scholarly journals Ankyrin G restricts ion channel diffusion at the axonal initial segment before the establishment of the diffusion barrier

2010 ◽  
Vol 191 (2) ◽  
pp. 383-395 ◽  
Author(s):  
Anna Brachet ◽  
Christophe Leterrier ◽  
Marie Irondelle ◽  
Marie-Pierre Fache ◽  
Victor Racine ◽  
...  
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Diffusion Barrier ◽  
Hippocampal Neurons ◽  
Initial Segment ◽  
Specific Binding ◽  
Single Particle Tracking ◽  
Binding Motif ◽  
Ankyrin G ◽  
Axonal Initial Segment

In mammalian neurons, the precise accumulation of sodium channels at the axonal initial segment (AIS) ensures action potential initiation. This accumulation precedes the immobilization of membrane proteins and lipids by a diffusion barrier at the AIS. Using single-particle tracking, we measured the mobility of a chimeric ion channel bearing the ankyrin-binding motif of the Nav1.2 sodium channel. We found that ankyrin G (ankG) limits membrane diffusion of ion channels when coexpressed in neuroblastoma cells. Site-directed mutants with decreased affinity for ankG exhibit increased diffusion speeds. In immature hippocampal neurons, we demonstrated that ion channel immobilization by ankG is regulated by protein kinase CK2 and occurs as soon as ankG accumulates at the AIS of elongating axons. Once the diffusion barrier is formed, ankG is still required to stabilize ion channels. In conclusion, our findings indicate that specific binding to ankG constitutes the initial step for Nav channel immobilization at the AIS membrane and precedes the establishment of the diffusion barrier.

scholarly journals Developmental formation of a diffusion barrier in the plasma membrane of the axonal initial segment : single lipid molecule approach

2000 ◽  
Vol 40 (supplement) ◽  
pp. S212
Author(s):  
C. Nakada ◽  
M. Nozaki ◽  
H. Yamashita ◽  
K. Yamaguchi ◽  
Ken Ritchie ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Diffusion Barrier ◽  
Initial Segment ◽  
Lipid Molecule ◽  
Axonal Initial Segment

scholarly journals Nanoscopic compartmentalization of membrane protein motion at the axon initial segment

2016 ◽  
Author(s):  
David Albrecht ◽  
Christian M. Winterflood ◽  
Thomas Tschager ◽  
Helge Ewers
Keyword(s):  
Membrane Protein ◽  
Particle Tracking ◽  
Single Particle ◽  
Diffusion Barrier ◽  
High Speed ◽  
Initial Segment ◽  
Single Particle Tracking ◽  
Axon Initial Segment ◽  
Periodic Pattern ◽  
Protein Motion

AbstractThe axon initial segment (AIS) is enriched in specific adaptor, cytoskeletal and transmembrane molecules. During AIS establishment, a membrane diffusion barrier is formed between the axon and the somatodendritic domain. Recently, an axonal periodic pattern of actin, spectrin and ankyrin forming 190 nm distanced, ring-like structures has been discovered. However, whether this structure is related to the diffusion barrier function is unclear.Here, we performed single particle tracking timecourse experiments on hippocampal neurons during AIS development. We analyzed the mobility of lipid-anchored molecules by high-speed single particle tracking and correlated positions of membrane molecules with the nanoscopic organization of the AIS cytoskeleton.We observe a strong reduction in mobility early in AIS development. Membrane protein motion in the AIS plasma membrane is confined to a repetitive pattern of ~190 nm spaced segments along the AIS axis as early as DIV4 and this pattern alternates with actin rings. Our data provide a new model for the mechanism of the AIS diffusion barrier.

scholarly journals Developmental formation of a diffusion barrier in the axonal initial segment membrane of the neuron ; a single molecule approach.

2001 ◽  
Vol 41 (supplement) ◽  
pp. S121
Author(s):  
C. Nakada ◽  
Kenneth Ritchie ◽  
T. Fujiwara ◽  
Y. Hotta ◽  
R. Iino ◽  
...  
Keyword(s):  
Single Molecule ◽  
Diffusion Barrier ◽  
Initial Segment ◽  
Axonal Initial Segment

scholarly journals Elucidating distinct ion channel populations on the surface of hippocampal neurons via single-particle tracking recurrence analysis

2017 ◽  
Vol 96 (6) ◽  
Author(s):  
Grzegorz Sikora ◽  
Agnieszka Wyłomańska ◽  
Janusz Gajda ◽  
Laura Solé ◽  
Elizabeth J. Akin ◽  
...  
Keyword(s):  
Ion Channel ◽  
Particle Tracking ◽  
Single Particle ◽  
Hippocampal Neurons ◽  
Single Particle Tracking ◽  
Recurrence Analysis

scholarly journals Endocytotic elimination and domain-selective tethering constitute a potential mechanism of protein segregation at the axonal initial segment

2004 ◽  
Vol 166 (4) ◽  
pp. 571-578 ◽  
Author(s):  
Marie-Pierre Fache ◽  
Anissa Moussif ◽  
Fanny Fernandes ◽  
Pierre Giraud ◽  
Juan José Garrido ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Hippocampal Neurons ◽  
Initial Segment ◽  
Distal Part ◽  
Potential Mechanism ◽  
Reporter Protein ◽  
Linker Sequence ◽  
Axonal Initial Segment ◽  
Cellular Polarization

The axonal initial segment is a unique subdomain of the neuron that maintains cellular polarization and contributes to electrogenesis. To obtain new insights into the mechanisms that determine protein segregation in this subdomain, we analyzed the trafficking of a reporter protein containing the cytoplasmic II–III linker sequence involved in sodium channel targeting and clustering (Garrido, J.J., P. Giraud, E. Carlier, F. Fernandes, A. Moussif, M.P. Fache, D. Debanne, and B. Dargent. 2003. Science. 300:2091–2094). Here, we show that this reporter protein is preferentially inserted in the somatodendritic domain and is trapped at the axonal initial segment by tethering to the cytoskeleton, before its insertion in the axonal tips. The nontethered population in dendrites, soma, and the distal part of axons is subsequently eliminated by endocytosis. We provide evidence for the involvement of two independent determinants in the II–III linker of sodium channels. These findings indicate that endocytotic elimination and domain-selective tethering constitute a potential mechanism of protein segregation at the axonal initial segment of hippocampal neurons.

scholarly journals Nanoscopic compartmentalization of membrane protein motion at the axon initial segment

2016 ◽  
Vol 215 (1) ◽  
pp. 37-46 ◽  
Author(s):  
David Albrecht ◽  
Christian M. Winterflood ◽  
Mohsen Sadeghi ◽  
Thomas Tschager ◽  
Frank Noé ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Particle Tracking ◽  
Single Particle ◽  
Diffusion Barrier ◽  
Initial Segment ◽  
Time Course ◽  
Single Particle Tracking ◽  
Axon Initial Segment ◽  
Periodic Pattern ◽  
Protein Motion

The axon initial segment (AIS) is enriched in specific adaptor, cytoskeletal, and transmembrane molecules. During AIS establishment, a membrane diffusion barrier is formed between the axonal and somatodendritic domains. Recently, an axonal periodic pattern of actin, spectrin, and ankyrin forming 190-nm-spaced, ring-like structures has been discovered. However, whether this structure is related to the diffusion barrier function is unclear. Here, we performed single-particle tracking time-course experiments on hippocampal neurons during AIS development. We analyzed the mobility of lipid-anchored molecules by high-speed single-particle tracking and correlated positions of membrane molecules with the nanoscopic organization of the AIS cytoskeleton. We observe a strong reduction in mobility early in AIS development. Membrane protein motion in the AIS plasma membrane is confined to a repetitive pattern of ∼190-nm-spaced segments along the AIS axis as early as day in vitro 4, and this pattern alternates with actin rings. Mathematical modeling shows that diffusion barriers between the segments significantly reduce lateral diffusion along the axon.

scholarly journals Protein kinase CK2 contributes to the organization of sodium channels in axonal membranes by regulating their interactions with ankyrin G

2008 ◽  
Vol 183 (6) ◽  
pp. 1101-1114 ◽  
Author(s):  
Aline Bréchet ◽  
Marie-Pierre Fache ◽  
Anna Brachet ◽  
Géraldine Ferracci ◽  
Agnés Baude ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Protein Kinase Ck2 ◽  
Binding Motif ◽  
Nodes Of Ranvier ◽  
Ankyrin G ◽  
Axonal Initial Segment ◽  
Kinase Ck2

In neurons, generation and propagation of action potentials requires the precise accumulation of sodium channels at the axonal initial segment (AIS) and in the nodes of Ranvier through ankyrin G scaffolding. We found that the ankyrin-binding motif of Nav1.2 that determines channel concentration at the AIS depends on a glutamate residue (E1111), but also on several serine residues (S1112, S1124, and S1126). We showed that phosphorylation of these residues by protein kinase CK2 (CK2) regulates Nav channel interaction with ankyrins. Furthermore, we observed that CK2 is highly enriched at the AIS and the nodes of Ranvier in vivo. An ion channel chimera containing the Nav1.2 ankyrin-binding motif perturbed endogenous sodium channel accumulation at the AIS, whereas phosphorylation-deficient chimeras did not. Finally, inhibition of CK2 activity reduced sodium channel accumulation at the AIS of neurons. In conclusion, CK2 contributes to sodium channel organization by regulating their interaction with ankyrin G.

scholarly journals Ankyrin G membrane partners drive the establishment and maintenance of the axon initial segment

2016 ◽  
Author(s):  
Christophe Leterrier ◽  
Nadine Clerc ◽  
Fanny Rueda-Boroni ◽  
Audrey Montersino ◽  
Bénédicte Dargent ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Adhesion Molecules ◽  
Hippocampal Neurons ◽  
Initial Segment ◽  
Neuronal Development ◽  
Axon Initial Segment ◽  
Membrane Anchor ◽  
Ankyrin G ◽  
Membrane Components ◽  
Cultured Hippocampal Neurons

The axon initial segment (AIS) is a specialized neuronal compartment that plays a key role in neuronal development and excitability. It concentrates multiple ion channels and cell adhesion molecules. The anchoring of these AIS membrane components is known to be highly dependent of the scaffold protein ankyrin G (ankG) but whether ankG membrane partners play a reciprocal role in ankG targeting and stabilization has not been studied yet. In cultured hippocampal neurons and cortical organotypic slices, we found that shRNA-mediated knockdown of ankG membrane partners led to a decrease of ankG concentration and perturbed the AIS formation and maintenance. These perturbations were rescued by expressing an AIS-targeted sodium channel, or a minimal construct containing the ankyrin-binding domain of Nav1.2 and a membrane anchor. We thus demonstrate that a tight and precocious association of ankG to its membrane partners is crucial for the establishment and maintenance of the AIS.

scholarly journals Axonal spectrins: All-purpose fences

2013 ◽  
Vol 203 (3) ◽  
pp. 381-383 ◽  
Author(s):  
Yael Eshed-Eisenbach ◽  
Elior Peles
Keyword(s):  
Adhesion Molecules ◽  
Diffusion Barrier ◽  
Initial Segment ◽  
Nodes Of Ranvier ◽  
Myelinated Axons ◽  
Membrane Cytoskeleton ◽  
Cell Biol ◽  
Axonal Initial Segment ◽  
Membrane Barrier

A membrane barrier important for assembly of the nodes of Ranvier is found at the paranodal junction. This junction is comprised of axonal and glial adhesion molecules linked to the axonal actin–spectrin membrane cytoskeleton through specific adaptors. In this issue, Zhang et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201308116) show that axonal βII spectrin maintains the diffusion barrier at the paranodal junction. Thus, βII spectrin serves to compartmentalize the membrane of myelinated axons at specific locations that are determined either intrinsically (i.e., at the axonal initial segment), or by axoglial contacts (i.e., at the paranodal junction).

scholarly journals TOK Homologue in Neurospora crassa: First Cloning and Functional Characterization of an Ion Channel in a Filamentous Fungus

2003 ◽  
Vol 2 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Stephen K. Roberts
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Neurospora Crassa ◽  
Filamentous Fungus ◽  
Single Channel ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Channel Activity ◽  
Biophysical Properties ◽  
Patch Clamp Technique

ABSTRACT In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the “filamentous” polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K+ channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K+ channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K+ efflux. NcTOKA channel gating was sensitive to extracellular K+ such that channel activation was dependent on the reversal potential for K+. However, expression of NcTOKA was able to overcome the K+ auxotrophy of a yeast mutant missing the K+ uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K+ influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K+ currents at potentials negative of EK. NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca2+, verapamil, quinine, and TEA+ but was insensitive to Cs+, 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.

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