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 KCNE3 Truncation Mutants Reveal a Bipartite Modulation of KCNQ1 K+ Channels

2004 ◽  
Vol 124 (6) ◽  
pp. 759-771 ◽  
Author(s):  
Steven D. Gage ◽  
William R. Kobertz
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Channel Gating ◽  
Terminal Region ◽  
Type I ◽  
Long Qt ◽  
Transmembrane Peptides ◽  
K Channels ◽  
Voltage Gated ◽  
La Luna

The five KCNE genes encode a family of type I transmembrane peptides that assemble with KCNQ1 and other voltage-gated K+ channels, resulting in potassium conducting complexes with varied channel-gating properties. It has been recently proposed that a triplet of amino acids within the transmembrane domain of KCNE1 and KCNE3 confers modulation specificity to the peptide, since swapping of these three residues essentially converts the recipient KCNE into the donor (Melman, Y.F., A. Domenech, S. de la Luna, and T.V. McDonald. 2001. J. Biol. Chem. 276:6439–6444). However, these results are in stark contrast with earlier KCNE1 deletion studies, which demonstrated that a COOH-terminal region, highly conserved between KCNE1 and KCNE3, was responsible for KCNE1 modulation of KCNQ1 (Tapper, A.R., and A.L. George. 2000 J. Gen. Physiol. 116:379–389.). To ascertain whether KCNE3 peptides behave similarly to KCNE1, we examined a panel of NH2- and COOH-terminal KCNE3 truncation mutants to directly determine the regions required for assembly with and modulation of KCNQ1 channels. Truncations lacking the majority of their NH2 terminus, COOH terminus, or mutants harboring both truncations gave rise to KCNQ1 channel complexes with basal activation, a hallmark of KCNE3 modulation. These results demonstrate that the KCNE3 transmembrane domain is sufficient for assembly with and modulation of KCNQ1 channels and suggests a bipartite model for KCNQ1 modulation by KCNE1 and KCNE3 subunits. In this model, the KCNE3 transmembrane domain is active in modulation and overrides the COOH terminus' contribution, whereas the KCNE1 transmembrane domain is passive and reveals COOH-terminal modulation of KCNQ1 channels. We furthermore test the validity of this model by using the active KCNE3 transmembrane domain to functionally rescue a nonconducting, yet assembly and trafficking competent, long QT mutation located in the conserved COOH-terminal region of KCNE1.

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.

Voltage-gated potassium channel Kv1.3 regulates GLUT4 trafficking to the plasma membrane via a Ca2+-dependent mechanism

2006 ◽  
Vol 290 (2) ◽  
pp. C345-C351 ◽  
Author(s):  
Yanyan Li ◽  
Peili Wang ◽  
Jianchao Xu ◽  
Gary V. Desir
Keyword(s):  
Plasma Membrane ◽  
Insulin Sensitivity ◽  
Protein Translocation ◽  
Channel Activity ◽  
Voltage Gated ◽  
White Adipocytes ◽  
Kv1.3 Channel ◽  
Channel Inhibition ◽  
Intracellular Ca ◽  
Insulin Dependent

Kv1.3 is a voltage-gated K+ channel expressed in insulin-sensitive tissues. We previously showed that gene inactivation or pharmacological inhibition of Kv1.3 channel activity increased peripheral insulin sensitivity independently of body weight by augmenting the amount of GLUT4 at the plasma membrane. In the present study, we further examined the effect Kv1.3 on GLUT4 trafficking and tested whether it occurred via an insulin-dependent pathway. We found that Kv1.3 inhibition by margatoxin (MgTX) stimulated glucose uptake in adipose tissue and skeletal muscle and that the effect of MgTX on glucose transport was additive to that of insulin. Furthermore, whereas the increase in uptake was wortmannin insensitive, it was completely inhibited by dantrolene, a blocker of Ca2+ release from intracellular Ca2+ stores. In white adipocytes in primary culture, channel inhibition by Psora-4 increased GLUT4 translocation to the plasma membrane. In these cells, GLUT4 protein translocation was unaffected by the addition of wortmannin but was significantly inhibited by dantrolene. Channel inhibition depolarized the membrane voltage and led to sustained, dantrolene-sensitive oscillations in intracellular Ca2+ concentration. These results indicate that the apparent increase in insulin sensitivity observed in association with inhibition of Kv1.3 channel activity is mediated by an increase in GLUT4 protein at the plasma membrane, which occurs largely through a Ca2+-dependent process.

Structure in three dimensions of H,K-ATPase by Electron Microscopy and image processing

1992 ◽  
Vol 50 (1) ◽  
pp. 444-445
Author(s):  
Manijeh Mohraz ◽  
Sonal Sathe ◽  
P.R. Smith
Keyword(s):  
Electron Microscopy ◽  
Image Processing ◽  
Amino Acids ◽  
Plasma Membrane ◽  
Gastric Mucosa ◽  
Sequence Homology ◽  
Concentration Gradient ◽  
Atp Hydrolysis ◽  
Three Dimensions ◽  
Β Subunit

H,K-ATPase is an integral protein of the plasma membrane of parietal cells in the gastric mucosa. It is believed to constitute the pump responsible for secretion of acid into the stomach. It utilizes energy from ATP hydrolysis to transport H+ out of the cell and K+ into the cell against an H+ concentration gradient of one million-fold. The catalytic subunit (Mr 110,000) of H,K-ATPase shows striking sequence homology to those of other ion-transporting ATPases. The enzyme has a second subunit, a glycoprotein of ca 300 amino acids, which is homologous to the β subunit of the Na, K-ATPase.

scholarly journals Translocatable voltage-gated Ca2+ channel β subunits in α1–β complexes reveal competitive replacement yet no spontaneous dissociation

2018 ◽  
Vol 115 (42) ◽  
pp. E9934-E9943 ◽  
Author(s):  
Jun-Hee Yeon ◽  
Cheon-Gyu Park ◽  
Bertil Hille ◽  
Byung-Chang Suh
Keyword(s):  
Plasma Membrane ◽  
Channel Gating ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intracellular Loop ◽  
Drug Induced ◽  
Interaction Domain ◽  
Intact Cells ◽  
Voltage Gated ◽  
The Stability

β subunits of high voltage-gated Ca2+ (CaV) channels promote cell-surface expression of pore-forming α1 subunits and regulate channel gating through binding to the α-interaction domain (AID) in the first intracellular loop. We addressed the stability of CaV α1B–β interactions by rapamycin-translocatable CaV β subunits that allow drug-induced sequestration and uncoupling of the β subunit from CaV2.2 channel complexes in intact cells. Without CaV α1B/α2δ1, all modified β subunits, except membrane-tethered β2a and β2e, are in the cytosol and rapidly translocate upon rapamycin addition to anchors on target organelles: plasma membrane, mitochondria, or endoplasmic reticulum. In cells coexpressing CaV α1B/α2δ1 subunits, the translocatable β subunits colocalize at the plasma membrane with α1B and stay there after rapamycin application, indicating that interactions between α1B and bound β subunits are very stable. However, the interaction becomes dynamic when other competing β isoforms are coexpressed. Addition of rapamycin, then, switches channel gating and regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipid. Thus, expression of free β isoforms around the channel reveals a dynamic aspect to the α1B–β interaction. On the other hand, translocatable β subunits with AID-binding site mutations are easily dissociated from CaV α1B on the addition of rapamycin, decreasing current amplitude and PI(4,5)P2 sensitivity. Furthermore, the mutations slow CaV2.2 current inactivation and shift the voltage dependence of activation to more positive potentials. Mutated translocatable β subunits work similarly in CaV2.3 channels. In sum, the strong interaction of CaV α1B–β subunits can be overcome by other free β isoforms, permitting dynamic changes in channel properties in intact cells.

Structural Study of H,K-ATPase by Electron Microscopy and Image Reconstruction

1990 ◽  
Vol 48 (1) ◽  
pp. 94-95
Author(s):  
Manijeh Mohraz ◽  
Sonal Sathe ◽  
P.R. Smith
Keyword(s):  
Electron Microscopy ◽  
Amino Acids ◽  
Plasma Membrane ◽  
Image Reconstruction ◽  
Sequence Homology ◽  
Membrane Vesicles ◽  
Two Dimensional ◽  
Β Subunit ◽  
Transport Atpases ◽  
Protein Rich

H,K-ATPase is an integral protein of the plasma membrane of parietal cells. It is believed to constitute the pump responsible for secretion of acid into stomach. Its catalytic subunit (Mr 110,000) shows striking sequence homology to those of other transport ATPases. Recent studies suggest that there is also a glycoprotein (ca 300 amino acids) associated with the H,K pump, which is very homologous to the β subunit of the Na,K-ATPase.The enzyme is isolated in protein-rich membrane vesicles from hog stomachs. Formation of two-dimensional crystals is induced in suspensions of the enzyme by methods that had proved successful for crystallization of the Na,K-ATPase.

A basolateral sorting signal is encoded in the α-subunit of Na-K-ATPase

1998 ◽  
Vol 274 (3) ◽  
pp. C688-C696 ◽  
Author(s):  
T. R. Muth ◽  
C. J. Gottardi ◽  
D. L. Roush ◽  
M. J. Caplan
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Surface Expression ◽  
Apical Surface ◽  
Amino Acid Residues ◽  
Β Subunit ◽  
Α Subunit ◽  
Sorting Signal ◽  
Apical Sorting ◽  
Gastric Parietal Cells

Na-K-ATPase and H-K-ATPase are highly homologous ion pumps that exhibit distinct plasma membrane distributions in epithelial cells. We have studied the α-subunits of these heterodimeric pumps to identify the protein domains responsible for their polarized sorting. A chimeric α-subunit construct (N519H) was generated in which the first 519 amino acid residues correspond to the Na-K-ATPase sequence and the remaining 500 amino acids are derived from the H-K-ATPase sequence. In stably transfected LLC-PK1cell lines, we found that the N519H chimera is restricted to the basolateral surface under steady-state conditions, suggesting that residues within the NH2-terminal 519 amino acids of the Na-K-ATPase α-subunit contain a basolateral sorting signal. H-K-ATPase β-subunit expressed alone in LLC-PK1cells accumulates at the apical surface. When coexpressed with N519H, the H-K-ATPase β-subunit assembles with this chimera and accompanies it to the basolateral surface. Thus the NH2-terminal basolateral signal in the Na-K-ATPase α-subunit masks or is dominant over any apical sorting information present in the β-polypeptide. In gastric parietal cells, the H-K-ATPase β-subunit targets the H-K-ATPase to an intracellular vesicular compartment which fuses with the plasma membrane in response to secretagogue stimulation. To test whether the chimera-H-K-ATPase β-subunit complex is directed to a similar compartment in LLC-PK1cells, we treated transfected cells with drugs that raise intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) levels. Elevation of cytosolic cAMP increased the surface expression of both the N519H chimera and the H-K-ATPase β-subunit. This increase in surface expression, however, appears to be the result of transcriptional upregulation and not recruitment of chimera to the surface from a cAMP-inducible compartment.

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