Conserved functional consequences of disease-associated mutations in the slide helix of Kir6.1 and Kir6.2 subunits of the ATP-sensitive potassium channel

Paige E Cooper,Conor Mcclenaghan,Xingyu Chen,Anna Stary-Weinzinger,Colin G Nichols

doi:10.1074/jbc.m117.804971

Paige E Cooper, Conor Mcclenaghan + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m117.804971

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Abstract

Cantu syndrome (CS) is a condition characterized by a range of anatomical defects, including cardiomegaly, hyperflexibility of the joints, hypertrichosis, and craniofacial dysmorphology. CS is associated with multiple missense mutations in the genes encoding the regulatory sulfonylurea receptor 2 (SUR2) subunits of the ATP-sensitive K+ (KATP) channel as well as two mutations (V65M and C176S) in the Kir6.1 (KCNJ8) subunit. Previous analysis of leucine and alanine substitutions at the Val-65-equivalent site (Val-64) in Kir6.2 indicated no major effects on channel function. In this study, we characterized the effects of both valine-to-methionine and valine-to-leucine substitutions at this position in both Kir6.1 and Kir6.2 using ion flux and patch clamp techniques. We report that methionine substitution, but not leucine substitution, results in increased open state stability and hence significantly reduced ATP sensitivity and a marked increase of channel activity in the intact cell irrespective of the identity of the coassembled SUR subunit. Sulfonylurea inhibitors, such as glibenclamide, are potential therapies for CS. However, as a consequence of the increased open state stability, both Kir6.1(V65M) and Kir6.2(V64M) mutations essentially abolish high-affinity sensitivity to the KATP blocker glibenclamide in both intact cells and excised patches. This raises the possibility that, at least for some CS mutations, sulfonylurea therapy may not prove to be successful and highlights the need for detailed pharmacogenomic analyses of CS mutations.

Highlights

Cantu syndrome (CS) is a condition characterized by a range of anatomical defects, including cardiomegaly, hyperflexibility of the joints, hypertrichosis, and craniofacial dysmorphology
Maximum KATP-dependent efflux rates in MI were significantly higher for V65M than for WT when expressed with SUR2A (Fig. 3C)
The recent identification of multiple missense mutations in sulfonylurea receptor 2 (SUR2) (ABCC9) and Kir6.1 (KCNJ8), which all result in KATP gain of function (GOF), demonstrates that CS arises primarily from KATP channel GOF [2, 10]

Summary

Results

To study recombinant channels containing mutant subunits, COSm6 cells were transfected with WT or mutant Kir6.1 and either SUR1 or SUR2A. Maximum KATP-dependent efflux rates in MI were significantly higher for V65M than for WT when expressed with SUR2A (Fig. 3C) This suggests that V65M increases maximal conductance, this analysis does not distinguish between increased channel density or more complete activation of available channels. We examined the sensitivity of Kir6.2 ϩ SUR2A and Kir6.2(V64M) ϩ SUR2A channels to glibenclamide inhibition in inside-out patch clamp recordings (Fig. 8, C and D). In Rbϩ flux experiments, the effect of 10 ␮M glibenclamide on KATPdependent efflux was reduced from ϳ50% for WT Kir6.1containing channels to ϳ30% for V65M-containing channels (Fig. 9, C and D), and glibenclamide sensitivity was markedly reduced in Kir6.2/Kir6.1(V65M) channels in inside-out patch clamp recordings (Fig. 9, E and F)

Discussion

Experimental procedures

Excised patch clamp