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

2020 ◽  
Author(s):  
Colin H. Peters ◽  
Mallory E. Myers ◽  
Julie Juchno ◽  
Charlie Haimbaugh ◽  
Hicham Bichraoui ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Ion Channels ◽  
Membrane Protein ◽  
Cyclic Nucleotide ◽  
Voltage Dependence ◽  
Cardiac Pacemaker ◽  
Pacemaker Cells ◽  
Inositol Trisphosphate Receptor ◽  
Kinase Substrate ◽  
Trisphosphate Receptor

AbstractIon 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.

scholarly journals Evidence for the involvement of a small subregion of the endoplasmic reticulum in the inositol trisphosphate receptor-induced activation of Ca2+ inflow in rat hepatocytes

1999 ◽  
Vol 341 (2) ◽  
pp. 401 ◽  
Author(s):  
Roland B. GREGORY ◽  
Robert A. WILCOX ◽  
Leise A. BERVEN ◽  
Nicole C.R. VAN STRATEN ◽  
Gijs A. VAN DER MAREL ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Inositol Trisphosphate ◽  
Rat Hepatocytes ◽  
Inositol Trisphosphate Receptor ◽  
Trisphosphate Receptor

scholarly journals cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice

2015 ◽  
Vol 290 (34) ◽  
pp. 20880-20892 ◽  
Author(s):  
Hongwei Ma ◽  
Michael R. Butler ◽  
Arjun Thapa ◽  
Josh Belcher ◽  
Fan Yang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Er Stress ◽  
Cyclic Nucleotide ◽  
Protein Kinase G ◽  
Cng Channel ◽  
Inositol 1,4,5 Trisphosphate ◽  
Gated Channel ◽  
Trisphosphate Receptor ◽  
Deficient Mice

Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca2+ channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP3R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3−/−/Nrl−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP3R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca2+ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency.

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

2021 ◽  
Vol 118 (16) ◽  
pp. e2004253118
Author(s):  
Ching-On Wong ◽  
Nicholas E. Karagas ◽  
Jewon Jung ◽  
Qiaochu Wang ◽  
Morgan A. Rousseau ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Potential ◽  
Neuronal Excitability ◽  
Atp Synthesis ◽  
Inositol Trisphosphate Receptor ◽  
Atp Production ◽  
Glutamatergic Neurons ◽  
Reciprocal Influence ◽  
Underlying Mechanisms ◽  
Trisphosphate Receptor

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.

scholarly journals IRAG1 Deficient Mice Develop PKG1β Dependent Pulmonary Hypertension

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2280
Author(s):  
Siladitta Biswas ◽  
Baktybek Kojonazarov ◽  
Stefan Hadzic ◽  
Michael Majer ◽  
Ganimete Bajraktari ◽  
...  
Keyword(s):  
Systolic Pressure ◽  
Right Heart Catheterization ◽  
Heart Catheterization ◽  
Inositol Trisphosphate Receptor ◽  
Kinase Substrate ◽  
Signaling Complex ◽  
Hypoxic Conditions ◽  
Pulmonary Artery Smooth Muscle ◽  
Trisphosphate Receptor

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.

scholarly journals Statistical analysis of modal gating in ion channels

2014 ◽  
Vol 470 (2166) ◽  
pp. 20140030 ◽  
Author(s):  
Ivo Siekmann ◽  
James Sneyd ◽  
Edmund J. Crampin
Keyword(s):  
Ion Channels ◽  
Single Channel ◽  
Channel Activity ◽  
Experimental Conditions ◽  
Inositol Trisphosphate Receptor ◽  
Statistical Framework ◽  
Modal Gating ◽  
Opening And Closing ◽  
Trisphosphate Receptor ◽  
Different Levels

Ion channels regulate the concentrations of ions within cells. By stochastically opening and closing its pore, they enable or prevent ions from crossing the cell membrane. However, rather than opening with a constant probability, many ion channels switch between several different levels of activity even if the experimental conditions are unchanged. This phenomenon is known as modal gating: instead of directly adapting its activity, the channel seems to mix sojourns in active and inactive modes in order to exhibit intermediate open probabilities. Evidence is accumulating that modal gating rather than modulation of opening and closing at a faster time scale is the primary regulatory mechanism of ion channels. However, currently, no method is available for reliably calculating sojourns in different modes. In order to address this challenge, we develop a statistical framework for segmenting single-channel datasets into segments that are characteristic for particular modes. The algorithm finds the number of mode changes, detects their locations and infers the open probabilities of the modes. We apply our approach to data from the inositol-trisphosphate receptor. Based upon these results, we propose that mode changes originate from alternative conformational states of the channel protein that determine a certain level of channel activity.

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

2020 ◽  
Vol 117 (30) ◽  
pp. 18079-18090
Author(s):  
Colin H. Peters ◽  
Mallory E. Myers ◽  
Julie Juchno ◽  
Charlie Haimbaugh ◽  
Hicham Bichraoui ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Voltage Dependence ◽  
Cardiac Pacemaker ◽  
Cell Types ◽  
The Body ◽  
Loss Of Function ◽  
Membrane Excitability ◽  
Homologous Proteins ◽  
Endogenous Expression ◽  
Specific Regulation

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 (ER) transmembrane proteins that associate with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, and 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 in response to the binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage dependence in the absence of cAMP. The mechanisms of action of LRMP and IRAG 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 current. Our results suggest important roles for LRMP and IRAG in the regulation of cellular excitability, as tools for advancing mechanistic understanding of HCN4 channel function, and as possible scaffolds for coordination of signaling pathways.

A second S4 movement opens hyperpolarization-activated HCN channels

2021 ◽  
Vol 118 (37) ◽  
pp. e2102036118
Author(s):  
Xiaoan Wu ◽  
Rosamary Ramentol ◽  
Marta E. Perez ◽  
Sergei Yu Noskov ◽  
H. Peter Larsson
Keyword(s):  
Sea Urchin ◽  
Cyclic Nucleotide ◽  
Voltage Dependence ◽  
Rhythmic Activity ◽  
Hcn Channels ◽  
Molecular Models ◽  
Transmembrane Segment ◽  
Pacemaker Cells ◽  
Gate Opening ◽  
Channel Opening

Rhythmic activity in pacemaker cells, as in the sino-atrial node in the heart, depends on the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. As in depolarization-activated K+ channels, the fourth transmembrane segment S4 functions as the voltage sensor in hyperpolarization-activated HCN channels. But how the inward movement of S4 in HCN channels at hyperpolarized voltages couples to channel opening is not understood. Using voltage clamp fluorometry, we found here that S4 in HCN channels moves in two steps in response to hyperpolarizations and that the second S4 step correlates with gate opening. We found a mutation in sea urchin HCN channels that separate the two S4 steps in voltage dependence. The E356A mutation in S4 shifts the main S4 movement to positive voltages, but channel opening remains at negative voltages. In addition, E356A reveals a second S4 movement at negative voltages that correlates with gate opening. Cysteine accessibility and molecular models suggest that the second S4 movement opens up an intracellular crevice between S4 and S5 that would allow radial movement of the intracellular ends of S5 and S6 to open HCN channels.

scholarly journals SLC26A9 is selected for endoplasmic reticulum associated degradation (ERAD) via Hsp70-dependent targeting of the soluble STAS domain

2021 ◽  
Author(s):  
Patrick G Needham ◽  
Jennifer L Goeckeler-Fried ◽  
Casey Zhang ◽  
Zhihao Sun ◽  
Adam R Wetzel ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Ion Channels ◽  
Membrane Protein ◽  
Secretory Pathway ◽  
Expression System ◽  
Chloride Ions ◽  
Substrate Selection ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Stas Domain

SLC26A9, a member of the solute carrier protein family, transports chloride ions across various epithelia. SLC26A9 also associates with other ion channels and transporters linked to human health, and in some cases these heterotypic interactions are essential to support the biogenesis of both proteins. Therefore, understanding how this complex membrane protein is initially folded might provide new therapeutic strategies to overcome deficits in the function of SLC26A9 partners, one of which is associated with Cystic Fibrosis. To this end, we developed a novel yeast expression system for SLC26A9. This facile system has been used extensively with other ion channels and transporters to screen for factors that oversee protein folding checkpoints. As commonly observed for other channels and transporters, we first noted that a substantial fraction of SLC26A9 is targeted for endoplasmic reticulum associated degradation (ERAD), which destroys folding-compromised proteins in the early secretory pathway. We next discovered that ERAD selection requires the Hsp70 chaperone, which can play a vital role in ERAD substrate selection. We then created SLC26A9 mutants and found that the transmembrane-rich domain of SLC26A9 was quite stable, whereas the soluble cytosolic STAS domain was responsible for Hsp70-dependent ERAD. To support data obtained in the yeast model, we were able to recapitulate Hsp70-facilitated ERAD of the STAS domain in human tissue culture cells. These results indicate that a critical barrier to nascent membrane protein folding can reside within a specific soluble domain, one that is monitored by components associated with the ERAD machinery.

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