scholarly journals Rab11-dependent recycling of calcium channels is mediated by auxiliary subunit α2δ-1 but not α2δ-3

2021 ◽  
Author(s):  
James O. Meyer ◽  
Annette C. Dolphin

AbstractN-type voltage-gated calcium channels (CaV2.2) are predominantly expressed at presynaptic terminals, and their function is regulated by auxiliary α2δ and β subunits. All four mammalian α2δ subunits enhance calcium currents through CaV1 and CaV2 channels, and this increase is attributed, in part, to increased CaV expression at the plasma membrane. In the present study we provide evidence that α2δ-1, like α2δ-2, is recycled to the plasma membrane through a Rab11a-dependent endosomal recycling pathway. Using a dominant-negative Rab11a mutant, Rab11a(S25N), we show that α2δ-1 increases plasma membrane CaV2.2 expression by increasing the rate and extent of net forward CaV2.2 trafficking in a Rab11a-dependent manner. Dominant-negative Rab11a also reduces the ability of α2δ-1 to increase CaV2.2 expression on the cell-surface of hippocampal neurites. In contrast, α2δ-3 does not enhance rapid forward CaV2.2 trafficking, regardless of whether Rab11a(S25N) is present. In addition, whole-cell CaV2.2 currents are reduced by co-expression of Rab11a(S25N) in the presence of α2δ-1, but not α2δ-3. Taken together these data suggest that α2δ subtypes participate in distinct trafficking pathways which in turn influence the localisation and function of CaV2.2.Summary statementThe calcium channel auxiliary subunit α2δ-1 but not α2δ-3 participates in Rab11a-dependent recycling, which in turn influences the localisation and function of CaV2.2.

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
James O. Meyer ◽  
Annette C. Dolphin

AbstractN-type voltage-gated calcium channels (CaV2.2) are predominantly expressed at presynaptic terminals, and their function is regulated by auxiliary α2δ and β subunits. All four mammalian α2δ subunits enhance calcium currents through CaV1 and CaV2 channels, and this increase is attributed, in part, to increased CaV expression at the plasma membrane. In the present study we provide evidence that α2δ-1, like α2δ-2, is recycled to the plasma membrane through a Rab11a-dependent endosomal recycling pathway. Using a dominant-negative Rab11a mutant, Rab11a(S25N), we show that α2δ-1 increases plasma membrane CaV2.2 expression by increasing the rate and extent of net forward CaV2.2 trafficking in a Rab11a-dependent manner. Dominant-negative Rab11a also reduces the ability of α2δ-1 to increase CaV2.2 expression on the cell-surface of hippocampal neurites. In contrast, α2δ-3 does not enhance rapid forward CaV2.2 trafficking, regardless of whether Rab11a(S25N) is present. In addition, whole-cell CaV2.2 currents are reduced by co-expression of Rab11a(S25N) in the presence of α2δ-1, but not α2δ-3. Taken together these data suggest that α2δ subtypes participate in distinct trafficking pathways which in turn influence the localisation and function of CaV2.2.


2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Vendula Ficelova ◽  
Ivana A. Souza ◽  
Leos Cmarko ◽  
Maria A. Gandini ◽  
Robin N. Stringer ◽  
...  

Abstract Low-voltage-activated T-type calcium channels are important contributors to nervous system function. Post-translational modification of these channels has emerged as an important mechanism to control channel activity. Previous studies have documented the importance of asparagine (N)-linked glycosylation and identified several asparagine residues within the canonical consensus sequence N-X-S/T that is essential for the expression and function of Cav3.2 channels. Here, we explored the functional role of non-canonical N-glycosylation motifs in the conformation N-X-C based on site directed mutagenesis. Using a combination of electrophysiological recordings and surface biotinylation assays, we show that asparagines N345 and N1780 located in the motifs NVC and NPC, respectively, are essential for the expression of the human Cav3.2 channel in the plasma membrane. Therefore, these newly identified asparagine residues within non-canonical motifs add to those previously reported in canonical sites and suggest that N-glycosylation of Cav3.2 may also occur at non-canonical motifs to control expression of the channel in the plasma membrane. It is also the first study to report the functional importance of non-canonical N-glycosylation motifs in an ion channel.


2005 ◽  
Vol 280 (16) ◽  
pp. 16076-16087 ◽  
Author(s):  
Claire Jacob ◽  
Graeme S. Cottrell ◽  
Daphne Gehringer ◽  
Fabien Schmidlin ◽  
Eileen F. Grady ◽  
...  

Mechanisms that arrest G-protein-coupled receptor (GPCR) signaling prevent uncontrolled stimulation that could cause disease. Although uncoupling from heterotrimeric G-proteins, which transiently arrests signaling, is well described, little is known about the mechanisms that permanently arrest signaling. Here we reported on the mechanisms that terminate signaling by protease-activated receptor 2 (PAR2), which mediated the proinflammatory and nociceptive actions of proteases. Given its irreversible mechanism of proteolytic activation, PAR2is a model to study the permanent arrest of GPCR signaling. By immunoprecipitation and immunoblotting, we observed that activated PAR2was mono-ubiquitinated. Immunofluorescence indicated that activated PAR2translocated from the plasma membrane to early endosomes and lysosomes where it was degraded, as determined by immunoblotting. Mutant PAR2lacking intracellular lysine residues (PAR2Δ14K/R) was expressed at the plasma membrane and signaled normally but was not ubiquitinated. Activated PAR2Δ14K/R internalized but was retained in early endosomes and avoided lysosomal degradation. Activation of wild type PAR2stimulated tyrosine phosphorylation of the ubiquitin-protein isopeptide ligase c-Cbl and promoted its interaction with PAR2at the plasma membrane and in endosomes in an Src-dependent manner. Dominant negative c-Cbl lacking the ring finger domain inhibited PAR2ubiquitination and induced retention in early endosomes, thereby impeding lysosomal degradation. Although wild type PAR2was degraded, and recovery of agonist responses required synthesis of new receptors, lysine mutation and dominant negative c-Cbl impeded receptor ubiquitination and degradation and allowed PAR2to recycle and continue to signal. Thus, c-Cbl mediated ubiquitination and lysosomal degradation of PAR2to irrevocably terminate signaling by this and perhaps other GPCRs.


2001 ◽  
Vol 95 (2) ◽  
pp. 515-524 ◽  
Author(s):  
Amadou K. S. Camara ◽  
Zeljana Begic ◽  
Wai-Meng Kwok ◽  
Zeljko J. Bosnjak

Background Volatile anesthetics exert their negative chronotropic and inotropic effects, in part by depressing the L- and T-type calcium channels. This study examines and compares the dose-dependent effects of isoflurane on atrial L- and T-type calcium currents (I(Ca,L) and I(Ca,T)) and ventricular I(Ca,L). Methods Whole cell I(Ca) was recorded from enzymatically isolated guinea pig cardiomyocytes. Current-voltage relations for atrial and ventricular I(Ca,L) was obtained from holding potentials of -90 and -50 mV to test a potential of +60 mV in 10-mV increments. Atrial I(Ca,T) was determined by subtraction of currents obtained from holding potentials of -50 and -90 mV. Steady state inactivation was determined using standard two-pulse protocols, and data were fitted with the Boltzmann equation. Results Isoflurane depressed I(Ca) in a dose-dependent manner, with Kd values of 0.23+/-0.03, 0.34+/-0.03, and 0.71+/-0.02 mM of anesthetic for atrial I(Ca,T) and I(Ca,L) and ventricular (ICa,L), respectively, and caused a significant (P < 0.05) hyperpolarizing shift in steady state inactivation. At 1.2 and 1.6 mm, isoflurane caused a significant (P < 0.05) depolarizing shift in the steady state activation in ventricular I(Ca,L) but not in atrial I(Ca,L) or I(Ca,T). In addition to the depression of I(Ca,L), isoflurane also induced a hyperpolarizing shift in the reversal potential of I(Ca) for both atrial and ventricular L-type calcium channels. Conclusion The results show that atrial I(Ca,T) is more sensitive to isoflurane than atrial I(Ca,L), and ventricular I(Ca,L) was the least responsive to the anesthetic. These differential sensitivities of the calcium channels in the atrial and ventricular chambers might reflect phenotypic differences in the calcium channels or differences in modulation by the anesthetic.


Endocrinology ◽  
2010 ◽  
Vol 151 (10) ◽  
pp. 4894-4907 ◽  
Author(s):  
Masha Dobkin-Bekman ◽  
Liat Rahamim Ben-Navi ◽  
Boris Shterntal ◽  
Ludmila Sviridonov ◽  
Fiorenza Przedecki ◽  
...  

GnRH is the first key hormone of reproduction. The role of protein kinase C (PKC) isoforms in GnRH-stimulated MAPK [ERK and Jun N-terminal kinase (JNK)] was examined in the αT3-1 and LβT2 gonadotrope cells. Incubation of the cells with GnRH resulted in a protracted activation of ERK1/2 and a slower and more transient activation of JNK1/2. Gonadotropes express conventional PKCα and conventional PKCβII, novel PKCδ, novel PKCε, and novel PKCθ, and atypical PKC-ι/λ. The use of green fluorescent protein-PKC constructs revealed that GnRH induced rapid translocation of PKCα and PKCβII to the plasma membrane, followed by their redistribution to the cytosol. PKCδ and PKCε localized to the cytoplasm and Golgi, followed by the rapid redistribution by GnRH of PKCδ to the perinuclear zone and of PKCε to the plasma membrane. Interestingly, PKCα, PKCβII, and PKCε translocation to the plasma membrane was more pronounced and more prolonged in phorbol-12-myristate-13-acetate (PMA) than in GnRH-treated cells. The use of selective inhibitors and dominant-negative plasmids for the various PKCs has revealed that PKCβII, PKCδ, and PKCε mediate ERK2 activation by GnRH, whereas PKCα, PKCβII, PKCδ, and PKCε mediate ERK2 activation by PMA. Also, PKCα, PKCβII, PKCδ, and PKCε are involved in GnRH and PMA stimulation of JNK1 in a cell-context-dependent manner. We present preliminary evidence that persistent vs. transient redistribution of selected PKCs or redistribution of a given PKC to the perinuclear zone vs. the plasma membrane may dictate its selective role in ERK or JNK activation. Thus, we have described the contribution of selective PKCs to ERK and JNK activation by GnRH.


2017 ◽  
Vol 216 (10) ◽  
pp. 3275-3290 ◽  
Author(s):  
Chris MacDonald ◽  
Robert C. Piper

Endocytosed cell surface membrane proteins rely on recycling pathways for their return to the plasma membrane. Although endosome-to-plasma membrane recycling is critical for many cellular processes, much of the required machinery is unknown. We discovered that yeast has a recycling route from endosomes to the cell surface that functions efficiently after inactivation of the sec7-1 allele of Sec7, which controls transit through the Golgi. A genetic screen based on an engineered synthetic reporter that exclusively follows this pathway revealed that recycling was subject to metabolic control through the Rag GTPases Gtr1 and Gtr2, which work downstream of the exchange factor Vam6. Gtr1 and Gtr2 control the recycling pathway independently of TORC1 regulation through the Gtr1 interactor Ltv1. We further show that the early-endosome recycling route and its control though the Vam6>Gtr1/Gtr2>Ltv1 pathway plays a physiological role in regulating the abundance of amino acid transporters at the cell surface.


2002 ◽  
Vol 13 (1) ◽  
pp. 317-335 ◽  
Author(s):  
Ray Mc Dermott ◽  
Umit Ziylan ◽  
Danièle Spehner ◽  
Huguette Bausinger ◽  
Dan Lipsker ◽  
...  

Birbeck granules are unusual rod-shaped structures specific to epidermal Langerhans cells, whose origin and function remain undetermined. We investigated the intracellular location and fate of Langerin, a protein implicated in Birbeck granule biogenesis, in human epidermal Langerhans cells. In the steady state, Langerin is predominantly found in the endosomal recycling compartment and in Birbeck granules. Langerin internalizes by classical receptor-mediated endocytosis and the first Birbeck granules accessible to endocytosed Langerin are those connected to recycling endosomes in the pericentriolar area, where Langerin accumulates. Drug-induced inhibition of endocytosis results in the appearance of abundant open-ended Birbeck granule-like structures appended to the plasma membrane, whereas inhibition of recycling induces Birbeck granules to merge with a tubular endosomal network. In mature Langerhans cells, Langerin traffic is abolished and the loss of internal Langerin is associated with a concomitant depletion of Birbeck granules. Our results demonstrate an exchange of Langerin between early endosomal compartments and the plasma membrane, with dynamic retention in the endosomal recycling compartment. They show that Birbeck granules are not endocytotic structures, rather they are subdomains of the endosomal recycling compartment that form where Langerin accumulates. Finally, our results implicate ADP-ribosylation factor proteins in Langerin trafficking and the exchange between Birbeck granules and other endosomal membranes.


2010 ◽  
Vol 299 (5) ◽  
pp. E772-E785 ◽  
Author(s):  
James P. Lopez ◽  
Jerrold R. Turner ◽  
Louis H. Philipson

A key step in regulating insulin secretion is insulin granule trafficking to the plasma membrane. Using live-cell time-lapse confocal microscopy, we observed a dynamic association of insulin granules with filamentous actin and PIP2-enriched structures. We found that the scaffolding protein family ERM, comprising ezrin, radixin, and moesin, are expressed in β-cells and target both F-actin and PIP2. Furthermore, ERM proteins are activated via phosphorylation in a glucose- and calcium-dependent manner. This activation leads to a translocation of the ERM proteins to sites on the cell periphery enriched in insulin granules, the exocyst complex docking protein Exo70, and lipid rafts. ERM scaffolding proteins also participate in insulin granule trafficking and docking to the plasma membrane. Overexpression of a truncated dominant-negative ezrin construct that lacks the ERM F-actin binding domain leads to a reduction in insulin granules near the plasma membrane and impaired secretion. Conversely, overexpression of a constitutively active ezrin results in more granules near the cell periphery and an enhancement of insulin secretion. Diabetic mouse islets contain less active ERM, suggestive of a novel mechanism whereby impairment of insulin granule trafficking to the membrane through a complex containing F-actin, PIP2, Exo70, and ERM proteins contributes to defective insulin secretion.


2021 ◽  
Author(s):  
Svenja C. Saile ◽  
Frank M. Ackermann ◽  
Sruthi Sunil ◽  
Jutta Keicher ◽  
Adam Bayless ◽  
...  

2018 ◽  
Author(s):  
Thomas D. Williams ◽  
Sew-Yeu Peak-Chew ◽  
Peggy Paschke ◽  
Robert R. Kay

AbstractMacropinocytosis is an actin-driven process of large-scale, non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum. In Dictyostelium, macropinocytic cups are organised by ‘macropinocytic patches’ in the plasma membrane. These contain activated Ras, Rac and PI(3,4,5)P3 and direct actin polymerisation to their periphery. Here, we show that a classical (PkbA) and a variant (PkbR1) Akt protein kinase acting downstream of PI(3,4,5)P3 are together are near-essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Akt targets identified by phosphoproteomics were highly enriched in small G-protein regulators, including the RhoGAP GacG. GacG knockout mutants make few macropinosomes but instead redeploy their cytoskeleton from macropinocytosis to motility, moving rapidly but taking up little fluid. The function of Akt in cell feeding through control of macropinosome size has implications for cancer cell biology.Summary statementDictyostelium amoebae feed by macropinocytosis in a PIP3 dependent manner. In the absence of PI3-kinases or the downstream Akt protein kinases, cells have smaller macropinosomes and nearly abolished fluid uptake.


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