Fluorescence measurements reveal stoichiometry of K
            <sup>+</sup>
            channels formed by modulatory and delayed rectifier α-subunits

Kerschensteiner, Daniel; Soto, Florentina; Stocker, Martin

doi:10.1073/pnas.0500468102

Cited by 57 publications

(66 citation statements)

References 45 publications

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“…24 h after transfection cells were fixed and mounted on slides as described previously (28). A Zeiss LSM510 META ConfoCor3 system with a 63 ϫ 1.4 numerical aperture oil-immersion objective was used for image acquisition.…”

Section: Methodsmentioning

confidence: 99%

The Oligomeric State of the Active BM2 Ion Channel Protein of Influenza B Virus

Balannik

Lamb

Pinto

2008

Journal of Biological Chemistry

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Influenza A virus and influenza B virus particles both contain small integral membrane proteins (A/M2 and BM2, respectively) that function as a pH-sensitive proton channel and are essential for virus replication. The mechanism of action of the M2 channels is a subject of scientific interest particularly as A/M2 channel was shown to be a target for the action of the antiviral drug amantadine. Unfortunately, an inhibitor of the BM2 channel activity is not known. Thus, knowledge of the structural and functional properties of the BM2 channel is essential for the development of potent antiviral drugs. The characterization of the oligomeric state of the BM2 channel is an essential first step in the understanding of channel function. Here we describe determination of the stoichiometry of the BM2 proton channel by utilizing three different approaches. 1) We demonstrated that BM2 monomers can be chemically cross-linked to yield species consistent with dimers, trimers, and tetramers. 2) We studied electrophysiological and biochemical properties of mixed oligomers consisting of wild-type and mutated BM2 subunits and related these data to predicted binomial distribution models. 3) We used fluorescence resonance energy transfer (FRET) in combination with biochemical measurements to estimate the relationships between BM2 channel subunits expressed in the plasma membrane. Our experimental data are consistent with a tetrameric structure of the BM2 channel. Finally, we demonstrated that BM2 transmembrane domain is responsible for the channel oligomerization.Influenza A and B viruses are enveloped viruses with a genome consisting of eight segments of negative-strand RNA. RNA segment 7 of both influenza A and B viruses encodes the M2 integral membrane protein (A/M2 and BM2, respectively) that functions as a pH-sensitive proton channel and is essential for virus replication (1-5). A/M2 and BM2 proteins both consist of a small N-terminal ectodomain (24 and 7 residues, respectively), a single transmembrane domain, 19 amino acids long, and a cytoplasmic tail (54 and 83 residues, respectively) (5). Although A/M2 and BM2 proteins have similar structural and functional properties, the only homology between their amino acid sequences is found in the HXXXW motif of the membrane-spanning region. This motif plays a critical role in the ion channel activity (4, 6 -8). The A/M2 channel activity is inhibited by the antiviral drug amantadine (9, 10) but unfortunately, amantadine does not inhibit the BM2 channel activity (5, 11).The oligomeric state of the A/M2 channel had been investigated by several approaches. First, A/M2 was shown to form homo-oligomers, most likely homotetramers, by cross-linking and sedimentation experiments (12, 13). Site-specific mutagenesis studies demonstrated that the oligomeric structure of A/M2 channels is stabilized by disulfide bonds (14). Finally, by functional analysis of mixed oligomers of known composition, the active oligomeric form of A/M2 protein was shown to be a tetramer (15). Most recently the atomic stru...

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Section: Methodsmentioning

confidence: 99%

The Oligomeric State of the Active BM2 Ion Channel Protein of Influenza B Virus

Balannik

Lamb

Pinto

2008

Journal of Biological Chemistry

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“…To confirm that the 75-kDa and 150-kDa complexes are dimers and tetramers constituted by 3a proteins only, FRET was used (10,11). 3aCFP and 3aYFP recombinant plasmids were constructed and used to transfect HeLa cells.…”

Section: Confirmation Of 3a Proteinmentioning

confidence: 99%

Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release

Zheng

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

285

395

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Fourteen ORFs have been identified in the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) genome. ORF 3a of SARS-CoV codes for a recently identified transmembrane protein, but its function remains unknown. In this study we confirmed the 3a protein expression and investigated its localization at the surface of SARS-CoV-infected or 3a-cDNA-transfected cells. Our experiments showed that recombinant 3a protein can form a homotetramer complex through interprotein disulfide bridges in 3a-cDNA-transfected cells, providing a clue to ion channel function. The putative ion channel activity of this protein was assessed in 3a-complement RNA-injected Xenopus oocytes by two-electrode voltage clamp. The results suggest that 3a protein forms a potassium sensitive channel, which can be efficiently inhibited by barium. After FRhK-4 cells were transfected with an siRNA, which is known to suppress 3a expression, followed by infection with SARS-CoV, the released virus was significantly decreased, whereas the replication of the virus in the infected cells was not changed. Our observation suggests that SARS-CoV ORF 3a functions as an ion channel that may promote virus release. This finding will help to explain the highly pathogenic nature of SARS-CoV and to develop new strategies for treatment of SARS infection. caused alarm all over the world. The newly discovered human coronavirus named SARS-associated coronavirus (SARS-CoV) was identified as the causative agent for this disease (1, 2). SARSCoV has a large single-positive-strand RNA genome that contains 14 ORFs. Some of these ORFs encode viral structural proteins, such as spike protein, membrane protein, small envelope protein, and nucleocapsid protein, as well as viral replicase and protease (3). Those proteins play important roles in viral infection and replication. However, functions for other ORFs are not clear. Therefore, identification and characterization of new functional proteins from the ORFs will be helpful for understanding the pathogenesis of SARS-CoV. Up to now there are still no effective drugs or vaccines against SARS-CoV. The identification of new viral proteins and the elucidation of their functions will provide potential targets for design of drugs or vaccines against SARS.Our previous work has revealed that ORF 3a of SARS-CoV is such a viral protein (4). Since then, other publications have concurred in this observation and have shown that it is a structural protein (5-8). ORF 3a is located between the S and E protein loci and encodes a protein of 274 aa. The only available information based on proteomics and immunoblotting suggests that 3a protein is structural in nature, but its localization, topology, and biological function have not been identified.A computed biology analysis of the amino acid sequence of the 3a protein revealed that it has low similarity with any other known protein. Its C-terminal region shares Ϸ50% similarity to Plasmodium calcium pump protein and to the Shewanella outer-membrane porin. Interestingly, comparison of ORFs ...

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“…Our experimental design and rationale for determining subunit stoichiometry closely followed that of Zheng and colleagues [16,17], Staruschenko and colleagues [13], and Kerschensteiner and colleagues [19]. Zheng et al (2002) and Kerschensteiner et al (2005) successfully used a FRET method in the determination of a fixed stoichiometry of 3:1 for the rod cyclic-nucleotide gated channels and heteromeric Kv2.1/Kv9.3 channels, respectively.…”

Section: Stoichiometry Of Asic Channelmentioning

confidence: 99%

“…Zheng et al (2002) and Kerschensteiner et al (2005) successfully used a FRET method in the determination of a fixed stoichiometry of 3:1 for the rod cyclic-nucleotide gated channels and heteromeric Kv2.1/Kv9.3 channels, respectively. Staruschenko and colleagues (2004) also used this method to establish that epithelial Na + channels (ENaC) have a 3α:3β:3γ stoichiometry.…”

Section: Stoichiometry Of Asic Channelmentioning

confidence: 99%

“…The efficiency of energy transfer falls off with the sixth power of the distance between the donor and acceptor molecules [14]. FRET has been successfully employed as a molecular ruler in investigations of the stoichiometry, assembly, and conformational arrangement of many types of receptors and channels [13,[15][16][17][18][19]. Another advantage of FRET over biochemistry approaches such as co-IP is that FRET measurement is non-invasive, allowing determination of subunit stoichiometry from intact channels in the cell membrane.…”

Section: Introductionmentioning

confidence: 99%

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Fluorescence resonance energy transfer analysis of subunit assembly of the ASIC channel

Gao

Liu

Qiu

et al. 2007

Biochemical and Biophysical Research Communications

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Acid-sensing ion channels (ASICs) are believed to be homo-or heteromeric complexes, which have been verified by classical methods such as co-immunoprecipitation or electrophysiological assays. However, the exact subunit combinations of ASICs in living cells have not been established yet. Here, we apply assays based on fluorescence resonance energy transfer (FRET) between GFP color mutants CFP and YFP to investigate ASIC assembly directly in living cells. Homomerization as well as heteromerization of different combinations of ASIC subunits were found. In addition, our results suggest the formation of heteromeric 1a/2a channels of stoichiometry consisting of at least two 1a subunits and two 2a subunits. Similar stoichiometry was observed from heteromeric 1a/2b and 2a/ 2b channels. Our results imply that these heteromeric ASIC channels contain at least four subunits. KeywordsFRET; ASIC; Assembly; Homomer; Heteromer; Stoichiometry; CHO Acid-sensing ion channels (ASICs), a class of ligand-gated cation channels activated by protons and highly expressed in both peripheral sensory neurons and central neurons, play important roles in mechanosensation, nociception, learning-memory, and brain ischemia [1,2]. ASICs belong to the ENaC/DEG superfamily, which also includes the amiloride-sensitive epithelial sodium channels (ENaCs), the mechanically gated degenerins of Caenorhabditis elegans (DEGs), and a neuropeptide-gated channel of Helix aspersa (FaNaC). The transmembrane topology of all channels in this superfamily features cytosolic amino and carboxy termini, two hydrophobic transmembrane segments and a large cysteine-rich extracellular domain [3]. So far, six ASIC subunits encoded by four genes have been cloned, ASIC1a (BNaC2α), ASIC1b(BNaC2β), ASIC2a (MDEG1), ASIC2b (MDEG2), ASIC3 (DRASIC) and ASIC4 (SPASIC). Homo-and heteromeric ASIC complexes have been verified by classical methods such as co-immunoprecipitation, or functional assays applying electrophysiology [4][5][6][7][8][9]. However, it is at present largely unknown how many subunits are Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. To directly determine the assembly of ASIC subunits of intact channels in living cells, we have used a biophysical approach based on fluorescence resonance energy transfer (FRET). FRET is a physical phenomenon in which energy absorbed by a fluorophore (CFP, the donor) is transferred to another fluorophore (YFP, the acceptor) through a nonradiative process. Because distances closer than about 10 nm between appropriate fluorophores are required to elicit FRET, it is a sensitive measure o...

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Fluorescence measurements reveal stoichiometry of K ⁺ channels formed by modulatory and delayed rectifier α-subunits

Cited by 57 publications

References 45 publications

The Oligomeric State of the Active BM2 Ion Channel Protein of Influenza B Virus

The Oligomeric State of the Active BM2 Ion Channel Protein of Influenza B Virus

Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release

Fluorescence resonance energy transfer analysis of subunit assembly of the ASIC channel

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Fluorescence measurements reveal stoichiometry of K + channels formed by modulatory and delayed rectifier α-subunits

Cited by 57 publications

References 45 publications

The Oligomeric State of the Active BM2 Ion Channel Protein of Influenza B Virus

The Oligomeric State of the Active BM2 Ion Channel Protein of Influenza B Virus

Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release

Fluorescence resonance energy transfer analysis of subunit assembly of the ASIC channel

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Product

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Fluorescence measurements reveal stoichiometry of K ⁺ channels formed by modulatory and delayed rectifier α-subunits