TMEM16A–TMEM16B chimaeras to investigate the structure–function relationship of calcium-activated chloride channels

2013 ◽  
Vol 452 (3) ◽  
pp. 443-455 ◽  
Author(s):  
Paolo Scudieri ◽  
Elvira Sondo ◽  
Emanuela Caci ◽  
Roberto Ravazzolo ◽  
Luis J. V. Galietta
Keyword(s):  
Chloride Channels ◽  
Transmembrane Domain ◽  
Amino Acid Residues ◽  
Intracellular Loop ◽  
Transmembrane Segments ◽  
C Terminus ◽  
Third Intracellular Loop ◽  
Cl Channels ◽  
Function Relationship ◽  
Relationship Of

TMEM16A and TMEM16B proteins are CaCCs (Ca2+-activated Cl− channels) with eight putative transmembrane segments. As shown previously, expression of TMEM16B generates CaCCs characterized by a 10-fold lower Ca2+ affinity and by faster activation and deactivation kinetics with respect to TMEM16A. To investigate the basis of the different properties, we generated chimaeric proteins in which different domains of the TMEM16A protein were replaced by the equivalent domains of TMEM16B. Replacement of the N-terminus, TMD (transmembrane domain) 1–2, the first intracellular loop and TMD3–4 did not change the channel's properties. Instead, replacement of intracellular loop 3 decreased the apparent Ca2+ affinity by nearly 8-fold with respect to wild-type TMEM16A. In contrast, the membrane currents derived from chimaeras containing TMD7–8 or the C-terminus of TMEM16B showed higher activation and deactivation rates without a change in Ca2+ sensitivity. Significantly accelerated kinetics were also found when the entire C-terminus of the TMEM16A protein (77 amino acid residues) was deleted. Our findings indicate that the third intracellular loop of TMEM16A and TMEM16B is the site involved in Ca2+-sensitivity, whereas the C-terminal part, including TMD7–8, affect the rate of transition between the open and the closed state.

scholarly journals Pleiotropic functions of the transmembrane domain 6 of human melanocortin-4 receptor

2012 ◽  
Vol 49 (3) ◽  
pp. 237-248 ◽  
Author(s):  
Hui Huang ◽  
Ya-Xiong Tao
Keyword(s):  
Cell Surface ◽  
Structure Function ◽  
Transmembrane Domain ◽  
Mapk Pathway ◽  
Surface Expression ◽  
Camp Signaling ◽  
Cell Surface Expression ◽  
Melanocortin 4 Receptor ◽  
Function Relationship ◽  
Relationship Of

The melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis and has emerged as a premier target for obesity treatment. Numerous mutations in transmembrane domain 6 (TM6) of MC4R resulting in functional alterations have been identified in obese patients. Several mutagenesis studies also provided some data suggesting the importance of this domain in receptor function. To gain a better understanding of the structure–function relationship of the receptor, we performed alanine-scanning mutagenesis in TM6 to determine the functions of side chains. Of the 31 residues, two were important for cell surface expression, five were indispensable for α-melanocyte-stimulating hormone (α-MSH) and β-MSH binding, and six were important for signaling in the Gs–cAMP–PKA pathway. H264A, targeted normally to the plasma membrane, was undetectable by competitive binding assay and severely defective in basal and stimulated cAMP production and ERK1/2 phosphorylation. Nine mutants had decreased basal cAMP signaling. Seven mutants were constitutively active in cAMP signaling and their basal activities could be inhibited by two MC4R inverse agonists, Ipsen 5i and ML00253764. Five mutants were also constitutively active in the MAPK pathway with enhanced basal ERK1/2 phosphorylation. In summary, our study provided comprehensive data on the structure–function relationship of the TM6 of MC4R. We identified residues that are important for cell surface expression, ligand binding, cAMP generation, and residues for maintaining the WT receptor in active conformation. We also reported constitutive activation of the MAPK pathway and biased signaling. These data will be useful for rationally designing MC4R agonists and antagonists for treatment of eating disorders.

scholarly journals Localization of regions of a Bordetella pertussis autotransporter, Vag8, interacting with C1 inhibitor

2020 ◽  
Author(s):  
Naoki Onoda ◽  
Yukihiro Hiramatsu ◽  
Shihono Teruya ◽  
Koichiro Suzuki ◽  
Yasuhiko Horiguchi
Keyword(s):  
Amino Acid ◽  
Bordetella Pertussis ◽  
Serine Proteases ◽  
Whooping Cough ◽  
Amino Acid Residues ◽  
C1 Inhibitor ◽  
Passenger Domain ◽  
Complement Regulator ◽  
Function Relationship ◽  
Relationship Of

AbstractBordetella pertussis is the causative agent of pertussis (whooping cough), a contagious respiratory disease that has recently seen a resurgence despite high vaccination coverage, necessitating improvement of current pertussis vaccines. An autotransporter of B. pertussis, virulence-associated gene 8 (Vag8), has been proposed as an additional component to improve pertussis vaccines. Vag8 is known to play a role in evasion of the complement system and activation of the contact system by inactivating the complement regulating factor, C1 inhibitor (C1 Inh), which inhibits serine proteases, such as plasma kallikrein (PK). However, the nature of the molecular interaction between Vag8 and C1 Inh remains to be determined. In the present study, we attempted to determine the minimum region of Vag8 that interacts with C1 Inh by examining the differently–truncated Vag8 derivatives for the ability to bind and inactivate C1 Inh. The region of Vag8 from amino–acid residues 102 to 548 was found to bind C1 Inh and cancel its inhibitory action on the protease activity of PK at the same level as a Vag8 fragment from amino–acid residues 52 to 648 covering the passenger domain, which carries its extracellular function. In contrast, the truncated Vag8 containing amino–acid residues 102 – 479 or 202 – 648 barely interacted with C1 Inh. These results indicated that the two separate regions of amino–acid residues 102 – 202 and 479 – 548 are likely required for the interaction with C1 Inh.ImportancePertussis is currently reemerging worldwide, and is still one of the greatest disease burdens in infants. B. pertussis produces a number of virulence factors, including toxins, adhesins, and autotransporters. One of the autotransporters, Vag8, which binds and inactivates the complement regulator C1 Inh, is considered to contribute to the establishment of B. pertussis infection. However, the nature of the interaction between Vag8 and C1 Inh remains to be explored. In this study, we narrowed down the region of Vag8 that interacts with C1 Inh and demonstrated that at least two separate regions of Vag8 are necessary for the interaction with C1 Inh. Our results provide insight into the structure–function relationship of the Vag8 molecule and information to determine its potential role in the pathogenesis of B. pertussis.

Both the wild type and a functional isoform of CFTR are expressed in kidney

1996 ◽  
Vol 270 (6) ◽  
pp. F1038-F1048 ◽  
Author(s):  
M. M. Morales ◽  
T. P. Carroll ◽  
T. Morita ◽  
E. M. Schwiebert ◽  
O. Devuyst ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Transmembrane Domain ◽  
Renal Medulla ◽  
Regulatory Domain ◽  
Wild Type ◽  
Transmembrane Segments ◽  
Binding Domains ◽  
Cl Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) consists of five domains, two transmembrane-spanning domains, each composed of six transmembrane segments, a regulatory domain, and two nucleotide-binding domains (NBDs). CFTR is expressed in kidney, but its role in overall renal function is not well understood, because mutations in CFTR found in patients with cystic fibrosis are not associated with renal dysfunction. To learn more about the distribution and functional forms of CFTR in kidney, we used a combination of molecular, cell biological, and electrophysiological approaches. These include an evaluation of CFTR mRNA and protein expression, as well as both two-electrode and patch clamping of CFTR expressed either in Xenopus oocytes or mammalian cells. In addition to wild-type CFTR mRNA, an alternate form containing only the first transmembrane domain (TMD), the first NBD, and the regulatory domain (TNR-CFTR) is expressed in kidney. Although missing the second set of TMDs and the second NBD, when expressed in Xenopus oocytes, TNR-CFTR has cAMP-dependent protein kinase A (PKA)-stimulated single Cl- channel characteristics and regulation of PKA activation of outwardly rectifying Cl- channels that are very similar to those of wild-type CFTR. TNR-CFTR mRNA is produced by an unusual mRNA processing mechanism and is expressed in a tissue-specific manner primarily in renal medulla.

scholarly journals Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

2008 ◽  
Vol 132 (6) ◽  
pp. 681-692 ◽  
Author(s):  
Qinghuan Xiao ◽  
Andrew Prussia ◽  
Kuai Yu ◽  
Yuan-yuan Cui ◽  
H. Criss Hartzell
Keyword(s):  
Amino Acids ◽  
Density Functional ◽  
Transmembrane Domain ◽  
Channel Gating ◽  
Regulatory Domain ◽  
Acidic Amino Acids ◽  
C Terminus ◽  
Ef Hand ◽  
Cl Channels

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl− ion channels that are activated by intracellular Ca2+. At present, the structures and mechanisms responsible for Ca2+ sensing remain unknown. Here, we have used a combination of molecular modeling, density functional–binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca2+ sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca2+ sensing by hBest1. The cluster of five amino acids (293–308) is crucial for normal channel gating by Ca2+ because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca2+. Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca2+ with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca2+-binding loop (312–323) reduced the apparent Ca2+ affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca2+-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350–390) that is required for Ca2+ activation and plays a role in current rundown. These experiments identify a region of hBest1 (312–323) that is involved in the gating of hBest1 by Ca2+ and suggest a model in which Ca2+ binding to EF1 activates the channel in a process that requires the acidic domain (293–308) and another regulatory domain (350–390). Many of the ∼100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca2+ sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca2+.

Cysteine-directed cross-linking localizes regions of the human erythrocyte anion-exchange protein (AE1) relative to the dimeric interface

2001 ◽  
Vol 359 (3) ◽  
pp. 661-668 ◽  
Author(s):  
Andrew M. TAYLOR ◽  
Quansheng ZHU ◽  
Joseph R. CASEY
Keyword(s):  
Protein Complex ◽  
Human Erythrocyte ◽  
Anion Channel ◽  
Cross Linking ◽  
Intracellular Loop ◽  
Dimeric Interface ◽  
Anion Exchange Protein ◽  
Dimeric Protein ◽  
Function Relationship ◽  
Relationship Of

The human erythrocyte anion-exchanger isoform 1 (AE1) is a dimeric membrane protein that exchanges chloride for bicarbonate across the erythrocyte plasma membrane. Crystallographic studies suggest that the transmembrane anion channel lies at the interface between the two monomers, whereas kinetic analysis provides evidence that each monomer contains an anion channel. We have studied the structure–function relationship of residues at the dimeric interface of AE1 by cysteine-directed cross-linking. Single cysteine mutations were introduced in 16 positions of putative loop regions throughout AE1. The ability of these residues to be chemically cross-linked to their partner within the dimeric protein complex was assessed by mobility of the protein on immunoblots. Introduced cysteine residues in extracellular loops (ECs) 1–4 and intracellular loop 1 formed disulphide cross-linked dimers. Treatment with homobifunctional maleimide cross-linkers of different lengths (6, 10 and 16 Å; 1Å≡0.1nm) also cross-linked AE1 with introduced cysteines in EC5 and close to the start of transmembrane segment (TM) 1. On the basis of these data, tentative positional constraints of TMs 1–4 and 6 relative to the dimeric interface are proposed. Neither disulphide- nor maleimide-mediated cross-linking perturbed AE1 transport function, suggesting that loop–loop contacts across the dimeric interface are not primarily responsible for allosteric interactions between monomers within the functional dimeric protein complex.

Structure and Function Relationship of Murine Insulin-like Peptide 5 (INSL5): Free C-Terminus Is Essential for RXFP4 Receptor Binding and Activation

Biochemistry ◽  
2011 ◽  
Vol 50 (39) ◽  
pp. 8352-8361 ◽  
Author(s):  
Alessia Belgi ◽  
Mohammed A. Hossain ◽  
Fazel Shabanpoor ◽  
Linda Chan ◽  
Suode Zhang ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Structure And Function ◽  
C Terminus ◽  
Function Relationship ◽  
Structure And Function Relationship ◽  
And Function ◽  
Relationship Of

scholarly journals The Extreme C-Terminal Region of Gαs Differentially Couples to the Luteinizing Hormone and β2-Adrenergic Receptors

2011 ◽  
Vol 25 (8) ◽  
pp. 1416-1430 ◽  
Author(s):  
Geneva DeMars ◽  
Francesca Fanelli ◽  
David Puett
Keyword(s):  
G Protein ◽  
Comparative Modeling ◽  
Cell System ◽  
Amino Acid Residues ◽  
Intracellular Loop ◽  
C Terminus ◽  
Model Cell ◽  
Rigid Body Docking ◽  
Molecular Landscape ◽  
Dynamics Simulations

The mechanisms of G protein coupling to G protein-coupled receptors (GPCR) share general characteristics but may exhibit specific interactions unique for each GPCR/G protein partnership. The extreme C terminus (CT) of G protein α-subunits has been shown to be important for association with GPCR. Hypothesizing that the extreme CT of Gαs is an essential component of the molecular landscape of the GPCR, human LH receptor (LHR), and β2-adrenergic receptor (β2-AR), a model cell system was created for the expression and manipulation of Gαs subunits in LHR+ s49 ck cells that lack endogenous Gαs. On the basis of studies involving truncations, mutations, and chain extensions of Gαs, the CT was found to be necessary for LHR and β2-AR signaling. Some general similarities were found for the responses of the two receptors, but significant differences were also noted. Computational modeling was performed with a combination of comparative modeling, molecular dynamics simulations, and rigid body docking. The resulting models, focused on the Gαs CT, are supported by the experimental observations and are characterized by the interaction of the four extreme CT amino acid residues of Gαs with residues in LHR and β2-AR helix 3, (including R of the DRY motif), helix 6, and intracellular loop 2. This portion of Gαs recognizes the same regions of the two GPCR, although with differences in the details of selected interactions. The predicted longer cytosolic extensions of helices 5 and 6 of β2-AR are expected to contribute significantly to differences in Gαs recognition by the two receptors.

The C-terminus of the third intracellular loop of the H2 receptor is important in the regulation of c-fos and cell growth

2000 ◽  
Vol 118 (4) ◽  
pp. A180
Author(s):  
Lidong Wang ◽  
Meizhi Wang ◽  
Kenneth Butler ◽  
Ira Gantz ◽  
Edward Grand ◽  
...  
Keyword(s):  
Cell Growth ◽  
Intracellular Loop ◽  
H2 Receptor ◽  
The Third ◽  
C Terminus ◽  
Third Intracellular Loop

scholarly journals Amino acid residues in third intracellular loop of melanocortin 1 receptor are involved in G-protein coupling

IUBMB Life ◽  
1998 ◽  
Vol 46 (5) ◽  
pp. 913-922
Author(s):  
Per-Anders Frändberg ◽  
Marina Doufexis ◽  
Supriya Kapas ◽  
Vijay Chhajlani
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
Amino Acid Residues ◽  
Intracellular Loop ◽  
G Protein Coupling ◽  
Melanocortin 1 Receptor ◽  
Protein Coupling ◽  
Third Intracellular Loop
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