Reference #5 (Donaldson MR et al.): D71N
- These included six novel mutations (D71N , T75R , G146D , R189I , G300D , and R312C) as well as previously reported mutations R67W and R218W
- Each mutation changed an amino acid of the Kir2.1 protein as follows: R67W (c427t) , D71N (g439a) , T75R (c452g) , G146D (g665a) , R189I (g794t) , R218W (c880t) , G300D (g1127a) , and R312C (c1162t)
- As shown in figure 1 , three sporadic de novo mutations (T75R , D71N , and R67W) were found and three were familial (G146D , R189I , R218W)
- This GYG (amino acids 144 to 146) sequence is vital for potassium selectivity in inward-rectifying and other potassium channels.18 A mutation changing the first glycine of this motif (G144S) in another kindred was shown to have a severe dominant negative effect on Kir2.1 current.6 T75R and D71N change residues in the N terminus with unknown contributions to channel function
- Interestingly , a combination of syndactyly and a previously unreported dramatic shortening of toes was observed in the proband with D71N (figure 2A )
- (A) Feet of proband from K7770 with mutation D71N
- Mutations R67W , D71N , T75R , G146D , R189I , R218W , G300V , and R312C are shown in bold italic type
- As shown in table 2 , patients with mutations in PIP2-related residues were not phenotypically distinct from those with mutations in residues that either had no in vitro involvement in PIP2 binding (D71N)17 or contribute to channel function through a different mechanism (G146D)
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Reference #6 (Bendahhou S et al.): D71V
- Confocal microscopy fluorescence revealed three patterns of channel expression in the cell: 1) mutations D71V , N216H , R218Q , and pore mutations co-assemble and co-localize to the membrane with the WT and exert a dominant-negative effect on the WT channels ; 2) mutation V302M leads to channels that lose their ability to co-assemble with WT and traffic to the cell surface ; 3) deletions {Delta}95-98 and {Delta}314-315 lead to channels that do not traffic to the membrane but retain their ability to co-assemble with WT channels
- All ATS missense mutations (filled circles , R67W , D71V / N , T75R , S136F , G144S , G146D , P186L , R189I , N216H , R218Q / W , G300V / D , V302M , E303K , R312C) and deletions (triangles , {Delta}95-98 and {Delta}314-315) are represented on the channel
- WT cDNA was fused to DsRed fluorescent protein (DsRed) and mutant clones (D71V , T75R , S136F , G144S , N216H , G300V , V302M , E303K , amino acid deletion 95-98 , {Delta}SWLF , deletion 314-315 , {Delta}SY) were fused to EGFP and subcloned in the pDsRed and pEGFP vectors
- To better understand the pathogenesis of this disease , we carried out mammalian cell expression studies of previously identified mutations D71V , {Delta}95-98 , S136F , G144S , N216H , R218Q , G300V , V302M , E303K , and {Delta}314-315
- WT cDNA was fused to Ds red fluorescent protein (DsRed) and all mutant clones (D71V , S136F , G144S , N216H , R218Q , G300V , V302M , E303K , amino acid deletion 95-98 , {Delta}SWLF , and deletion 314-315 , {Delta}SY) were fused to enhanced green fluorescent protein (EGFP) and subcloned in the pDsRed and pEGFP vectors
- RESULTS (image)TOP (image)ABSTRACT (image)INTRODUCTION (image)MATERIALS AND METHODS (image)RESULTS (image)DISCUSSION (image)REFERENCES Andersen-Tawil Syndrome Mutants Do Not Produce Current in HEK293 Cells—To correlate functional expression data with confocal microscopy fluorescence imaging , KCNJ2 wild type (WT) or mutant cDNAs (D71V , G144S , N216H , R218Q , G300V , V302M , E303K , amino acid deletion {Delta}95-98 , and {Delta}314-315) were fused to DsRed or EGFP at the COOH-terminal end and subcloned into the pIRES vector (Fig. 1 )
- While D71V , G144S , N216H , R218Q , G300V , and E303K (-EGFP) co-localize to the membrane with the WT-DsRed producing a yellow color , {Delta}95-98-EGFP and {Delta}314-315-EGFP co-localize in the cytoplasm with WT-DsRed and also produce a yellow color (Fig. 4 )
- Notice that fluorescence is localized to the membrane for WT , D71V , G144S , N216H , R218Q , G300V , and E303K
- Three of these mutations are located in the NH2 terminus (R67W , D71V / N , and T75R) , one deletion occurs in the first transmembrane segment M1 ({Delta}95-98) , three mutations occur in the pore region (S136F , G144S and G146D) , and the remaining mutations reside in the COOH terminus of the Kir 2.1 channel (P186L , R189I , N216H , R218Q / W , G300V / D , V302M , E303K , R312C , and {Delta}314-315)
- D71V , N216H , R218Q , R218W , G300V , and E303K mutant channels seem to co-assemble with the Kir2.1-WT channels and to co-localize to the plasma membrane in HEK293 cells
- The D71V mutation does not alter channel assembly or trafficking or seem to be degraded , suggesting that the slide helix plays a role in the Kir2.1 channel gating
- Protein extracts from HEK293 cells transfected with EGFP alone , WTKir2.1-EGFP , D71V-EGFP , {Delta}95-98-EGFP , {Delta}314-315-EGFP , and mock-transfected were analyzed by Western im munoblotting using anti-GFP antibody
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2005, KChannelDB.
cmbi.ru.nl), 17-Aug-2005