scholarly journals Distinct domains of the sodium channel β3-subunit modulate channel-gating kinetics and subcellular location

2005 ◽  
Vol 392 (3) ◽  
pp. 519-526 ◽  
Author(s):  
Esther J. Yu ◽  
Seong-Hoon Ko ◽  
Paul W. Lenkowski ◽  
Alena Pance ◽  
Manoj K. Patel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sodium Channel ◽  
Secretory Pathway ◽  
Function Analysis ◽  
Channel Gating ◽  
Subcellular Location ◽  
Disulphide Bond ◽  
Inactivation Kinetics ◽  
Α Subunit ◽  
Ion Conducting

Electrical excitability in neurons depends on the expression and activity of voltage-gated sodium channels in the neuronal plasma membrane. The ion-conducting α-subunit of the channel is associated with auxiliary β-subunits of which there are four known types. In the present study, we describe the first detailed structure/function analysis of the β3-subunit. We correlate the effect of point mutations and deletions in β3 with the functional properties of the sodium channel and its membrane-targeting behaviour. We show that the extracellular domain influences sodium channel gating properties, but is not required for the delivery of β3 to the plasma membrane when expressed with the α-subunit. In contrast, the intracellular domain is essential for correct subunit targeting. Our results reveal the crucial importance of the Cys21–Cys96 disulphide bond in maintaining the functionally correct β3 structure and establish a role for a second putative disulphide bond (Cys2–Cys24) in modulating channel inactivation kinetics. Surprisingly, our results imply that the wild-type β3 molecule can traverse the secretory pathway independently of the α-subunit.

scholarly journals Recognition of a Single Transmembrane Degron by Sequential Quality Control Checkpoints

2003 ◽  
Vol 14 (3) ◽  
pp. 1268-1278 ◽  
Author(s):  
Laurence Fayadat ◽  
Ron R. Kopito
Keyword(s):  
Quality Control ◽  
Plasma Membrane ◽  
Disulfide Bonds ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Proteasome Inhibitors ◽  
Integral Membrane Protein ◽  
Type I ◽  
Α Subunit ◽  
Influenza Hemagglutinin

To understand the relationship between conformational maturation and quality control–mediated proteolysis in the secretory pathway, we engineered the well-characterized degron from the α-subunit of the T-cell antigen receptor (TCRα) into the α-helical transmembrane domain of homotrimeric type I integral membrane protein, influenza hemagglutinin (HA). Although the membrane degron does not appear to interfere with acquisition of native secondary structure, as assessed by the formation of native intrachain disulfide bonds, only ∼50% of nascent mutant HA chains (HA++) become membrane-integrated and acquire complex N-linked glycans indicative of transit to a post-ER compartment. The remaining ∼50% of nascent HA++ chains fail to integrate into the lipid bilayer and are subject to proteasome-dependent degradation. Site-specific cleavage by extracellular trypsin and reactivity with conformation-specific monoclonal antibodies indicate that membrane-integrated HA++ molecules are able to mature to the plasma membrane with a conformation indistinguishable from that of HAwt. These apparently native HA++ molecules are, nevertheless, rapidly degraded by a process that is insensitive to proteasome inhibitors but blocked by lysosomotropic amines. These data suggest the existence in the secretory pathway of at least two sequential quality control checkpoints that recognize the same transmembrane degron, thereby ensuring the fidelity of protein deployment to the plasma membrane.

scholarly journals The HOOK region of voltage-gated Ca2+ channel β subunits senses and transmits PIP2 signals to the gate

2017 ◽  
Vol 149 (2) ◽  
pp. 261-276 ◽  
Author(s):  
Cheon-Gyu Park ◽  
Yongsoo Park ◽  
Byung-Chang Suh
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Binding Assay ◽  
Channel Gating ◽  
Inactivation Kinetics ◽  
Β Subunit ◽  
Voltage Gated ◽  
Anionic Phospholipids ◽  
Channel Inhibition ◽  
Important Regulator

The β subunit of voltage-gated Ca2+ (CaV) channels plays an important role in regulating gating of the α1 pore-forming subunit and its regulation by phosphatidylinositol 4,5-bisphosphate (PIP2). Subcellular localization of the CaV β subunit is critical for this effect; N-terminal–dependent membrane targeting of the β subunit slows inactivation and decreases PIP2 sensitivity. Here, we provide evidence that the HOOK region of the β subunit plays an important role in the regulation of CaV biophysics. Based on amino acid composition, we broadly divide the HOOK region into three domains: S (polyserine), A (polyacidic), and B (polybasic). We show that a β subunit containing only its A domain in the HOOK region increases inactivation kinetics and channel inhibition by PIP2 depletion, whereas a β subunit with only a B domain decreases these responses. When both the A and B domains are deleted, or when the entire HOOK region is deleted, the responses are elevated. Using a peptide-to-liposome binding assay and confocal microscopy, we find that the B domain of the HOOK region directly interacts with anionic phospholipids via polybasic and two hydrophobic Phe residues. The β2c-short subunit, which lacks an A domain and contains fewer basic amino acids and no Phe residues in the B domain, neither associates with phospholipids nor affects channel gating dynamically. Together, our data suggest that the flexible HOOK region of the β subunit acts as an important regulator of CaV channel gating via dynamic electrostatic and hydrophobic interaction with the plasma membrane.

scholarly journals Differential Sialylation Modulates Voltage-gated Na+ Channel Gating throughout the Developing Myocardium

2006 ◽  
Vol 127 (3) ◽  
pp. 253-265 ◽  
Author(s):  
Patrick J. Stocker ◽  
Eric S. Bennett
Keyword(s):  
Sialic Acid ◽  
Sodium Channel ◽  
Channel Gating ◽  
Sialic Acids ◽  
Na Channel ◽  
Voltage Gated Sodium Channel ◽  
Α Subunit ◽  
Rat Cardiomyocytes ◽  
Voltage Gated ◽  
Α Subunits

Voltage-gated sodium channel function from neonatal and adult rat cardiomyocytes was measured and compared. Channels from neonatal ventricles required an ∼10 mV greater depolarization for voltage-dependent gating events than did channels from neonatal atria and adult atria and ventricles. We questioned whether such gating shifts were due to developmental and/or chamber-dependent changes in channel-associated functional sialic acids. Thus, all gating characteristics for channels from neonatal atria and adult atria and ventricles shifted significantly to more depolarized potentials after removal of surface sialic acids. Desialylation of channels from neonatal ventricles did not affect channel gating. After removal of the complete surface N-glycosylation structures, gating of channels from neonatal atria and adult atria and ventricles shifted to depolarized potentials nearly identical to those measured for channels from neonatal ventricles. Gating of channels from neonatal ventricles were unaffected by such deglycosylation. Immunoblot gel shift analyses indicated that voltage-gated sodium channel α subunits from neonatal atria and adult atria and ventricles are more heavily sialylated than α subunits from neonatal ventricles. The data are consistent with approximately 15 more sialic acid residues attached to each α subunit from neonatal atria and adult atria and ventricles. The data indicate that differential sialylation of myocyte voltage-gated sodium channel α subunits is responsible for much of the developmental and chamber-specific remodeling of channel gating observed here. Further, cardiac excitability is likely impacted by these sialic acid–dependent gating effects, such as modulation of the rate of recovery from inactivation. A novel mechanism is described by which cardiac voltage-gated sodium channel gating and subsequently cardiac rhythms are modulated by changes in channel-associated sialic acids.

Revealing a subclinical salt-losing phenotype in heterozygous carriers of the novel S562P mutation in the α subunit of the epithelial sodium channel

2009 ◽  
Vol 70 (2) ◽  
pp. 252-258 ◽  
Author(s):  
Felix G. Riepe ◽  
Miguel X. P. Van Bemmelen ◽  
Francois Cachat ◽  
Hansjörg Plendl ◽  
Ivan Gautschi ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Epithelial Sodium Channel ◽  
The Novel ◽  
Α Subunit ◽  
Heterozygous Carriers

scholarly journals Vesicular Transport Is Not Required for the Cytoplasmic Pool of Cholera Toxin To Interact with the Stimulatory Alpha Subunit of the Heterotrimeric G Protein

2004 ◽  
Vol 72 (12) ◽  
pp. 6826-6835 ◽  
Author(s):  
Ken Teter ◽  
Michael G. Jobling ◽  
Randall K. Holmes
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cholera Toxin ◽  
G Protein ◽  
Vesicular Transport ◽  
Cytotoxic Action ◽  
Anterograde Transport ◽  
Heterotrimeric G Protein ◽  
Α Subunit ◽  
Vesicular Traffic

ABSTRACT Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic A1 polypeptide of CT (CTA1) then crosses the ER membrane, enters the cytosol, ADP-ribosylates the stimulatory α subunit of the heterotrimeric G protein (Gsα) at the cytoplasmic face of the plasma membrane, and activates adenylate cyclase. The cytosolic pool of CTA1 may reach the plasma membrane and its Gsα target by traveling on anterograde-directed transport vesicles. We examined this possibility with the use of a plasmid-based transfection system that directed newly synthesized CTA1 to either the ER lumen or the cytosol of CHO cells. Such a system allowed us to bypass the CT retrograde trafficking itinerary from the cell surface to the ER. Previous work has shown that the ER-localized pool of CTA1 is rapidly exported from the ER to the cytosol. Expression of CTA1 in either the ER or the cytosol led to the activation of Gsα, and Gsα activation was not inhibited in transfected cells exposed to drugs that inhibit vesicular traffic. Thus, anterograde transport from the ER to the plasma membrane is not required for the cytotoxic action of CTA1.

scholarly journals pH-dependent Cargo Sorting from the Golgi

2011 ◽  
Vol 286 (12) ◽  
pp. 10058-10065 ◽  
Author(s):  
Chunjuan Huang ◽  
Amy Chang
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Atpase Activity ◽  
Secretory Pathway ◽  
Ph Dependence ◽  
Glucose Deprivation ◽  
Ph Homeostasis ◽  
Plasma Membrane Proteins ◽  
Cytosolic Ph ◽  
Cargo Sorting

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.

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