Participation of Nucleoside-diphosphate Kinase in Muscarinic K+ Channel Activation Does Not Involve GTP Formation

Xu, Long; Murphy, Joseph F.; Otero, Angela de S.

doi:10.1074/jbc.271.35.21120

Cited by 17 publications

(17 citation statements)

References 38 publications

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“…1B and 3); this is expected, since the enzyme from frog tissues tends to aggregate when heated in SDS. 3 Nevertheless, the NDP kinase band was still clearly detectable in these eluates. This result demonstrates that the copurification of the other proteins with NDP kinase depends on native structures; furthermore, when denatured, these proteins do not share epitopes with NDP kinase and do not bind directly to the antibodies.…”

Section: Resultsmentioning

confidence: 99%

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Copurification of Vimentin, Energy Metabolism Enzymes, and a MER5 Homolog with Nucleoside Diphosphate Kinase

Otero

1997

Journal of Biological Chemistry

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Chromatography on immobilized antibodies specific to nucleoside diphosphate (NDP) kinase was utilized for affinity purification of this enzyme from detergent extracts of frog heart post-mitochondrial fractions. SDSpolyacrylamide gel electrophoresis analysis of eluates from these supports shows that five polypeptides copurify with nucleoside diphosphate (NDP) kinase. Tryptic digests of each band were analyzed by mass spectrometric microsequencing. Data base searches by peptide mass matching and sequence homology led to the identification of these proteins as glyceraldehyde-3-phosphate dehydrogenase (40 kDa), creatine kinase (45 kDa), vimentin (55 kDa), pyruvate kinase (60 kDa), and a putative member of the antioxidant protein family (28 kDa). Distinct protein compositions were found in eluates of lung and liver extracts processed in a like manner. The 28-kDa band and vimentin were associated with NDP kinase from all tissues, but co-purification of pyruvate kinase was seen only in liver, while creatine kinase and glyceraldehyde-3-phosphate dehydrogenase were absent from eluates from lung and liver. The results suggest that while NDP kinase is associated with vimentin intermediate filaments and an antioxidant protein in most tissues, it interacts with energy metabolism enzymes in a tissue-specific manner.In recent years NDP kinase 1 has been found to have unexpected roles, in addition to its well known ability to catalyze transfer of phosphate groups from trinucleotides to dinucleotides (1). NDP kinases are involved in growth, differentiation, development, tumor progression, metastasis, and apoptosis (reviewed in Ref. 2). NDP kinase participates in muscarinic K ϩ channel opening (3), is a transcription factor for c-myc (4) and a protein kinase as well (5, 6). These varied functions seem to be accomplished by a limited number of gene products: to date, complete cDNAs encoding 4 human NDP kinases (nm23-H1, nm23-H2, DR-nm23, and nm23-H4) have been identified (7-9).Also, in Dictyostelium a separate nuclear gene encodes a mitochondrial NDP kinase (10). Although other NDP kinase genes may be identified in the future, it is unlikely that each of the multiple functions associated with this protein is performed by a distinct isoform. Differential distribution of NDP kinase isoforms (11) may account for adaptation to the requirements of specific cell types. Additionally, interaction of distinct isoforms with cell-specific factors, conceivably other proteins, could govern NDP kinase function in different tissues. This in turn might explain apparently contradictory findings regarding the connection between NDP kinase and cancer (2): while in some cell types, studies of NDP kinase expression suggest that it has a metastasis suppressor function, in other systems this relation between expression of NDP kinase and metastatic potential is either nonexistent or operates in the opposite direction.The objective of the present work was to identify proteins that interact with NDP kinase in cardiac muscle. Immobilized antibodies to frog ...

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Section: Resultsmentioning

confidence: 99%

“…2). NDP kinase participates in muscarinic K ϩ channel opening (3), is a transcription factor for c-myc (4) and a protein kinase as well (5,6). These varied functions seem to be accomplished by a limited number of gene products: to date, complete cDNAs encoding 4 human NDP kinases (nm23-H1, nm23-H2, DR-nm23, and nm23-H4) have been identified (7)(8)(9).…”

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confidence: 99%

Copurification of Vimentin, Energy Metabolism Enzymes, and a MER5 Homolog with Nucleoside Diphosphate Kinase

Otero

1997

Journal of Biological Chemistry

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“…Single channel activity in excised, inside-out atrial membrane patches was measured, stored, and analyzed essentially as described previously (10). The external (pipette) solution contained 0.1 M KCl, 2.5 mM CaCl 2 , and 20 mM HEPES, pH 7.2 with KOH; carbamylcholine (10 M) or atropine (1 M) was added when noted.…”

Section: Methodsmentioning

confidence: 99%

“…Pertussis toxin was activated as described previously (10) and diluted to a final toxin concentration of 1 g/ml with bath solution containing 1 mM NAD before application to patches.…”

Section: Methodsmentioning

confidence: 99%

Receptor-independent Activation of Atrial Muscarinic Potassium Channels in the Absence of Nucleotides

Otero

et al. 1998

Journal of Biological Chemistry

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The removal of nucleotides from the solution bathing the inner face of excised patches of frog atrial membranes was found to activate muscarinic K ؉ channels in the absence of agonists. Channel activation was also observed in Mg 2؉-free solutions and blocked by low (0.1-10 M) concentrations of GDP or GTP. After full activation was achieved, channel openings were abolished by the application of GDP-bound G␣ i2 but were not affected by exogenous G␤␥ dimers, suggesting that effector activation is a consequence of the liberation of ␤␥ subunits from endogenous G proteins. The process of channel activation in the absence of nucleotides seems to be receptor-independent, because it is not influenced by muscarinic receptor agonists and antagonists or by treatment with uncoupling agents such as pertussis toxin or N-ethyl maleimide. Taken together, these results suggest that the loss of GDP from the G protein nucleotide binding site promotes its uncoupling from receptors and destabilizes the G␣ (empty) ␤␥ heterotrimer. Therefore, the nucleotide-free form of G proteins has some of the characteristics of the GTP-bound, activated form.The muscarinic K ϩ channel (K ACh ) 1 is an inwardly rectifying, potassium-selective channel whose activation by agonistbound receptors is mediated by the ␤␥ subunits of a pertussis toxin (PTX)-sensitive GTP-binding protein (reviewed in Ref. 1). In the heart, the primary components of this system of signal transduction have been identified. These include the muscarinic m 2 receptor, heterotrimeric G proteins of the G i subfamily, and the heterotetrameric channel formed by K ir 3.1 (GIRK1) and K ir 3.4 (GIRK4 or CIR) subunits (2-5). Several neurotransmitter-regulated channels share the basic structural and functional features of atrial K ACh channels; therefore, the latter are considered a prototype for the study of G protein-channel interactions (6). In addition, because patch clamp methods allow the study of signal transduction with high time resolution and under circumstances in which the composition of the milieu can be strictly controlled, atrial muscarinic K ϩ channels can also be viewed as rapid, selective, and sensitive monitors of G protein function (7,8). In the present work, we used excised, inside-out patches of atrial membranes to examine how G proteins might be affected by the total absence of intracellular nucleotides. Our results provide new insights into the behavior of a nucleotide-free form of G proteins that appears regardless of receptor occupancy and suggest that the empty ␣ subunit shares some characteristics of the activated, GTP-bound form. EXPERIMENTAL PROCEDURESAtrial myocytes were obtained from bullfrog hearts by digestion with collagenase and trypsin (9). Pertussis toxin was a gift from Dr. Erik Hewlett (University of Virginia). G protein subunits ␣ i2 -GDP and ␤␥ were a gift from Dr. S. Graber (West Virginia University). Protease inhibitors and nucleotides were from Boehringer Manheim, except for Mg⅐ATP, which was from Sigma. All other chemicals were of the highest p...

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“…Indeed, experimental data have been reported already in the late 1980s, which support such a complex of membranous NDPK with the stimulatory G protein of adenylyl cyclase (G s ; Kimura andShimada 1988a,b, 1990) or the retinal G protein transducin (G t ; Orlov et al 1996;Klinker and Seifert 1999). Nevertheless, the contribution of local GTP formation to effector regulation was disputed or not detected in other systems (Xu et al 1996;Sorota et al 1998). The major drawback in all those studies was that the enzymatic activity of NDPK was used as a major read-out for detection of the presence of the enzyme.…”

Section: Introductionmentioning

confidence: 99%

Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein βγ dimers: consequences on G protein activation and stability

Wieland

2007

Naunyn-Schmied Arch Pharmacol

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Participation of Nucleoside-diphosphate Kinase in Muscarinic K+ Channel Activation Does Not Involve GTP Formation

Cited by 17 publications

References 38 publications

Copurification of Vimentin, Energy Metabolism Enzymes, and a MER5 Homolog with Nucleoside Diphosphate Kinase

Copurification of Vimentin, Energy Metabolism Enzymes, and a MER5 Homolog with Nucleoside Diphosphate Kinase

Receptor-independent Activation of Atrial Muscarinic Potassium Channels in the Absence of Nucleotides

Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein βγ dimers: consequences on G protein activation and stability

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