Homeostatic Depression Shows Heightened Sensitivity to Synaptic Calcium

Synapses and circuits rely on homeostatic forms of regulation in order to transmit meaningful information. The Drosophila melanogaster neuromuscular junction (NMJ) is a well-studied synapse that shows robust homeostatic control of function. Most prior studies of homeostatic plasticity at the NMJ have centered on presynaptic homeostatic potentiation (PHP). PHP happens when postsynaptic muscle neurotransmitter receptors are impaired, triggering retrograde signaling that causes an increase in presynaptic neurotransmitter release. As a result, normal levels of evoked excitation are maintained. The counterpart to PHP at the NMJ is presynaptic homeostatic depression (PHD). Overexpression of the Drosophila vesicular glutamate transporter (VGlut) causes an increase in the amplitude of spontaneous events. PHD happens when the synapse responds to the challenge by decreasing quantal content (QC) during evoked neurotransmission—again, resulting in normal levels of postsynaptic excitation. We hypothesized that there may exist a class of molecules that affects both PHP and PHD. Impairment of any such molecule could hurt a synapse’s ability to respond to any significant homeostatic challenge. We conducted an electrophysiology-based screen for blocks of PHD. We did not observe a block of PHD in the genetic conditions screened, but we found loss-of-function conditions that led to a substantial deficit in evoked amplitude when combined with VGlut overexpression. The conditions causing this phenotype included a double heterozygous loss-of-function condition for genes encoding the inositol trisphosphate receptor (IP3R —itpr) and ryanodine receptor (RyR). IP3Rs and RyRs gate calcium release from intracellular stores. Pharmacological agents targeting IP3R and RyR recapitulated the genetic losses of these factors, as did lowering calcium levels from other sources. Our data are consistent with the idea that the homeostatic signaling process underlying PHD is especially sensitive to levels of calcium at the presynapse.

Regulation of longevity by depolarization-induced activation of PLC-β–IP3R signaling in neurons

Mitochondrial ATP production is a well-known regulator of neuronal excitability. The reciprocal influence of plasma-membrane potential on ATP production, however, remains poorly understood. Here, we describe a mechanism by which depolarized neurons elevate the somatic ATP/ADP ratio in Drosophila glutamatergic neurons. We show that depolarization increased phospholipase-Cβ (PLC-β) activity by promoting the association of the enzyme with its phosphoinositide substrate. Augmented PLC-β activity led to greater release of endoplasmic reticulum Ca2+ via the inositol trisphosphate receptor (IP3R), increased mitochondrial Ca2+ uptake, and promoted ATP synthesis. Perturbations that decoupled membrane potential from this mode of ATP synthesis led to untrammeled PLC-β–IP3R activation and a dramatic shortening of Drosophila lifespan. Upon investigating the underlying mechanisms, we found that increased sequestration of Ca2+ into endolysosomes was an intermediary in the regulation of lifespan by IP3Rs. Manipulations that either lowered PLC-β/IP3R abundance or attenuated endolysosomal Ca2+ overload restored animal longevity. Collectively, our findings demonstrate that depolarization-dependent regulation of PLC-β–IP3R signaling is required for modulation of the ATP/ADP ratio in healthy glutamatergic neurons, whereas hyperactivation of this axis in chronically depolarized glutamatergic neurons shortens animal lifespan by promoting endolysosomal Ca2+ overload.

Inositol Trisphosphate Receptors and Nuclear Calcium in Atrial Fibrillation

Rationale: The mechanisms underlying atrial fibrillation (AF), the most common clinical arrhythmia, are poorly understood. Nucleoplasmic Ca 2+ regulates gene-expression, but the nature and significance of nuclear Ca 2+ -changes in AF are largely unknown. Objective: To elucidate mechanisms by which AF alters atrial cardiomyocyte (CM) nuclear Ca 2+ ([Ca 2+ ] Nuc ) and Ca 2+ /calmodulin-dependent protein kinase-II (CaMKII)-related signaling. Methods and Results: Atrial CMs were isolated from control and AF-dogs (kept in AF by atrial tachypacing [600 bpm x 1 week]). [Ca 2+ ] Nuc and cytosolic [Ca 2+ ] (Ca 2+ ] Cyto ) were recorded via confocal microscopy. Diastolic [Ca 2+ ] Nuc was greater than [Ca 2+ ] Cyto under control conditions, while resting [Ca 2+ ] Nuc was similar to [Ca 2+ ] Cyto ; both diastolic and resting [Ca 2+ ] Nuc increased with AF. Inositol-trisphosphate-receptor (IP 3 R) stimulation produced larger [Ca 2+ ] Nuc increases in AF versus control CMs, and IP 3 R-blockade suppressed the AF-related [Ca 2+ ] Nuc -differences. AF upregulated nuclear protein-expression of IP 3 R-type 1 (IP 3 R1) and of phosphorylated CaMKII (immunohistochemistry and immunoblot), while decreasing the nuclear/cytosolic expression-ratio for histone deacetylase type-4 (HDAC4). Isolated atrial CMs tachypaced at 3 Hz for 24 hours mimicked AF-type [Ca 2+ ] Nuc changes and L-type calcium current (ICaL) decreases versus 1-Hz-paced CMs; these changes were prevented by IP3R knockdown with short-interfering RNA directed against IP 3 R1. Nuclear/cytosolic HDAC4 expression-ratio was decreased by 3-Hz pacing, while nuclear CaMKII and HDAC4 phosphorylation were increased. Either CaMKII-inhibition (by autocamtide-2-related peptide) or IP 3 R-knockdown prevented the CaMKII-hyperphosphorylation and nuclear-to-cytosolic HDAC4 shift caused by 3-Hz pacing. In human atrial CMs from AF patients, nuclear IP 3 R1-expression was significantly increased, with decreased nuclear/non-nuclear HDAC4 ratio. MicroRNA-26a was predicted to target ITPR1 (confirmed by Luciferase assay) and was downregulated in AF atrial CMs; microRNA-26a silencing reproduced AF-induced IP3R1 upregulation and nuclear diastolic Ca 2+ -loading. Conclusions: AF increases atrial CM nucleoplasmic Ca 2+ -handling by IP 3 R1-upregulation involving miR-26a, leading to enhanced IP 3 R1-CaMKII-HDAC4 signaling and I CaL -downregulation.

IRAG1 Deficient Mice Develop PKG1β Dependent Pulmonary Hypertension

PKGs are serine/threonine kinases. PKG1 has two isoforms—PKG1α and β. Inositol trisphosphate receptor (IP3R)-associated cGMP-kinase substrate 1 (IRAG1) is a substrate for PKG1β. IRAG1 is also known to further interact with IP3RI, which mediates intracellular Ca2+ release. However, the role of IRAG1 in PH is not known. Herein, WT and IRAG1 KO mice were kept under normoxic or hypoxic (10% O2) conditions for five weeks. Animals were evaluated for echocardiographic variables and went through right heart catheterization. Animals were further sacrificed to prepare lungs and right ventricular (RV) for immunostaining, western blotting, and pulmonary artery smooth muscle cell (PASMC) isolation. IRAG1 is expressed in PASMCs and downregulated under hypoxic conditions. Genetic deletion of IRAG1 leads to RV hypertrophy, increase in RV systolic pressure, and RV dysfunction in mice. Absence of IRAG1 in lung and RV have direct impacts on PKG1β expression. Attenuated PKG1β expression in IRAG1 KO mice further dysregulates other downstream candidates of PKG1β in RV. IRAG1 KO mice develop PH spontaneously. Our results indicate that PKG1β signaling via IRAG1 is essential for the homeostasis of PASMCs and RV. Disturbing this signaling complex by deleting IRAG1 can lead to RV dysfunction and development of PH in mice.

A class of synaptic signaling molecules required for homeostatic potentiation also tunes homeostatic depression

Synapses and circuits rely on homeostatic forms of regulation in order to transmit meaningful information. The Drosophila melanogaster neuromuscular junction (NMJ) is a well-studied synapse that shows robust homeostatic control of function. Most prior studies of homeostatic plasticity at the NMJ have centered on presynaptic homeostatic potentiation (PHP). PHP happens when postsynaptic muscle neurotransmitter receptors are impaired, triggering retrograde signaling that causes an increase in presynaptic neurotransmitter release. As a result, normal levels of evoked excitation are maintained. The counterpart to PHP at the NMJ is presynaptic homeostatic depression (PHD). Overexpression of the Drosophila vesicular glutamate transporter (VGlut) causes an increase in the amplitude of spontaneous events. PHD happens when the synapse responds to the challenge by decreasing quantal content during evoked neurotransmission, again, resulting in normal levels of postsynaptic excitation. We hypothesized that there may exist a class of molecules that affects both PHP and PHD. Impairment of any such molecule could hurt the ability of a synapse to respond to any significant homeostatic challenge. We conducted an electrophysiology-based screen for blocks of PHD. While we did not observe a block of PHD in the genetic conditions screened, we instead found loss-of-function conditions that led to excess depression, i.e., a substantial deficit in evoked amplitude when combined with VGlut overexpression. The conditions causing this phenotype included a double heterozygous loss-of-function condition for genes encoding the inositol trisphosphate receptor (IP3R, itpr) and ryanodine receptor (RyR). IP3Rs and RyRs gate calcium release from intracellular stores. Pharmacological agents targeting IP3R and RyR recapitulated the genetic losses of these factors, as did lowering calcium levels from other sources. Our data are consistent with the idea that some factors required for homeostatic potentiation are also required for the synapse to achieve appropriate levels of homeostatic depression. Loss of such factors may disorient compensatory plasticity signals.

Isoform-specific regulation of HCN4 channels by a family of endoplasmic reticulum proteins

Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary proteins. Here, we report the discovery of a family of endoplasmic reticulum transmembrane proteins that interact with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. Despite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depolarizing shift in the voltage-dependence of HCN4 activation in response to binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage-dependence of activation in the absence of cAMP. The mechanisms of action of LRMP and IRAG are novel; they are independent of trafficking and cAMP binding, and they are specific to the HCN4 isoform. We also found that IRAG is highly expressed in the mouse sinoatrial node where computer modeling predicts that its presence increases HCN4 availability. Our results suggest important roles for LRMP and IRAG in regulation of cellular excitability and as tools for advancing mechanistic understanding of HCN4 channel function.Significance statementThe pore-forming subunits of ion channels are regulated by auxiliary interacting proteins. Hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channels are critical determinants of electrical excitability in many types of cells including neurons and cardiac pacemaker cells. Here we report the discovery of two novel HCN4 regulatory proteins. Despite their homology, the two proteins — lymphoid-restricted membrane protein (LRMP) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG) — have opposing effects on HCN4, causing loss- and gain-of-function, respectively. LRMP and IRAG are expected to play critical roles in regulation of physiological processes ranging from wakefulness to heart rate through their modulation of HCN4 channel function.

Extraocular muscle function is impaired in ryr3−/− mice

Calcium is an ubiquitous second messenger mediating numerous physiological processes, including muscle contraction and neuronal excitability. Ca2+ is stored in the ER/SR and is released into the cytoplasm via the opening of intracellular inositol trisphosphate receptor and ryanodine receptor calcium channels. Whereas in skeletal muscle, isoform 1 of the RYR is the main channel mediating calcium release from the SR leading to muscle contraction, the function of ubiquitously expressed ryanodine receptor 3 (RYR3) is far from clear; it is not known whether RYR3 plays a role in excitation–contraction coupling. We recently reported that human extraocular muscles express high levels of RYR3, suggesting that such muscles may be useful to study the function of this isoform of the Ca2+ channel. In the present investigation, we characterize the visual function of ryr3−/− mice. We observe that ablation of RYR3 affects both mechanical properties and calcium homeostasis in extraocular muscles. These changes significantly impact vision. Our results reveal for the first time an important role for RYR3 in extraocular muscle function.

Functional coupling of GABAA/B receptors and the channel TRPV4 mediates rapid progesterone signaling in the oviduct

The molecular mechanism by which progesterone (P4) modulates the transport of ova and embryos along the oviduct is not fully resolved. We report a rapid response to P4 and agonists of γ-aminobutyric acid receptors A and B (GABAA/B) in the mouse oviduct that was characterized by oscillatory Ca2+ signals and increased ciliary beat frequency (CBF). Pharmacological manipulation, genetic ablation, and siRNA-mediated knockdown in oviductal cells, as well as overexpression experiments in HEK 293T cells, confirmed the participation of the cationic channel TRPV4, different subunits of GABAA (α1 to α3, β2, and β3), and GABAB1 in P4-induced responses. TRPV4-mediated Ca2+ entry in close proximity to the inositol trisphosphate receptor was required to initiate and maintain Ca2+ oscillations after P4 binding to GABAA and transactivation of Gi/o protein–coupled GABAB receptors. Coimmunoprecipitation experiments and imaging of native tissue and HEK 293T cells demonstrated the close association of GABAA and GABAB1 receptors and the activation of Gi/o proteins in response to P4 and GABA receptor agonists, confirming a molecular mechanism in which P4 and GABAergic agonists cooperatively stimulate cilial beating.