Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+ homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+ and PO43- ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ ions stabilize the active state, PO43- ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.DOI: http://dx.doi.org/10.7554/eLife.13662.001
Obesity and high saturated fat intake increase the risk of heart failure and arrhythmias. The molecular mechanisms are poorly understood. We hypothesized that physiologic levels of saturated fat could increase mitochondrial reactive oxygen species (ROS) in cardiomyocytes, leading to abnormalities of calcium homeostasis and mitochondrial function. We investigated the effect of saturated fat on mitochondrial function and calcium homeostasis in isolated ventricular myocytes. The saturated fatty acid palmitate causes a decrease in mitochondrial respiration in cardiomyocytes. Palmitate, but not the monounsaturated fatty acid oleate, causes an increase in both total cellular ROS and mitochondrial ROS. Palmitate depolarizes the mitochondrial inner membrane and causes mitochondrial calcium overload by increasing sarcoplasmic reticulum calcium leak. Inhibitors of PKC or NOX2 prevent mitochondrial dysfunction and the increase in ROS, demonstrating that PKC-NOX2 activation is also required for amplification of palmitate induced-ROS. Cardiomyocytes from mice with genetic deletion of NOX2 do not have palmitate-induced ROS or mitochondrial dysfunction. We conclude that palmitate induces mitochondrial ROS that is amplified by NOX2, causing greater mitochondrial ROS generation and partial depolarization of the mitochondrial inner membrane. The abnormal sarcoplasmic reticulum calcium leak caused by palmitate could promote arrhythmia and heart failure. NOX2 inhibition is a potential therapy for heart disease caused by diabetes or obesity.
The use of selective serotonin reuptake inhibitors (SSRIs) has been associated with an increased risk of bone fracture, raising concerns about their increasingly broader usage. This deleterious effect is poorly understood and thus strategies to avoid this side effect remain elusive. We show here that fluoxetine (Flx), one of the most prescribed SSRI, acts on bone remodeling through two distinct mechanisms. Peripherally, Flx has antiresorptive properties, directly impairing osteoclast differentiation and function through a serotonin reuptake-independent Ca2+-calmodulin-NFATc1-dependent mechanism. With time, however, Flx also triggers a brain serotonin-dependent rise in sympathetic output that increases bone resorption sufficiently to counteract its local antiresorptive effect; thus leading to a net effect of impaired bone formation and bone loss. Accordingly, neutralizing this second mode of action through co-treatment with the β-blocker propranolol, while leaving the peripheral effect intact, prevents Flx-induced bone loss in mice. Hence, this study identifies a dual mode of action of SSRIs on bone remodeling and suggests a therapeutic strategy to block the deleterious effect on bone homeostasis from their chronic use.
Auxiliary  subunits modulate current properties and mediate the functional membrane expression of voltage-gated Ca 2؉ channels in heterologous cells. In brain, all four  isoforms are widely expressed, yet little is known about their specific roles in neuronal functions. Here, we investigated the expression and targeting properties of  subunits and their role in membrane expression of Ca V 1.2 ␣ 1 subunits in cultured hippocampal neurons. Quantitative reverse transcription-PCR showed equal expression, and immunofluorescence showed a similar distribution of all endogenous  subunits throughout dendrites and axons. High resolution microscopy of hippocampal neurons transfected with six different V5 epitope-tagged  subunits demonstrated that all  subunits were able to accumulate in synaptic terminals and to colocalize with postsynaptic Ca V 1.2, thus indicating a great promiscuity in ␣ 1 - interactions. In contrast, restricted axonal targeting of  1 and weak colocalization of  4b with Ca V 1.2 indicated isoform-specific differences in local channel complex formation. Membrane expression of external hemagglutinin epitope-tagged Ca V 1.2 was strongly enhanced by all  subunits in an isoform-specific manner. Conversely, mutating the ␣-interaction domain of Ca V 1.2 (W440A) abolished membrane expression and targeting into dendritic spines. This demonstrates that in neurons the interaction of a  subunit with the ␣-interaction domain is absolutely essential for membrane expression of ␣ 1 subunits, as well as for the subcellular localization of  subunits, which by themselves possess little or no targeting properties.Voltage-gated Ca 2ϩ channels (Ca V ) 3 provide key pathways for Ca 2ϩ entry into neurons and translate membrane depolarization into neurotransmitter secretion and gene regulation.Ca V s are composed of a pore-forming ␣ 1 subunit and the auxiliary ␣ 2 ␦ and  subunits (1). Whereas the ␣ 1 subunits are responsible for voltage sensing and ion conduction, the auxiliary subunits have been implicated in membrane targeting and modulation of channel properties (for review see Ref.2). Presynaptic Ca V s regulate neurotransmitter release (3), and postsynaptic Ca V s activate the transcriptional regulators cAMP-response element-binding protein (CREB) and nuclear factor of activated T-cells (NFAT) (4, 5) and thus modulate long term potentiation (6). These functions reflect both the diversity of Ca V isoforms expressed in brain (7-11) and their differential subcellular localization in neurons (12-15).Four distinct  isoforms have been identified (16 -19), all of which are expressed in brain (20 -23). They contain an Src homology 3 domain and a guanylate kinase domain (24 -27). However, the guanylate kinase fold is modified so that it can bind with high affinity to the so-called ␣-interaction domain (AID) in the intracellular I-II linker of Ca V ␣ 1 subunits (28, 29). The Src homology 3 and the guanylate kinase-like domains are highly conserved among the four genes encoding  subunits (Cacnb1-b4; Fig. 1C), whereas t...
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