ABCMdb

PMID: 10748197

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 [PubMed]

Sentences

No. Mutations Sentence Comment

53 ABCC7 p.Lys593* ABCC7 p.Asp835* 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. Login to comment 58 ABCC7 p.Lys593* ABCC7 p.Asp835* 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). Login to comment 61 ABCC7 p.Lys593* ABCC7 p.Asp835* The oligonucleotides used for mutagenesis were CFTR-K593X, 5Ј-CTGTTAACTGATGGCT- AGCAAACTAGG-3Ј and CFTR-D835X, 5ЈCACGAAAAGTGTCACTGG- CCCCTCAGGCAAACTTCGATATATTACTGTCCACAAGAGCTTAAT- TTTGTGC-3Ј. Login to comment 65 ABCC7 p.Lys593* ABCC7 p.Asp835* CFTR-D835X and CFTR-K593X are expressed at membrane level, in varying expression systems (9, 19). Login to comment 76 ABCC7 p.Lys593* When ROMK2 was co-expressed with a CFTR construct containing transmembrane domain 1 and NBF1 without an R domain (CFTR-K593X) and exposed to FSK/IBMX, the resultant Ba2ϩ -sensitive Kϩ current was inFIG. 1. Login to comment 77 ABCC7 p.Lys593* ABCC7 p.Asp835* 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. Login to comment 81 ABCC7 p.Lys593* Without cAMP stimulation glibenclamide inhibited 46 Ϯ 4.5% (n ϭ 6) of Kϩ current when ROMK2 and CFTR-K593X were co-expressed, which is similar to previous findings. Login to comment 84 ABCC7 p.Lys593* Furthermore, after cAMP stimulation there is a significant enhancement of glibenclamide effect on ROMK2/CFTR-K593X currents (Table I). Login to comment 85 ABCC7 p.Asp835* 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). Login to comment 86 ABCC7 p.Asp835* 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). Login to comment 91 ABCC7 p.Asp835* ABCC7 p.Asp835* 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. Login to comment 92 ABCC7 p.Lys593* However, in contrast to the ROMK2/CFTR-K593X currents, glibenclamide inhibition is not enhanced after cAMP stimulation. Login to comment 96 ABCC7 p.Lys593* ABCC7 p.Asp835* 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. Login to comment 103 ABCC7 p.Lys593* ABCC7 p.Asp835* 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. Login to comment 105 ABCC7 p.Lys593* ABCC7 p.Asp835* 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. Login to comment 120 ABCC7 p.Asp835* 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. Login to comment 121 ABCC7 p.Lys593* e p value Ͻ0.05 when comparing % glibenclamide inhibition of whole cell Kϩ current under basal conditions and after stimulation with FSK/IBMX for R2/K593X where there is a marked enhancement of inhibitory response with FSK/IBMX. Login to comment 122 ABCC7 p.Lys593* ABCC7 p.Asp835* 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. Login to comment 125 ABCC7 p.Asp835* 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. Login to comment 128 ABCC7 p.Asp835* 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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