ABCMdb

ABCC8 p.Arg74Lys

None has been submitted yet.

Publications

PMID: 21321069 [PubMed] Pratt EB et al: "N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2."

No. Sentence Comment

78 Surface expression for every R74X-mutant tested, except the charge-conserving R74K mutation, was significantly reduced to <40% of WT (Fig. 1 B). Login to comment
107 R74K, which had surface expression at 74 ± 4% of WT by chemiluminescence (Fig. 1 B), showed both upper and lower glycosylation bands. Login to comment
121 R74K had the highest level, with 70% of WT; all other R74 substitutions (alanine, phenylalanine, tyrosine, and tryptophan) resulted in significantly decreased TMD0 levels, with <25% of WT (Fig. 3, A and B), possibly a result of increased degradation. Login to comment
122 Next, we examined the surface expression of R74W and R74K fTMD0 when coexpressed with Kir6.2C36 using immunofluorescent obtain the IC50 for ATP inhibition (Fig. 2 B). Login to comment
123 As with trafficking, the charge-conserving mutant R74K had minimal effect on ATP inhibition (IC50 = 18 ± 2 and 17 ± 1 µM for WT and R74K, respectively). Login to comment
141 No surface fTMD0 was detected above background signal using anti-FLAG antibody for either mutant construct, even though the R74K mutation exhibited the greatest total TMD0 level (70% of WT; Fig. 3 B); in contrast, surface staining of cells expressing WT mini-KATP channels was clearly visible (Fig. 3 E). Login to comment
142 Consistently, in single-channel recordings from cells coexpressing R74K or R74W fTMD0 with Kir6.2C36, no channel kinetics distinct from those characteristic of channels formed by Kir6.2C36 alone were observed (unpublished data). Login to comment
149 (E) Cells cotransfected with WT, R74W, R74K, or E128K fTMD0 and Kir6.2C36 were probed with -FLAG antibody 48 h after transfection to detect surface expression of mini-KATP channels. Login to comment
150 A rim of surface staining was detected in WTand E128K-transfected cells (green, inset images), but not in either R74W- or R74K-transfected cells. Login to comment
258 It may seem surprising that although total R74K TMD0 protein remains at near WT levels (Fig. 3, A and B), no R74K mini-channels were observed at the cell surface either by immunostaining or single-channel recording. Login to comment
259 That the R74K mutation has a more pronounced effect on the surface expression of mini-KATP versus full-length channels (i.e., no detectable surface expression of mini-channels [Fig. 3 C] vs. 80% of WT for full-length channels [Fig. 1, B and C]) suggests a role for extra-TMD0 regions of SUR1 in channel protein folding and assembly. Login to comment
288 In addition, the ATP-dependent recovery from inactivation in the E128W mutant suggests that conformational changes in Kir6.2 tertiary structure of R74K TMD0 and its ability to assemble with Kir6.2 to form channels may be contingent upon the SUR1 structures downstream of TMD0. Login to comment