scholarly journals Cholesterol-Binding Sites in GIRK Channels: The Devil is in the Details

2018 ◽  
Vol 11 ◽  
pp. 117863531775407 ◽  
Author(s):  
Avia Rosenhouse-Dantsker
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Experimental Approach ◽  
Transmembrane Domain ◽  
Transmembrane Helices ◽  
Direct Role ◽  
Girk Channels ◽  
Inwardly Rectifying ◽  
The Devil ◽  
Cholesterol Binding

In recent years, it has become evident that cholesterol plays a direct role in the modulation of a variety of ion channels. In most cases, cholesterol downregulates channel activity. In contrast, our earlier studies have demonstrated that atrial G protein inwardly rectifying potassium (GIRK) channels are upregulated by cholesterol. Recently, we have shown that hippocampal GIRK currents are also upregulated by cholesterol. A combined computational-experimental approach pointed to putative cholesterol-binding sites in the transmembrane domain of the GIRK2 channel, the primary subunit in hippocampal GIRK channels. In particular, the principal cholesterol-binding site was located in the center of the transmembrane domain in between the inner and outer α-helices of 2 adjacent subunits. Further studies pointed to a similar cholesterol-binding site in GIRK4, a major subunit in atrial GIRK channels. However, a close look at a sequence alignment of the transmembrane helices of the 2 channels reveals surprising differences among the residues that interact with the cholesterol molecule in these 2 channels. Here, we compare the residues that form putative cholesterol-binding sites in GIRK2 and GIRK4 and discuss the similarities and differences among them.

scholarly journals Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABAA) receptors

2020 ◽  
Vol 295 (33) ◽  
pp. 11495-11512 ◽  
Author(s):  
Selwyn S. Jayakar ◽  
David C. Chiara ◽  
Xiaojuan Zhou ◽  
Bo Wu ◽  
Karol S. Bruzik ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Photoaffinity Labeling ◽  
Subunit Interface ◽  
Steroid Sensitivity ◽  
Steroid Binding ◽  
Definition Of

Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) with in vivo anesthetic, anxiolytic, and anti-convulsant effects. Mutational analysis, photoaffinity labeling, and structural studies have provided evidence for intersubunit and intrasubunit steroid-binding sites in the GABAAR transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1β3 and α1β3γ2 GABAARs by photoaffinity labeling with [3H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([3H]21-pTFDBzox-AP), a potent GABAAR PAM. Protein microsequencing established 3α5α-P inhibitable photolabeling of amino acids near the cytoplasmic end of the β subunit M4 (β3Pro-415, β3Leu-417, and β3Thr-418) and M3 (β3Arg-309) helices located at the base of a pocket in the β+–α− subunit interface that extends to the level of αGln-242, a steroid sensitivity determinant in the αM1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3α-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3α-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3α5α-P, as well as 3β-OH analogs that are GABAAR antagonists, bound with at least 1000-fold lower affinity than 3α5α-P. Similarly, for GABAAR PAMs with the C-20 carbonyl of 3α5α-P or pregnanolone reduced to a hydroxyl, binding affinity is reduced by 1,000-fold, whereas binding is retained after deoxygenation at the C-20 position. These results provide a first insight into the structure-activity relationship at the GABAAR β+–α− subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.

Fluorometric study on conformational changes in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase

1995 ◽  
Vol 15 (5) ◽  
pp. 317-326 ◽  
Author(s):  
Tohru Kanazawa ◽  
Hiroshi Suzuki ◽  
Takashi Daiho ◽  
Kazuo Yamasaki
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Conformational Changes ◽  
Binding Sites ◽  
Transmembrane Domain ◽  
Tryptophan Fluorescence ◽  
Cytoplasmic Domain ◽  
Catalytic Cycle ◽  
Atp Binding ◽  
Atp Binding Site

Changes in the fluoresence of N-acetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine (EDANS), being attached to Cys-674 of sarcoplasmic reticulum Ca2+-ATPase without affecting the catalytic activity, as well as changes in the intrinsic tryptophan fluorescence were followed throughout the catalytic cycle by the steady-state measurements and the stopped-flow spectrofluorometry. EDANS-fluorescence changes reflect conformational changes near the ATP binding site in the cytoplasmic domain, while tryptophan-fluorescence changes most probably reflect conformational changes in or near the transmembrane domain in which the Ca2+ binding sites are located. Formation of the phosphoenzyme intermediates (EP) was also followed by the continuous flow-rapid quenching method. The kinetic analysis of EDANS-fluorescence changes and EP formation revealed that, when ATP is added to the calcium-activated enzyme, conformational changes in the ATP binding site occur in three successive reaction steps; conformational change in the calcium enzyme substrate complex, formation of ADP-sensitive EP, and transition of ADP-sensitive EP to ADP-insensitive EP. In contrast, the ATP-induced tryptophan-fluorescence changes occur only in the latter two steps. Thus, we conclude that conformational changes in the ATP binding site in the cytoplasmic domain are transmitted to the Ca2+-binding sites in the transmembrane domain in these latter two steps.

scholarly journals Photolabelling the urotensin II receptor reveals distinct agonist- and partial-agonist-binding sites

2007 ◽  
Vol 402 (1) ◽  
pp. 51-61 ◽  
Author(s):  
Brian J. Holleran ◽  
Marie-Eve Beaulieu ◽  
Christophe D. Proulx ◽  
Pierre Lavigne ◽  
Emanuel Escher ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Binding Sites ◽  
Transmembrane Domain ◽  
Partial Agonist ◽  
Binding Pocket ◽  
Activation Mechanism ◽  
Urotensin Ii ◽  
Agonist Binding ◽  
Partial Agonists

The mechanism by which GPCRs (G-protein-coupled receptors) undergo activation is believed to involve conformational changes following agonist binding. We have used photoaffinity labelling to identify domains within GPCRs that make contact with various photoreactive ligands in order to better understand the activation mechanism. Here, a series of four agonist {[Bpa1]U-II (Bpa is p-benzoyl-L-phenylalanine), [Bpa2]U-II, [Bpa3]U-II and [Bpa4]U-II} and three partial agonist {[Bpa1Pen5D-Trp7Orn8]U-II (Pen is penicillamine), [Bpa2Pen5D-Trp7Orn8]U-II and [Pen5Bpa6D-Trp7Orn8]U-II} photoreactive urotensin II (U-II) analogues were used to identify ligand-binding sites on the UT receptor (U-II receptor). All peptides bound the UT receptor expressed in COS-7 cells with high affinity (Kd of 0.3–17.7 nM). Proteolytic mapping and mutational analysis led to the identification of Met288 of the third extracellular loop of the UT receptor as a binding site for all four agonist peptides. Both partial agonists containing the photoreactive group in positions 1 and 2 also cross-linked to Met288. We found that photolabelling with the partial agonist containing the photoreactive group in position 6 led to the detection of transmembrane domain 5 as a binding site for that ligand. Interestingly, this differs from Met184/Met185 of the fourth transmembrane domain that had been identified previously as a contact site for the full agonist [Bpa6]U-II. These results enable us to better map the binding pocket of the UT receptor. Moreover, the data also suggest that, although structurally related agonists or partial agonists may dock in the same general binding pocket, conformational changes induced by various states of activation may result in slight differences in spatial proximity within the cyclic portion of U-II analogues.

The Cleaved Peptide of PAR1 Results in a Redistribution of the Platelet Surface GPIb-IX-V Complex to the Surface-Connected Canalicular System

2000 ◽  
Vol 84 (11) ◽  
pp. 897-903 ◽  
Author(s):  
Mark Furman ◽  
Paquita Nurden ◽  
Michael Berndt ◽  
Alan Nurden ◽  
Stephen Benoit ◽  
...  
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Binding Sites ◽  
Transmembrane Domain ◽  
Surface Expression ◽  
Thrombin Receptor ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe only known function of the 41 amino acid cleaved peptide (TR1-41) of the seven transmembrane domain thrombin receptor (PAR1) is to activate platelets (as determined by aggregation, surface P-selectin, and fibrinogen binding to activated GPIIb-IIIa). We now demonstrate that TR1-41 results in a concentration-dependent decrease in the platelet surface expression of each component of the GPIb-IX-V complex, as determined by flow cytometry with a panel of monoclonal antibodies (including 6D1, directed against the von Willebrand factor binding site on GPIbα, and TM60, directed against the thrombin binding site on GPIbα). TR1-41 also decreased ristocetin-induced platelet agglutination. Immunoblotting after incubation of platelets with TR1-41 revealed neither a loss of platelet GPIb nor increase in supernatant GPIb fragments. As demonstrated by immunoelectron microscopy, TR1-41 resulted in a redistribution of GPIb, GPIX, and GPV from the platelet surface to the surface-connected canalicular system (SCCS). In summary, the cleaved peptide (TR1-41) of PAR1 results in a redistribution of the platelet surface GPIb-IX-V complex to the SCCS, thereby negatively regulating the GPIbα binding sites for von Willebrand factor and thrombin.

scholarly journals Regulation of mga Transcription in the Group A Streptococcus: Specific Binding of Mga within Its Own Promoter and Evidence for a Negative Regulator

1999 ◽  
Vol 181 (17) ◽  
pp. 5373-5383 ◽  
Author(s):  
Kevin S. McIver ◽  
Alec S. Thurman ◽  
June R. Scott
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Group A Streptococcus ◽  
Specific Binding ◽  
Virulence Gene ◽  
Negative Regulator ◽  
Mobility Shift ◽  
Direct Role ◽  
Group A

ABSTRACT Transcription of mga, encoding the multiple virulence gene regulator of the group A streptococcus, is positively autoregulated. This regulation requires a DNA region (Pmga) that contains both a promoter proximal to mga (P2) and a promoter located further upstream (P1). To determine if Mga has a direct role in this process, its ability to bind to specific sequences within Pmga was tested. A purified fusion of Mga to the C-terminal end of maltose-binding protein (MBP-Mga), encoded by malE-mga, was shown previously to bind to the promoter regions of Mga-regulated genes, including scpA and emm. We report here that MBP-Mga can function in vivo to regulate emm andmga. Electrophoretic mobility shift assays and DNase I footprinting were used to demonstrate specific binding of MBP-Mga to two ca. 59-bp binding sites in Pmga centered around bases −108 and −180 from the major P2 start of transcription. Mga binding sites from Pemm and PscpA were shown to compete for binding at the two Pmga sites, suggesting that the same domain of Mga interacts at all of these promoter targets. Deletion of the distal Pmga binding site (site I) in vivo resulted in loss of Mga-dependent transcription from the P2 start. However, the same lesion resulted in an increase in P1 transcription that was independent of Mga. This suggests the existence of a repressor of mga transcription with a binding site overlapping those of Mga.

scholarly journals Structural basis of GIRK2 channel modulation by cholesterol and PIP2

2020 ◽  
Author(s):  
Yamuna Kalyani Mathiharan ◽  
Ian W. Glaaser ◽  
Yulin Zhao ◽  
Michael J. Robertson ◽  
Georgios Skiniotis ◽  
...  
Keyword(s):  
G Protein ◽  
Transmembrane Domain ◽  
Structural Data ◽  
Structural Basis ◽  
Girk Channels ◽  
Channel Modulation ◽  
Cryo Electron Microscopy ◽  
Cellular Excitability ◽  
Cholesteryl Hemisuccinate ◽  
Inwardly Rectifying

ABSTRACTG protein-gated inwardly rectifying potassium (GIRK) channels play important roles in controlling cellular excitability in the heart and brain. While structural data begin to unravel the molecular basis for G protein and alcohol dependent activation of GIRK channels, little is known about the modulation by cholesterol. Here, we present cryo-electron microscopy (cryoEM) structures of GIRK2 in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS), and PIP2. The structures and their comparison reveal that CHS binds near PIP2 in lipid-facing hydrophobic pockets of the transmembrane domain (TMD). CHS potentiates the effects of PIP2, which stabilizes the inter-domain region and promotes the engagement of the cytoplasmic domain (CTD) onto the transmembrane region. The results suggest that CHS acts as a positive allosteric modulator and identify novel therapeutic sites for modulating GIRK channels in the brain.

Sign in / Sign up

Export Citation Format

Share Document