scholarly journals FGF14 modulates resurgent sodium current in mouse cerebellar Purkinje neurons

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Haidun Yan ◽  
Juan L Pablo ◽  
Chaojian Wang ◽  
Geoffrey S Pitt
Keyword(s):  
Sodium Current ◽  
Direct Interaction ◽  
Purkinje Neurons ◽  
Repetitive Firing ◽  
Spinocerebellar Ataxias ◽  
Channel Inactivation ◽  
C Terminus ◽  
N Terminus ◽  
Resurgent Current ◽  
Cerebellar Purkinje Neurons

Rapid firing of cerebellar Purkinje neurons is facilitated in part by a voltage-gated Na+ (NaV) ‘resurgent’ current, which allows renewed Na+ influx during membrane repolarization. Resurgent current results from unbinding of a blocking particle that competes with normal channel inactivation. The underlying molecular components contributing to resurgent current have not been fully identified. In this study, we show that the NaV channel auxiliary subunit FGF14 ‘b’ isoform, a locus for inherited spinocerebellar ataxias, controls resurgent current and repetitive firing in Purkinje neurons. FGF14 knockdown biased NaV channels towards the inactivated state by decreasing channel availability, diminishing the ‘late’ NaV current, and accelerating channel inactivation rate, thereby reducing resurgent current and repetitive spiking. Critical for these effects was both the alternatively spliced FGF14b N-terminus and direct interaction between FGF14b and the NaV C-terminus. Together, these data suggest that the FGF14b N-terminus is a potent regulator of resurgent NaV current in cerebellar Purkinje neurons.

CoCoA/CCAR1 Pair-Mediated Recruitment of Mediator Complex Indicates Novel Pathway for the Function of GATA1 in Erythroid Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1302-1302
Author(s):  
Chihiro Kaminaga ◽  
Shumpei Mizuta ◽  
Tomoya Minami ◽  
Kasumi Oda ◽  
Haruka Fujita ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Direct Interaction ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Luciferase Reporter ◽  
Zinc Finger Domain ◽  
C Terminus ◽  
N Terminus ◽  
Reporter Assays ◽  
Globin Promoter

Abstract Abstract 1302 The mammalian multi-protein complex Mediator, originally identified by ourselves as a nuclear receptor-specific coactivator complex, is a phylogenetically-conserved subcomplex of the RNA polymerase II holoenzyme and serves as an end-point integrator of diverse intracellular signals and transcriptional activators. The 220-kDa Mediator subunit MED1 is a specific coactivator not only for nuclear receptors but for GATA family activators, and serves as a GATA1-specific coactivator that is essential for optimal GATA1-mediated erythropoiesis. In this study, we show a novel nuclear signaling pathway for MED1 action in GATA1-induced transcriptional activation during erythroid differentiation. First, we identified the amino acid residues 681–715 of human MED1 (MED1(aa.681-715)) to be responsible for the direct interaction with GATA1. When MED1 in K562 human erythroleukemic cells was knocked down during hemin-induced erythroid differentiation, the erythroid differentiation was significantly attenuated as assessed by an erythroid differentiation score defined by the number of cells positive for benzidine staining, and the expressions of the GATA1-targeted and erythroid differentiation marker genes, β-globin, γ-globin, PBGD and ALAS-E, were prominently attenuated. However, overexpressions of the N-terminal MED1 truncations without and with nuclear receptor recognition motifs, MED1(aa.1–602) and MED1(aa.1–703), respectively, but neither of which could bind to GATA1 (above), prominently enhanced erythroid differentiation of K562 cells. Luciferase reporter assays by using the human γ-globin promoter and Med1−/− mouse embryonic fibroblasts (MEFs) showed that these N-terminal MED1 truncations rescued GATA1-mediated transactivation, indicating that MED1(a.a.1–602) served as the functional interaction surface for GATA1. Hence, a putative bypass for GATA1-MED1 pathway appears to exist, and is expected to interact with the N-terminus of MED1. As a candidate bypass system, we tested both the recently reported bypass molecule for a nuclear post-activator signaling, CCAR1, and its partner coactivator CoCoA. CCAR1 was reported by others to bypass the estrogen receptor-mediated transactivation by a simultaneous binding of CCAR1 with the estrogen receptor and the N-terminus of MED1. Functionally, serial luciferase reporter assays by using the γ-globin promoter and MEFs demonstrated cooperative transactivation by combinations of GATA1, CCAR1, CoCoA and/or the N-terminus of MED1, but the transactivation mediated by the N-terminus of MED1 was not as prominent as the one mediated by the full-length MED1. An overexperssion of CCAR1 or CoCoA in K562 cells prominently enhanced both the GATA1-mediated erythroid differentiation and the expressions of the GATA1-targeted genes. Next, the mechanisms underlying the CCAR1- and CoCoA-mediated GATA1 functions were analyzed by serial GST-pulldown and mammalian two-hybrid assays, and the following results were obtained. (i) The N-terminus of CCAR1 interacted with the C-terminus of CoCoA. (ii) The N-terminus of MED1 interacted with both the N- and C-termini of CCAR1. (iii) While the N-terminal zinc-finger domain of human GATA1 (GATA1(a.a.204–228)) is known to bind to the well-known GATA1 partner FOG1, intriguingly, the C-terminal zinc-finger domain of GATA1 (GATA1(a.a.258–272)) interacted with all three of the following cofactors; MED1 (MED1(aa.681–715)), CCAR1 (at the C-terminus) and CoCoA (at both the N- and C-termini). The affinity of CoCoA to bind to GATA1 appeared to be a little higher than the other. Thus, the GATA1(a.a.258-272) zinc finger appears to serve as a docking surface for multiple coactivating proteins, where both MED1 and CoCoA/CCAR1 pair can interact, probably in a competitive manner, or perhaps simultaneously. Here, both CoCoA/CCAR1 as a pair and CCAR1 by itself can serve as a bypass. Finally, ChIP assays of hemin-treated K562 cells showed that GATA1, CCAR1/CoCoA and MED1 were all recruited onto the γ-globin promoter during transactivation. Taken together, besides a direct interaction between GATA1 and MED1, the CoCoA/CCAR1 pair appears to relay the GATA1 signal to MED1. The multiple modes of mechanisms for transcription mediated by the GATA1-MED1 axis might contribute to a fine tuning of the GATA1 function, not only during erythropoiesis but also in other GATA1-mediated homeostasis events, within a living animal. Disclosures: No relevant conflicts of interest to declare.

Sodium Currents in Subthalamic Nucleus Neurons From Nav1.6-Null Mice

2004 ◽  
Vol 92 (2) ◽  
pp. 726-733 ◽  
Author(s):  
Michael Tri H. Do ◽  
Bruce P. Bean
Keyword(s):  
Subthalamic Nucleus ◽  
Sodium Channels ◽  
Sodium Current ◽  
Great Majority ◽  
Purkinje Neurons ◽  
Wild Type ◽  
Decay Kinetics ◽  
Persistent Currents ◽  
Resurgent Current ◽  
Resurgent Sodium Current

In some central neurons, including cerebellar Purkinje neurons and subthalamic nucleus (STN) neurons, TTX-sensitive sodium channels show unusual gating behavior whereby some channels open transiently during recovery from inactivation. This “resurgent” sodium current is effectively activated immediately after action potential-like waveforms. Earlier work using Purkinje neurons suggested that the great majority of resurgent current originates from Nav1.6 sodium channels. Here we used a mouse mutant lacking Nav1.6 to explore the contribution of these channels to resurgent, transient, and persistent components of TTX-sensitive sodium current in STN neurons. The resurgent current of STN neurons from Nav1.6−/− mice was reduced by 63% relative to wild-type littermates, a less dramatic reduction than that observed in Purkinje neurons recorded under identical conditions. The transient and persistent currents of Nav1.6−/− STN neurons were reduced by ∼40 and 55%, respectively. The resurgent current present in Nav1.6−/− null STN neurons was similar in voltage dependence to that in wild-type STN and Purkinje neurons, differing only in having somewhat slower decay kinetics. These results show that sodium channels other than Nav1.6 can make resurgent sodium current much like that from Nav1.6 channels.

scholarly journals Direct Interaction between the N- and C-Terminal Portions of the Herpes Simplex Virus Type 1 Origin Binding Protein UL9 Implies the Formation of a Head-to-Tail Dimer

2007 ◽  
Vol 81 (24) ◽  
pp. 13659-13667 ◽  
Author(s):  
Soma Chattopadhyay ◽  
Sandra K. Weller
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Binding ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Direct Interaction ◽  
C Terminus ◽  
N Terminus ◽  
Simplex Virus

ABSTRACT UL9, a superfamily II helicase, is a multifunctional protein required for herpes simplex virus type 1 replication in vivo. Although the C-terminal 317-amino-acid DNA binding domain of UL9 exists as a monomer, the full-length protein behaves as a dimer in solution. Thus, it has been assumed that the N-terminal 534 residues contain a region necessary for efficient dimerization and that UL9 dimers are in a head-to-head configuration. We recently showed, however, that residues in the N terminus could modulate the inhibitory properties of UL9 by decreasing the DNA binding ability of the C terminus (S. Chattopadhyay and S. K. Weller, J. Virol. 80:4491-4500, 2006). We suggested that a direct interaction between the N- and C-terminal portions of UL9 might exist and serve to modulate the DNA binding activities of the C terminus. In this study, we used a coimmunoprecipitation assay to show that the N-terminal portion of UL9 can indeed directly interact with the C terminus. A series of truncation mutant proteins were used to show that a region in the N terminus between residues 293 and 321 is necessary for efficient interaction. Similarly, a region in the C terminus between residues 600 and 800 is required for this interaction. The simplest model to explain these data is that UL9 dimers are oriented in a head-to-tail arrangement in which the N terminus is in contact with the C terminus.

scholarly journals The eag domain regulates hERG channel inactivation gating via a direct interaction

2013 ◽  
Vol 141 (2) ◽  
pp. 229-241 ◽  
Author(s):  
Ahleah S. Gustina ◽  
Matthew C. Trudeau
Keyword(s):  
Amino Acids ◽  
Steady State ◽  
Direct Interaction ◽  
Terminal Region ◽  
Herg Channel ◽  
Channel Inactivation ◽  
Large N ◽  
Recovery From Inactivation ◽  
Steady State Inactivation ◽  
Resurgent Current

Human ether-á-go-go (eag)-related gene (hERG) potassium channel kinetics are characterized by rapid inactivation upon depolarization, along with rapid recovery from inactivation and very slow closing (deactivation) upon repolarization. These factors combine to create a resurgent hERG current, where the current amplitude is paradoxically larger with repolarization than with depolarization. Previous data showed that the hERG N-terminal eag domain regulated deactivation kinetics by making a direct interaction with the C-terminal region of the channel. A primary mechanism for fast inactivation depends on residues in the channel pore; however, inactivation was also shown to be slower after deletion of a large N-terminal region. The mechanism for N-terminal region regulation of inactivation is unclear. Here, we investigated the contributions of the large N-terminal domains (amino acids 1–354), including the eag domain (amino acids 1–135), to hERG channel inactivation kinetics and steady-state inactivation properties. We found that N-deleted channels lacking just the eag domain (Δ2–135) or both the eag domain and the adjacent proximal domain (Δ2–354) had less rectifying current–voltage (I-V) relationships, slower inactivation, faster recovery from inactivation, and lessened steady-state inactivation. We coexpressed genetically encoded N-terminal fragments for the eag domain (N1–135) or the eag domain plus the proximal domain (N1–354) with N-deleted hERG Δ2–135 or hERG Δ2–354 channels and found that the resulting channels had more rectifying I-V relationships, faster inactivation, slower recovery from inactivation, and increased steady-state inactivation, similar to those properties measured for wild-type (WT) hERG. We also found that the eag domain–containing fragments regulated the time to peak and the voltage at the peak of a resurgent current elicited with a ramp voltage protocol. The eag domain–containing fragments effectively converted N-deleted channels into WT-like channels. Neither the addition of the proximal domain to the eag domain in N1–354 fragments nor the presence of the proximal domain in hERG Δ2–135 channels measurably affected inactivation properties; in contrast, the proximal region regulated steady-state activation in hERG Δ2–135 channels. The results show that N-terminal region-dependent regulation of channel inactivation and resurgent current properties are caused by a direct interaction of the eag domain with the rest of the hERG channel.

scholarly journals Intrinsic Mechanisms in the Gating of Resurgent Na+ Currents

2021 ◽  
Author(s):  
Joseph L. Ransdell ◽  
Jonathan D. Moreno ◽  
Druv Bhagavan ◽  
Jonathan R. Silva ◽  
Jeanne M. Nerbonne
Keyword(s):  
Voltage Clamp ◽  
Sodium Current ◽  
Neuronal Cell ◽  
Purkinje Neurons ◽  
Relative Distribution ◽  
Membrane Hyperpolarization ◽  
Voltage Gated ◽  
Nav Channels ◽  
Cerebellar Purkinje Neurons ◽  
Kinetics Of

ABSTRACTThe resurgent component of the voltage-gated sodium current (INaR) is a depolarizing conductance, revealed on membrane hyperpolarizations following brief depolarizing voltage steps, which has been shown to contribute to regulating the firing properties of numerous neuronal cell types throughout the central and peripheral nervous systems. Although mediated by the same voltage-gated sodium (Nav) channels that underlie the transient and persistent Nav current components, the gating mechanisms that contribute to the generation of INaR remain unclear. Here, we characterized Nav currents in mouse cerebellar Purkinje neurons, and used tailored voltage-clamp protocols to define how the voltage and the duration of the initial membrane depolarization affect the amplitudes and kinetics of INaR. Using the acquired voltage-clamp data, we developed a novel Markov kinetic state model with parallel (fast and slow) inactivation pathways and, we show that this model reproduces the properties of the resurgent, as well as the transient and persistent, Nav currents recorded in (mouse) cerebellar Purkinje neurons. Based on the acquired experimental data and the simulations, we propose that resurgent Na+ influx occurs as a result of fast inactivating Nav channels transitioning into an open/conducting state on membrane hyperpolarization, and that the decay of INaR reflects the slow accumulation of recovered/opened Nav channels into a second, alternative and more slowly populated, inactivated state. Additional simulations reveal that extrinsic factors that affect the kinetics of fast or slow Nav channel inactivation and/or impact the relative distribution of Nav channels in the fast- and slow-inactivated states, such as the accessory Navβ4 channel subunit, can modulate the amplitude of INaR.SUMMARYThe resurgent component of the voltage-gated sodium current (INaR) is revealed on membrane hyperpolarizations following brief depolarizing voltage steps that activate the rapidly activating and inactivating, transient Nav current (INaT). To probe the mechanisms contributing to the generation and properties of INaR, we combined whole-cell voltage-clamp recordings from mouse cerebellar Purkinje neurons with computational modeling to develop a novel, blocking particle-independent, model for the gating of INaR that involves two parallel inactivation pathways, and we show that this model recapitulates the detailed biophysical properties of INaR measured in mouse cerebellar Purkinje neurons.

scholarly journals The Spindle Pole Body Protein Cdc11p Links Sid4p to the Fission Yeast Septation Initiation Network

2002 ◽  
Vol 13 (4) ◽  
pp. 1203-1214 ◽  
Author(s):  
Gregory C. Tomlin ◽  
Jennifer L. Morrell ◽  
Kathleen L. Gould
Keyword(s):  
Spindle Pole Body ◽  
Direct Interaction ◽  
Spindle Pole ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Body Protein ◽  
Pole Body ◽  
C Terminus ◽  
N Terminus ◽  
Septation Initiation Network

The Schizosaccharomyces pombe septation initiation network (SIN) signals the onset of cell division from the spindle pole body (SPB) and is regulated by the small GTPase Spg1p. The localization of SIN components including Spg1p to the SPB is required for cytokinesis and is dependent on Sid4p, a constitutive resident of SPBs. However, a direct interaction between Sid4p and other members of the SIN has not been detected. To understand how Sid4p is linked to other SIN components, we have begun to characterize an S. pombe homolog of the Saccharomyces cerevisiaeSPB protein Nud1p. We have determined that this S. pombeNud1p homolog corresponds to Cdc11p, a previously uncharacterized SIN element. We report that Cdc11p is present constitutively at SPBs and that its function appears to be required for the localization of all other SIN components to SPBs with the exception of Sid4p. The Cdc11p C terminus localizes the protein to SPBs in a Sid4p-dependent manner, and we demonstrate a direct Cdc11p-Sid4p interaction. The N-terminus of Cdc11p is required for Spg1p binding to SPBs. Our studies indicate that Cdc11p provides a physical link between Sid4p and the Spg1p signaling pathway.

scholarly journals Physiological role of dendrotoxin sensitive k channels in the rat cerebellar Purkinje neurons

2007 ◽  
pp. 807-813 ◽  
Author(s):  
H Haghdoust ◽  
M Janahmadi ◽  
G Behzadi
Keyword(s):  
Action Potentials ◽  
Physiological Role ◽  
Neuronal Firing ◽  
Purkinje Neurons ◽  
Repetitive Firing ◽  
Neuronal Membrane ◽  
K Channels ◽  
Type K ◽  
Cerebellar Purkinje Neurons

To understand the contribution of potassium (K+) channels, particularly alpha-dendrotoxin (D-type)-sensitive K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits), to the generation of neuronal spike output we must have detailed information of the functional role of these channels in the neuronal membrane. Conventional intracellular recording methods in current clamp mode were used to identify the role of alpha-dendrotoxin (alpha-DTX)-sensitive K+ channel currents in shaping the spike output and modulation of neuronal properties of cerebellar Purkinje neurons (PCs) in slices. Addition of alpha-DTX revealed that D-type K+ channels play an important role in the shaping of Purkinje neuronal firing behavior. Repetitive firing capability of PCs was increased following exposure to artificial cerebrospinal fluid (aCSF) containing alpha-DTX, so that in response to the injection of 0.6 nA depolarizing current pulse of 600 ms, the number of action potentials insignificantly increased from 15 in the presence of 4-AP to 29 action potentials per second after application of DTX following pretreatment with 4-AP. These results indicate that D-type K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits) may contribute to the spike frequency adaptation in PCs. Our findings suggest that the activation of voltage-dependent K+ channels (D and A types) markedly affect the firing pattern of PCs.

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