglobulin (α₂M^⋆), a physiological ligand for the low-density lipoprotein receptor-related protein (LRP) that is abundantly expressed in the CNS. These α₂M^⋆/¹²⁵I-Aβ complexes are immunoreactive with both anti-Aβ and anti-α₂M IgG and are stable under various pH conditions, sodium dodecyl sulfate, reducing agents, and boiling. We demonstrate that α₂M^⋆/¹²⁵I-Aβ complexes can be degraded by glioblastoma cells and fibroblasts via LRP, because degradation is partially inhibited by receptor-associated protein (RAP), an antagonist of ligand interactions with LRP. In contrast, the degradation of free ¹²⁵I-Aβ is not inhibited by RAP and thus must be mediated via an LRP-independent pathway. These results suggest that LRP can function as a clearance receptor for Aβ via a physiological ligand.     

Fluctuation in O-GlcNAcylation inactivates STIM1 to reduce store-operated calcium ion entry via down-regulation of Ser621 phosphorylation

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca²⁺ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser⁶²¹ and Thr⁶²⁶ were O-GlcNAcylated and that Thr⁶²⁶ was O-GlcNAcylated in the steady state but Ser⁶²¹ was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser⁶²¹. Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser⁶²¹. These data suggest that both decrease in O-GlcNAcylation at Thr⁶²⁶ and increase in O-GlcNAcylation at Ser⁶²¹ in STIM1 lead to impairment of SOCE activity through decrease in Ser⁶²¹ phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.     

β-Lumicolchicine Interacts with the Benzodiazepine Binding Site to Potentiate GABAA Receptor-Mediated Currents

Abstract: An analogue of colchicine,β-lumicolchicine, does not bind tubulin or disrupt microtubules. However, this compound is not pharmacologically completely inactive. β-Lumicolchicine was found to competitively inhibit [³H]flunitrazepam binding and to enhance muscimol-stimulated ³⁶Cr-uptake in mouse cerebral cortical microsacs. It also markedly potentiated GABA responses in Xenopusoocytes expressing human α₁β₂γ_2S, but not α₁β₂, GABA_A receptor subunits; this potentiation was reversed by the benzodiazepine receptor antagonist flumazenil. These results strongly suggest a direct effect of β-Lumicolchicine on the GABA_A receptor/chloride channel complex and caution that it possesses pharmacological effects, despite its inability to disrupt microtubules. Furthermore, β-Lumicolchicine is structurally unrelated to benzodiazepines or quinolines and may provide a novel approach to the synthesis of ligands for this receptor.     

Galectin-1 attenuates cardiomyocyte hypertrophy through splice-variant specific modulation of CaV1.2 calcium channel

Pressure overload-induced cardiac hypertrophy occurs in response to chronic blood pressure increase, and dysfunction of Ca_V1.2 calcium channel involves in cardiac hypertrophic processes by perturbing intracellular calcium concentration ([Ca²⁺]_i) and calcium-dependent signaling. As a carbohydrate-binding protein, galectin-1 (Gal-1) is found to bind with Ca_V1.2 channel, which regulates vascular Ca_V1.2 channel functions and blood pressure. However, the potential roles of Gal-1 in cardiac Ca_V1.2 channel (Ca_V1.2_CM) and cardiomyocyte hypertrophy remain elusive. By whole-cell patch clamp, we find Gal-1 decreases the I_Ca,L with or without isoproterenol (ISO) application by reducing the channel membrane expression in neonatal rat ventricular myocytes (NRVMs). Moreover, Gal-1 could inhibit the current densities of Ca_V1.2_CM by an alternative exon 9*-dependent manner in heterologously expressed HEK293 cells. Of significance, overexpression of Gal-1 diminishes ISO or KCl-induced [Ca²⁺]_i elevation and attenuates ISO-induced hypertrophy in NRVMs. Mechanistically, Gal-1 decreases the ISO or Bay K8644-induced phosphorylation of intracellular calcium-dependent signaling proteins δCaMKII and HDAC4, and inhibits ISO-triggered translocation of HDAC4 in NRVMs. Pathologically, we observe that the expressions of Gal-1 and Ca_V1.2_E9* channels are synchronously increased in rat hypertrophic cardiomyocytes and hearts. Taken together, our study indicates that Gal-1 reduces the channel membrane expression to inhibit the currents of Ca_V1.2_CM in a splice-variant specific manner, which diminishes [Ca²⁺]_i elevation, and attenuates cardiomyocyte hypertrophy by inhibiting the phosphorylation of δCaMKII and HDAC4. Furthermore, our work suggests that dysregulated Gal-1 and Ca_V1.2 alternative exon 9* might be attributed to the pathological processes of cardiac hypertrophy, and provides a potential anti-hypertrophic target in the heart.     

Opioid-Induced Increase in [Ca2+]i in ND8-47 Neuroblastoma × Dorsal Root Ganglion Hybrid Cells Is Mediated Through G Protein-Coupled δ-Opioid Receptors and Desensitized by Chronic Exposure to Opioid

Abstract: δ-Receptor agonists induce a concentration-dependent increase in intracellular calcium concentration ([Ca²⁺]_i) in ND8-47 cells by activating dihydropyridine-sensitive Ca²⁺ channels. The role of G proteins in transducing the opioid effect has been studied. Pretreatment of cells with pertussis toxin (100 ng/ml, 24 h) almost completely blocked [d -Ser²,Leu⁵]enkephalin-Thr (DSLET)-induced increase in [Ca²⁺]_i. Cholera toxin (10 nM, 24 h) had no effect on DSLET-induced response. Pretreatment of the cells with 1 µM DSLET for 1 h resulted in a 30% inhibition of DSLET-induced increase in [Ca²⁺]_i and a 78% inhibition after exposure for 24 h. After 1 h of exposure to DSLET, there was a decrease in agonist affinity with no significant changes in receptor density. Cells exposed to 1 µM DSLET for 24 h demonstrate a nearly 90% decrease in [³H]diprenorphine binding, with a decrease in affinity for agonist at the remaining binding sites. G protein subunits α_i2, α_i3, α_s, and α_q were detected in ND8-47 cell membranes by western blot; α_o and α_i1 were not present. Chronic DSLET treatment had no significant effect on the quantity of each of the α-subunits. These results suggest that the DSLET-induced increase in [Ca²⁺]_i is mediated through pertussis toxin-sensitive G proteins (probably G_i2 or G_i3) and the attenuation of this response in chronically treated cells is associated with a relatively rapid reduction in receptor affinity to DSLET and a slow reduction in receptor density.