ABCC7 p.Asp835*
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[hide] Identification of the cystic fibrosis transmembran... J Biol Chem. 2000 Jun 2;275(22):16697-701. Cahill P, Nason MW Jr, Ambrose C, Yao TY, Thomas P, Egan ME
Identification of the cystic fibrosis transmembrane conductance regulator domains that are important for interactions with ROMK2.
J Biol Chem. 2000 Jun 2;275(22):16697-701., 2000-06-02 [PMID:10748197]
Abstract [show]
In addition to functioning as a cAMP-activated chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR) plays an important role in conferring regulatory properties on other ion channels. It is known, with respect to CFTR regulation of ROMK2 (renally derived K(ATP) channel), that the first transmembrane domain and the first nucleotide binding fold domain (NBF1) of CFTR are necessary for this interaction to occur. It has been shown that under conditions that promote phosphorylation, the ROMK2-CFTR interaction is attenuated. To elucidate the complex nature of this interaction, CFTR constructs were co-expressed with ROMK2 in Xenopus oocytes, and two microelectrode voltage clamp experiments were performed. Although the second half of CFTR can act as a functional chloride channel, our results suggest that it does not confer glibenclamide sensitivity on ROMK2, as does the first half of CFTR. The attenuation of the ROMK2-CFTR interaction under conditions that promote phosphorylation is dependent on at least the presence of the R domain of CFTR. We conclude that transmembrane domain 1, NBF1, and the R domain are the CFTR domains involved in the ROMK2-CFTR interaction and that NBF2 and transmembrane domain 2 are not essential. Lastly, the R domain of CFTR is necessary for the attenuation of the ROMK2-CFTR interaction under conditions that promote phosphorylation.
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No. Sentence Comment
53 These experiments were performed on oocytes expressing ROMK2/CFTR-K593X and ROMK2/CFTR-D835X and showed that the current was Ba2ϩ -sensitive Kϩ current and not stimulated Cl- current.
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ABCC7 p.Asp835* 10748197:53:87
status: NEW58 CFTR Constructs and Method of Mutagenesis-Cells were injected with ROMK2 alone, ROMK2/CFTR-WT, ROMK2/SUR, ROMK2/CFTR-D835X (a CFTR construct with an intact nucleotide binding fold and a functional R domain), ROMK2/CFTR-K593X (a CFTR construct with an intact nucleotide binding fold but no R domain), or ROMK2/ RT2N2CFTR (a CFTR construct containing the R domain, the second nucleotide binding fold domain, the second transmembrane domain, and the first 159 bases of CFTR-WT so as to include the Kozak methionine for translation initiation) (Fig. 1).
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ABCC7 p.Asp835* 10748197:58:117
status: NEW61 The oligonucleotides used for mutagenesis were CFTR-K593X, 5Ј-CTGTTAACTGATGGCT- AGCAAACTAGG-3Ј and CFTR-D835X, 5ЈCACGAAAAGTGTCACTGG- CCCCTCAGGCAAACTTCGATATATTACTGTCCACAAGAGCTTAAT- TTTGTGC-3Ј.
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ABCC7 p.Asp835* 10748197:61:116
status: NEW65 CFTR-D835X and CFTR-K593X are expressed at membrane level, in varying expression systems (9, 19).
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ABCC7 p.Asp835* 10748197:65:5
status: NEW77 Schematic representation of CFTR-WT (A), RT2N2CFTR (B), a CFTR mutant containing the R domain, transmembrane domain 2, NBF2, and the initial 159 bases of CFTR-WT so as to include the Kozak methionone for translation initiation, CFTR-K593X (C), a CFTR mutant truncated at residue 593, with an intact NBF1 and CFTR-D835X (D), a CFTR mutant truncated after the R domain.
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ABCC7 p.Asp835* 10748197:77:313
status: NEW85 However, when ROMK2 and CFTR-D835X (a CFTR construct with an intact nucleotide binding fold and a functional R domain) were co-expressed and stimulated with the cAMP- enhancing mixture, the resultant Kϩ current demonstrated an attenuated sulfonylurea response (26 Ϯ 5% inhibition, n ϭ 16) (Fig. 4).
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ABCC7 p.Asp835* 10748197:85:29
status: NEW86 In the absence of FSK/IBMX there was a 48 Ϯ 10% inhibition of Kϩ current when ROMK2 and CFTR-D835X were co-expressed (n ϭ 9) (Table I, Figs. 2-4).
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ABCC7 p.Asp835* 10748197:86:105
status: NEW91 This suggests that the ROMK2/SUR proteins remain functionally interactive under conditions that increase cAMP-dependent phosphorylation unlike the ROMK/CFTRWT and ROMK2/CFTR-D835X proteins.
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ABCC7 p.Asp835* 10748197:91:174
status: NEW96 Conditions that promote phosphorylation did not alter the glibenclamide insensitive FIG. 2. Effect of glibenclamide on whole cell Ba2ϩ -sensitive Kϩ currents for ROMK2 when co-expressed with CFTR-D835X, CFTR-K593X, or RT2N2CFTR under basal conditions (in the absence of FSK/IBMX) (A, B, and E) in Xenopus oocytes, using two-microelectrode voltage clamp techniques.
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ABCC7 p.Asp835* 10748197:96:208
status: NEW103 A representative family of whole cell currents from oocytes injected with either ROMK2/CFTR-D835X or ROMK2/ CFTR-K593X, and traces under basal conditions (in the absence of FSK/IBMX) and after stimulation with FSK/IBMX (100 M/1 mM) are compared.
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ABCC7 p.Asp835* 10748197:103:92
status: NEW105 It demonstrates that in ROMK2/ CFTR-K593X-injected oocytes, glibenclamide inhibition of whole cell Kϩ current remains prominent, despite application of FSK/IBMX, but that in ROMK2/CFTR-D835X-injected oocytes the FSK/IBMX mixture attenuates this inhibitory response.
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ABCC7 p.Asp835* 10748197:105:191
status: NEW120 d p value Ͻ0.05 when comparing % glibenclamide inhibition of whole cell Kϩ current under basal conditions and after stimulation with FSK/IBMX for R2/D835X where there is attenuation of inhibitory response with FSK/IBMX.
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ABCC7 p.Asp835* 10748197:120:161
status: NEW122 FIG. 4. Effect of glibenclamide on Ba2؉ -sensitive currents obtained from Xenopus oocytes using two-microelectrode voltage clamp techniques. Summary of data obtained for ROMK2, ROMK2/ CFTR-WT, ROMK2/CFTR-D835X, ROMK2/CFTR-K593X, and ROMK2/SUR, as indicated on the x axis. Represented on the y axis is the percentage of total barium-sensitive Kϩ current inhibited by glibenclamide.
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ABCC7 p.Asp835* 10748197:122:210
status: NEW125 FIG. 5. Effect of glibenclamide on Ba2؉ -sensitive currents obtained from Xenopus oocytes using two-microelectrode voltage clamp techniques. Summary of data obtained for ROMK2, ROMK2/ RT2N2CFTR, ROMK2/CFTR-WT, and ROMK2/CFTR-D835X as indicated on the x axis. Represented on the y axis is the percentage of total barium-sensitive Kϩ current inhibited by glibenclamide.
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ABCC7 p.Asp835* 10748197:125:231
status: NEW128 In contrast, the first half of the CFTR molecule (CFTR-D835X), when co-expressed with ROMK2, behaves in a similar pattern to ROMK2/CFTR-WT and assumes sensitivity to sulfonylureas.
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ABCC7 p.Asp835* 10748197:128:55
status: NEW