scholarly journals Regulation of the cardiac Na(+)-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca(2+)-binding domain.

1995 ◽  
Vol 105 (3) ◽  
pp. 403-420 ◽  
Author(s):  
S Matsuoka ◽  
D A Nicoll ◽  
L V Hryshko ◽  
D O Levitsky ◽  
J N Weiss ◽  
...  
Keyword(s):  
Binding Site ◽  
Mutational Analysis ◽  
Outward Current ◽  
Binding Domain ◽  
Kinetic Properties ◽  
Regulatory Site ◽  
Wild Type ◽  
Intracellular Loop ◽  
High Affinity ◽  
Reverse Mode

The sarcolemmal Na(+)-Ca2+ exchanger is regulated by intracellular Ca2+ at a high affinity Ca2+ binding site separate from the Ca2+ transport site. Previous data have suggested that the Ca2+ regulatory site is located on the large intracellular loop of the Na(+)-Ca2+ exchange protein, and we have identified a high-affinity 45Ca2+ binding domain on this loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Journal of Biological Chemistry. 269:22847-22852). We now use electrophysiological and mutational analyses to further define the Ca2+ regulatory site. Wild-type and mutant exchangers were expressed in Xenopus oocytes, and the exchange current was measured using the inside-out giant membrane patch technique. Ca2+ regulation was measured as the stimulation of reverse Na(+)-Ca2+ exchange (intracellular Na+ exchanging for extracellular Ca2+) by intracellular Ca2+. Single-site mutations within two acidic clusters of the Ca2+ binding domain lowered the apparent Ca2+ affinity at the regulatory site from 0.4 to 1.1-1.8 microM. Mutations had parallel effects on the affinity of the exchanger loop for 45Ca2+ binding (Levitsky et al., 1994) and for functional Ca2+ regulation. We conclude that we have identified the functionally important Ca2+ binding domain. All mutant exchangers with decreased apparent affinities at the regulatory Ca2+ binding site also have a complex pattern of altered kinetic properties. The outward current of the wild-type Na(+)-Ca2+ exchanger declines with a half time (th) of 10.8 +/- 3.2 s upon Ca2+ removal, whereas the exchange currents of several mutants decline with th values of 0.7-4.3 s. Likewise, Ca2+ regulation mutants respond more rapidly to Ca2+ application. Study of Ca2+ regulation has previously been possible only with the exchanger operating in the reverse mode as the regulatory Ca2+ and the transported Ca2+ are then on opposite sides of the membrane. The use of exchange mutants with low affinity for Ca2+ at regulatory sites also allows demonstration of secondary Ca2+ regulation with the exchanger in the forward or Ca2+ efflux mode. In addition, we find that the affinity of wild-type and mutant Na(+)-Ca2+ exchangers for intracellular Na+ decreases at low regulatory Ca2+. This suggests that Ca2+ regulation modifies transport properties and does not only control the fraction of exchangers in an active state.

scholarly journals Gating Competence of Constitutively Open CLC-0 Mutants Revealed by the Interaction with a Small Organic Inhibitor

2003 ◽  
Vol 122 (3) ◽  
pp. 295-306 ◽  
Author(s):  
Sonia Traverso ◽  
Laura Elia ◽  
Michael Pusch
Keyword(s):  
Chloride Channels ◽  
Bound State ◽  
Side Chain ◽  
Pore Channel ◽  
Kinetic Properties ◽  
Wild Type ◽  
High Affinity ◽  
Critical Residue ◽  
Fast Gating

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl−-sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at −140 mV ∼4 μM). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

scholarly journals Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing

1992 ◽  
Vol 12 (3) ◽  
pp. 1209-1217
Author(s):  
C F Hardy ◽  
D Balderes ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Binding Protein ◽  
Binding Domain ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Carboxy Terminal

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of an agonistic binding site for blockers of the M3 muscarinic acetylcholine receptor

2008 ◽  
Vol 412 (1) ◽  
pp. 103-112 ◽  
Author(s):  
Doreen Thor ◽  
Angela Schulz ◽  
Thomas Hermsdorf ◽  
Torsten Schöneberg
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
G Protein ◽  
Binding Sites ◽  
Expression System ◽  
Muscarinic Acetylcholine Receptor ◽  
Intracellular Loop ◽  
Inverse Agonists ◽  
Yeast Expression System ◽  
High Affinity

GPCRs (G-protein-coupled receptors) exist in a spontaneous equilibrium between active and inactive conformations that are stabilized by agonists and inverse agonists respectively. Because ligand binding of agonists and inverse agonists often occurs in a competitive manner, one can assume an overlap between both binding sites. Only a few studies report mutations in GPCRs that convert receptor blockers into agonists by unknown mechanisms. Taking advantage of a genetically modified yeast strain, we screened libraries of mutant M3Rs {M3 mAChRs [muscarinic ACh (acetylcholine) receptors)]} and identified 13 mutants which could be activated by atropine (EC50 0.3–10 μM), an inverse agonist on wild-type M3R. Many of the mutations sensitizing M3R to atropine activation were located at the junction of intracellular loop 3 and helix 6, a region known to be involved in G-protein coupling. In addition to atropine, the pharmacological switch was found for other M3R blockers such as scopolamine, pirenzepine and oxybutynine. However, atropine functions as an agonist on the mutant M3R only when expressed in yeast, but not in mammalian COS-7 cells, although high-affinity ligand binding was comparable in both expression systems. Interestingly, we found that atropine still blocks carbachol-induced activation of the M3R mutants in the yeast expression system by binding at the high-affinity-binding site (Ki ∼10 nM). Our results indicate that blocker-to-agonist converting mutations enable atropine to function as both agonist and antagonist by interaction with two functionally distinct binding sites.

scholarly journals Identification of the Magnesium-binding Domain of the High-affinity ATP-binding Site of theBacillus subtilisandEscherichia coliSecA Protein

1995 ◽  
Vol 270 (32) ◽  
pp. 18975-18982 ◽  
Author(s):  
Jeroen P. W. van der Wolk ◽  
Michael Klose ◽  
Janny G. de Wit ◽  
Tanneke den Blaauwen ◽  
Roland Freudl ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Domain ◽  
Atp Binding ◽  
Atp Binding Site ◽  
High Affinity ◽  
Magnesium Binding

scholarly journals Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing.

1992 ◽  
Vol 12 (3) ◽  
pp. 1209-1217 ◽  
Author(s):  
C F Hardy ◽  
D Balderes ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Binding Protein ◽  
Binding Domain ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Carboxy Terminal

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Functional Analysis of the CAAT Box in the Major Late Promoter of the Subgroup C Human Adenoviruses

1998 ◽  
Vol 72 (4) ◽  
pp. 3213-3220 ◽  
Author(s):  
Byeongwoon Song ◽  
C. S. H. Young
Keyword(s):  
Binding Site ◽  
Transcription Initiation ◽  
Mutational Analysis ◽  
Wild Type ◽  
Late Promoter ◽  
Phenotypic Effect ◽  
Partial Restoration ◽  
Wild Type Phenotype ◽  
Major Late Promoter

ABSTRACT Comparisons among sequences predicted to encode the major late promoter (MLP) of adenoviruses from a wide variety of host species show that an inverted CAAT box is among the most highly conserved transcription elements found in the putative MLPs. The high degree of conservation suggests that the CAAT box plays an important role in the function of the MLP in vivo, an idea supported by a previous mutational analysis of the core CCAAT sequence. To address the importance of the CAAT box, in terms both of quantitative levels of transcription and of specificity, a further set of mutations was created and examined in the context of the viral genome. One mutation, CAAT5, contains individual changes at five positions, four of which correspond to invariant residues in a CAAT box consensus derived either by computer analysis or empirically. The CAAT5 mutation had no discernible phenotype by itself but when coupled with the previously described USF0 mutation, which disrupts binding of the upstream stimulating factor (USF) but is otherwise phenotypically silent, gave rise to virus with a severe replication deficiency. Nuclear run-on assays showed that transcription initiation at the mutant MLP was significantly reduced compared with that of the wild type or the virus containing CAAT5 alone. Replication of the double mutant was lower than that of the previously described USF0::CCCAT virus, suggesting that the additional mutations in the CAAT box had further lowered the binding of transcription factor CP1 (also called CBF, NF-Y). Replacement of the CAAT box by an ATF binding site or an OCT1 binding site had no phenotypic effect in an otherwise wild-type background, but replacement in a USF0::CCCAT background led to only partial restoration of the wild-type phenotype. The failure to restore the functional redundancy normally exhibited by the CAAT box and the proximal upstream activating element is consistent with the idea that in the adenovirus MLP the CAAT box is preferred over others as the distal transcriptional element.

scholarly journals Characterization of the DNA-Binding Domain of the Avian Y-Box Protein, chkYB-2, and Mutational Analysis of Its Single-Strand Binding Motif in the Rous Sarcoma Virus Enhancer

1998 ◽  
Vol 72 (2) ◽  
pp. 900-909 ◽  
Author(s):  
Ashok Nambiar ◽  
S. K. Swamynathan ◽  
Jagannadha C. Kandala ◽  
Ramareddy V. Guntaka
Keyword(s):  
Dna Binding ◽  
Rous Sarcoma Virus ◽  
Mutational Analysis ◽  
Binding Domain ◽  
Single Strand ◽  
Dna Binding Domain ◽  
Single Stranded Dna ◽  
High Affinity ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT chkYB-2 is a sequence-specific, single-stranded DNA binding chicken Y-box protein that promotes Rous sarcoma virus long terminal repeat (RSV LTR)-driven transcription in avian fibroblasts. The DNA-binding domain of chkYB-2 has been mapped by characterizing the DNA binding properties of purified recombinant chkYB-2 mutant polypeptides. The data indicate that the invariant cold shock domain (CSD) is necessary but not sufficient for association with DNA and suggest that another conserved region, adjacent to the carboxyl boundary of the CSD, plays a role in high-affinity DNA binding. chkYB-2 binds to a tandem repeat of the 5′-GTACCACC-3′ motif on the RSV LTR. Mutational analysis of this recognition sequence revealed the requirement of an essentially unaltered template for both high-affinity binding by chkYB-2 as well as maximal transcriptional activity of the RSV LTR in vivo. The single-stranded DNA binding activity of chkYB-2 is augmented by Mg2+. The possible significance of this finding for transactivation by a single-strand DNA binding protein is discussed.

scholarly journals Mutational Analysis of the High-Affinity Zinc Binding Site Validates a Refined Human Dopamine Transporter Homology Model

2013 ◽  
Vol 9 (2) ◽  
pp. e1002909 ◽  
Author(s):  
Thomas Stockner ◽  
Therese R. Montgomery ◽  
Oliver Kudlacek ◽  
Rene Weissensteiner ◽  
Gerhard F. Ecker ◽  
...  
Keyword(s):  
Binding Site ◽  
Dopamine Transporter ◽  
Mutational Analysis ◽  
Homology Model ◽  
Zinc Binding ◽  
High Affinity ◽  
Zinc Binding Site
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