Molecular site for nucleotide binding on an ATP-sensitive renal K+ channel (ROMK2)

1996 ◽  
Vol 271 (2) ◽  
pp. F275-F285 ◽  
Author(s):  
C. M. McNicholas ◽  
Y. Yang ◽  
G. Giebisch ◽  
S. C. Hebert
Keyword(s):  
Channel Gating ◽  
Direct Interaction ◽  
K Channel ◽  
Amino Terminus ◽  
Atp Binding Site ◽  
Gating Mechanism ◽  
Alternatively Spliced ◽  
Carboxy Terminal ◽  
Apical Membranes

ATP-sensitive, inwardly rectifying K+ channels are present in apical membranes of the distal nephron and play a major role in K+ recycling and secretion. The cloned renal K+ channel, ROMK1, is a candidate for the renal epithelial K+ channel, since it shares many functional characteristics with the native channel. Additionally, ROMK1 contains a putative carboxy-terminal ATP-binding site. Although ROMK1 channel activity could be reactivated by cytosolic Mg-ATP after rundown, the role of nucleotides in channel gating was less certain. We now show that an alternatively spliced transcript of the ROMK channel gene, ROMK2, which encodes a K+ channel with a truncated amino terminus, expresses an ATP-regulated and ATP-sensitive K+ channel (IKATP). Differences in the amino terminus of ROMK isoforms alters the sensitivity of the channel-gating mechanism to ATP. To test whether ATP sensitivity of renal IKATP is mediated by direct interaction of nucleotide, point mutation of specific residues within the ROMK2 phosphate loop (P-loop) were investigated. These either enhanced or attenuated the sensitivity to both activation and inhibition by Mg-ATP, thus demonstrating a direct interaction of nucleotide with the channel-forming polypeptide.

scholarly journals Direct Interaction of a Brain Voltage-Gated K+Channel with Syntaxin 1A: Functional Impact on Channel Gating

2001 ◽  
Vol 21 (6) ◽  
pp. 1964-1974 ◽  
Author(s):  
Oded Fili ◽  
Itzhak Michaelevski ◽  
Yaniv Bledi ◽  
Dodo Chikvashvili ◽  
Dafna Singer-Lahat ◽  
...  
Keyword(s):  
Channel Gating ◽  
Direct Interaction ◽  
K Channel ◽  
Syntaxin 1A ◽  
Functional Impact ◽  
Voltage Gated

K+ channel gating mechanism proposed using EPR

1998 ◽  
Vol 5 (6) ◽  
pp. 418-420 ◽  
Author(s):  
Yeon-Kyun Shin
Keyword(s):  
Channel Gating ◽  
K Channel ◽  
Gating Mechanism

scholarly journals Interaction of internal anions with potassium channels of the squid giant axon.

1983 ◽  
Vol 82 (4) ◽  
pp. 429-448 ◽  
Author(s):  
D J Adams ◽  
G S Oxford
Keyword(s):  
Prolonged Exposure ◽  
Channel Gating ◽  
Reversal Potential ◽  
Inorganic Anions ◽  
K Channel ◽  
Delayed Rectifier ◽  
K Channels ◽  
Gating Mechanism ◽  
Inorganic Species ◽  
K Currents

The interaction of internal anions with the delayed rectifier potassium channel was studied in perfused squid axons. Changing the internal potassium salt from K+ glutamate- to KF produced a reversible decline of outward K currents and a marked slowing of the activation of K channels at all voltages. Fluoride ions exert a differential effect upon K channel gating kinetics whereby activation of IK during depolarizing steps is slowed dramatically, but the rate of closing after the step is not much altered. These effects develop with a slow time course (30-60 min) and are specific for K channels over Na channels. Both the amplitude and activation rate of IK were restored within seconds upon return to internal glutamate solutions. The fluoride effect is independent of the external K+ concentration and test membrane potential, and does not recover with repetitive application of depolarizing voltage steps. Of 11 different anions tested, all inorganic species induced similar decreases and slowing of IK, while K currents were maintained during extended perfusion with several organic anions. Anions do not alter the reversal potential or shape of the instantaneous current-voltage relation of open K channels. The effect of prolonged exposure to internal fluoride could be partially reversed by the addition of cationic K channel blocking agents such as TEA+, 4-AP+, and Cs+. The competitive antagonism between inorganic anions and internal cationic K channel blockers suggests that they may interact at a related site(s). These results indicate that inorganic anions modify part of the K channel gating mechanism (activation) at a locus near the inner channel surface.

scholarly journals Shaker-IR K Channel Gating in Heavy Water: Role of Structural Water Molecules in Inactivation

2016 ◽  
Vol 110 (3) ◽  
pp. 343a-344a
Author(s):  
Tibor G. Szanto ◽  
Szabolcs M. Gaal ◽  
Zoltan Varga ◽  
Gyorgy Panyi
Keyword(s):  
Heavy Water ◽  
Channel Gating ◽  
K Channel ◽  
Water Molecules ◽  
Structural Water

scholarly journals Localization of the ATP/Phosphatidylinositol 4,5 Diphosphate-binding Site to a 39-Amino Acid Region of the Carboxyl Terminus of the ATP-regulated K+Channel Kir1.1

2002 ◽  
Vol 277 (51) ◽  
pp. 49366-49373 ◽  
Author(s):  
Ke Dong ◽  
LieQi Tang ◽  
Gordon G. MacGregor ◽  
Steven C. Hebert
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Transmembrane Domain ◽  
Direct Interaction ◽  
Terminal Segment ◽  
K Channel ◽  
Atp Binding ◽  
Amino Acid Residues ◽  
Atp Binding Site ◽  
Arginine Residues

Intracellular ATP and membrane-associated phosphatidylinositol phospholipids, like PIP2(PI(4,5)P2), regulate the activity of ATP-sensitive K+(KATP) and Kir1.1 channels by direct interaction with the pore-forming subunits of these channels. We previously demonstrated direct binding of TNP-ATP (2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)-ATP) to the COOH-terminal cytosolic domains of the pore-forming subunits of Kir1.1 and Kir6.x channels. In addition, PIP2competed for TNP-ATP binding on the COOH termini of Kir1.1 and Kir6.x channels, providing a mechanism that can account for PIP2antagonism of ATP inhibition of these channels. To localize the ATP-binding site within the COOH terminus of Kir1.1, we produced and purified maltose-binding protein (MBP) fusion proteins containing truncated and/or mutated Kir1.1 COOH termini and examined the binding of TNP-ATP and competition by PIP2. A truncated COOH-terminal fusion protein construct, MBP_1.1CΔC170, containing the first 39 amino acid residues distal to the second transmembrane domain was sufficient to bind TNP-ATP with high affinity. A construct containing the remaining COOH-terminal segment distal to the first 39 amino acid residues did not bind TNP-ATP. Deletion of 5 or more amino acid residues from the NH2-terminal side of the COOH terminus abolished nucleotide binding to the entire COOH terminus or to the first 49 amino acid residues of the COOH terminus. PIP2competed TNP-ATP binding to MBP_1.1CΔC170 with an EC50of 10.9 μm. Mutation of any one of three arginine residues (R188A/E, R203A, and R217A), which are conserved in Kir1.1 and KATPchannels and are involved in ATP and/or PIP2effects on channel activity, dramatically reduced TNP-ATP binding to MBP_1.1ΔC170. In contrast, mutation of a fourth conserved residue (R212A) exhibited slightly enhanced TNP-ATP binding and increased affinity for PIP2competition of TNP-ATP (EC50= 5.7 μm). These studies suggest that the first 39 COOH-terminal amino acid residues form an ATP-PIP2binding domain in Kir1.1 and possibly the Kir6.x ATP-sensitive K+channels.

scholarly journals Revisiting the Role of Ca2+ in Shaker K+ Channel Gating

2001 ◽  
Vol 80 (5) ◽  
pp. 2216-2220 ◽  
Author(s):  
Kwang Hee Hong ◽  
Clay M. Armstrong ◽  
Christopher Miller
Keyword(s):  
Channel Gating ◽  
K Channel

scholarly journals Cer1p Functions as a Molecular Chaperone in the Endoplasmic Reticulum of Saccharomyces cerevisiae

1999 ◽  
Vol 19 (8) ◽  
pp. 5298-5307 ◽  
Author(s):  
T. Guy Hamilton ◽  
Tracy B. Norris ◽  
Pamela R. Tsuruda ◽  
Gregory C. Flynn
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Peptide Binding ◽  
Direct Interaction ◽  
Amino Acid Identity ◽  
Carboxypeptidase Y ◽  
Atp Binding Site ◽  
Hsp70 Family

ABSTRACT Cer1p/Lhs1p/Ssi1p is a novel Hsp70-related protein that is important for the translocation of a subset of proteins into the yeastSaccharomyces cerevisiae endoplasmic reticulum. Cer1p has very limited amino acid identity to the hsp70 chaperone family in the N-terminal ATPase domain but lacks homology to the highly conserved hsp70 peptide binding domain. The role of Cer1p in protein folding and translocation was assessed. Deletion of CER1 slowed the folding of reduced pro-carboxypeptidase Y (pro-CPY) approximately twofold in yeast. In wild-type yeast under reducing conditions, pro-CPY can be found in a complex with Cer1p, while partially purified Cer1p is able to bind directly to peptides. Together, this suggests that Cer1p has a chaperoning activity required for proper refolding of denatured pro-CPY which is mediated by direct interaction with the unfolded polypeptide. Cer1p peptide binding and oligomerization could be disrupted by addition of ATP, confirming that Cer1p possesses a functional ATP binding site, much like Kar2p and other members of the hsp70 family. Interestingly, replacing the signal sequence of aCER1-dependent protein with that of aCER1-independent protein did not relieve the requirement ofCER1 for import. This result suggests that an interaction with the mature portion of the protein also is important for the translocation role of Cer1p. The CER1 RNA levels increase at lower temperatures. In addition, the effects of deletion on folding and translocation are more severe at lower temperatures. Therefore, these results suggest that Cer1p provides an additional chaperoning activity in processes known to require Kar2p. However, there appears to be a greater requirement for Cer1p chaperone activity at lower temperatures.

The Ability of Fibrinogens, FI and FII, to Support ADP- Induced Platelet Aggregation

1983 ◽  
Vol 50 (02) ◽  
pp. 527-529 ◽  
Author(s):  
H M Phillips ◽  
A Mansouri ◽  
C A Perry
Keyword(s):  
Platelet Aggregation ◽  
Terminal Portion ◽  
Hepes Buffer ◽  
Carboxy Terminal

SummaryFibrinogen plays an integral part in ADP-induced platelet aggregation. Controversy exists in regard to the role of the carboxy termini of fibrinogen Aa chains in this reaction. We have attempted to clarify this problem in view of the availability of a highly purified FII fibrinogen fraction. Kabi fibrinogen or its purified fractions FI, FII and FIII-IV-V were added to washed platelets in the presence of Tyrode-HEPES buffer pH 7.4. Aggregation was initiated by the addition of calcium and ADP. These fibrinogen fractions equally promoted ADP-induced platelet aggregation. The major difference among these fractions is in their Aα chains. The FI fraction contains intact Aα chains while FII and FIH-IV-V fractions have one and two partially degraded Aα chains at the carboxy terminal portion respectively. We conclude that the carboxy terminal portion of the Aα chain does not play an important role in promoting ADP-induced platelet aggregation.

scholarly journals Structure of the full-length human Pannexin1 channel and insights into its role in pyroptosis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Sensen Zhang ◽  
Baolei Yuan ◽  
Jordy Homing Lam ◽  
Jun Zhou ◽  
Xuan Zhou ◽  
...  
Keyword(s):  
Md Simulation ◽  
Potential Role ◽  
Md Simulations ◽  
Full Length ◽  
Inflammasome Activation ◽  
Simulation Studies ◽  
Cryo Electron Microscopy ◽  
Gating Mechanism ◽  
Atp Efflux

AbstractPannexin1 (PANX1) is a large-pore ATP efflux channel with a broad distribution, which allows the exchange of molecules and ions smaller than 1 kDa between the cytoplasm and extracellular space. In this study, we show that in human macrophages PANX1 expression is upregulated by diverse stimuli that promote pyroptosis, which is reminiscent of the previously reported lipopolysaccharide-induced upregulation of PANX1 during inflammasome activation. To further elucidate the function of PANX1, we propose the full-length human Pannexin1 (hPANX1) model through cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulation studies, establishing hPANX1 as a homo-heptamer and revealing that both the N-termini and C-termini protrude deeply into the channel pore funnel. MD simulations also elucidate key energetic features governing the channel that lay a foundation to understand the channel gating mechanism. Structural analyses, functional characterizations, and computational studies support the current hPANX1-MD model, suggesting the potential role of hPANX1 in pyroptosis during immune responses.

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