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Differential mechanisms of Cantú syndrome-associated gain of function mutations in the ABCC9 (SUR2) subunit of the KATP channel.

Cooper PE, Sala-Rabanal M, Lee SJ, Nichols CG - J. Gen. Physiol. (2015)

Bottom Line: For P429L and A475V mutants, sensitivity to ATP inhibition was comparable to WT channels, but activation by MgADP was significantly greater.C1039Y-dependent channels were significantly less sensitive to inhibition by ATP or by glibenclamide, but MgADP activation was comparable to WT.The results indicate that these three CS mutations all lead to overactive K(ATP) channels, but at least two mechanisms underlie the observed gain of function: decreased ATP inhibition and enhanced MgADP activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology and Physiology, and Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, Saint Louis, MO 63110 Department of Cell Biology and Physiology, and Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, Saint Louis, MO 63110.

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Decreased sensitivity to glibenclamide inhibition in C1039Y channels. 86Rb+ efflux as a function of time was measured in GFP-transfected control cells and in cells transiently expressing KATP channels composed of Kir6.2 plus WT or mutant SUR2 subunits (A), as well as in cells expressing a 1:1 mixture of WT and mutant SUR2 subunits. (B) Experiments were performed in MI plus glibenclamide. The data represent the means ± SEM of three to six experiments. Data were fit with Eq. 2 to obtain rate constants for KATP-dependent efflux, k2. (C and D) k2 in glibenclamide plus MI divided by k2 in MI for each condition.
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fig10: Decreased sensitivity to glibenclamide inhibition in C1039Y channels. 86Rb+ efflux as a function of time was measured in GFP-transfected control cells and in cells transiently expressing KATP channels composed of Kir6.2 plus WT or mutant SUR2 subunits (A), as well as in cells expressing a 1:1 mixture of WT and mutant SUR2 subunits. (B) Experiments were performed in MI plus glibenclamide. The data represent the means ± SEM of three to six experiments. Data were fit with Eq. 2 to obtain rate constants for KATP-dependent efflux, k2. (C and D) k2 in glibenclamide plus MI divided by k2 in MI for each condition.

Mentions: Some NDM patients with GOF mutations in Kir6.2 or SUR1 have successfully been switched from insulin therapy to KATP inhibitors such as glibenclamide (Zung et al., 2004). However, a correlation between increased channel activity and diminished effectiveness of such drugs has been noted (Koster et al., 2005). Therefore, to test the effectiveness of glibenclamide on overactive P429L, A475V, or C1039Y channels, expressed both homomerically and heteromerically; 86Rb+ efflux experiments were performed in the presence of MI plus 10 µM glibenclamide (Fig. 10). The time course of Rb+ effluxes shows an unusual behavior in that although initial fluxes are markedly lower than in MI alone, the inhibition is not maintained through the time course of the assay. It is well understood that the inhibitory action of glibenclamide is a complex function of the metabolic conditions and decreases under conditions of MI (Findlay, 1994; Koster et al., 1999). To account for this behavior, the efflux data in glibenclamide were fit by Eq. 2, where positive values for k−2 now result in the rate of efflux actually increasing with time for A475V and P429L channels (see Table 3). From this analysis, the ratio of the k2 in MI plus glibenclamide to k2 in MI provides an estimate of the relative sensitivity to inhibition by glibenclamide (Fig. 10 C). Although the glibenclamide sensitivity of A475V and P429L channels was not different from WT, C1039Y channels appeared almost insensitive to the drug. Qualitatively similar results were obtained with channels expressed in heteromeric mixture with WT subunits (Fig. 10, B–D). As discussed below, this glibenclamide insensitivity is consistent with similar findings for SUR1 mutations that produce increased activity of resultant KATP channels.


Differential mechanisms of Cantú syndrome-associated gain of function mutations in the ABCC9 (SUR2) subunit of the KATP channel.

Cooper PE, Sala-Rabanal M, Lee SJ, Nichols CG - J. Gen. Physiol. (2015)

Decreased sensitivity to glibenclamide inhibition in C1039Y channels. 86Rb+ efflux as a function of time was measured in GFP-transfected control cells and in cells transiently expressing KATP channels composed of Kir6.2 plus WT or mutant SUR2 subunits (A), as well as in cells expressing a 1:1 mixture of WT and mutant SUR2 subunits. (B) Experiments were performed in MI plus glibenclamide. The data represent the means ± SEM of three to six experiments. Data were fit with Eq. 2 to obtain rate constants for KATP-dependent efflux, k2. (C and D) k2 in glibenclamide plus MI divided by k2 in MI for each condition.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4664827&req=5

fig10: Decreased sensitivity to glibenclamide inhibition in C1039Y channels. 86Rb+ efflux as a function of time was measured in GFP-transfected control cells and in cells transiently expressing KATP channels composed of Kir6.2 plus WT or mutant SUR2 subunits (A), as well as in cells expressing a 1:1 mixture of WT and mutant SUR2 subunits. (B) Experiments were performed in MI plus glibenclamide. The data represent the means ± SEM of three to six experiments. Data were fit with Eq. 2 to obtain rate constants for KATP-dependent efflux, k2. (C and D) k2 in glibenclamide plus MI divided by k2 in MI for each condition.
Mentions: Some NDM patients with GOF mutations in Kir6.2 or SUR1 have successfully been switched from insulin therapy to KATP inhibitors such as glibenclamide (Zung et al., 2004). However, a correlation between increased channel activity and diminished effectiveness of such drugs has been noted (Koster et al., 2005). Therefore, to test the effectiveness of glibenclamide on overactive P429L, A475V, or C1039Y channels, expressed both homomerically and heteromerically; 86Rb+ efflux experiments were performed in the presence of MI plus 10 µM glibenclamide (Fig. 10). The time course of Rb+ effluxes shows an unusual behavior in that although initial fluxes are markedly lower than in MI alone, the inhibition is not maintained through the time course of the assay. It is well understood that the inhibitory action of glibenclamide is a complex function of the metabolic conditions and decreases under conditions of MI (Findlay, 1994; Koster et al., 1999). To account for this behavior, the efflux data in glibenclamide were fit by Eq. 2, where positive values for k−2 now result in the rate of efflux actually increasing with time for A475V and P429L channels (see Table 3). From this analysis, the ratio of the k2 in MI plus glibenclamide to k2 in MI provides an estimate of the relative sensitivity to inhibition by glibenclamide (Fig. 10 C). Although the glibenclamide sensitivity of A475V and P429L channels was not different from WT, C1039Y channels appeared almost insensitive to the drug. Qualitatively similar results were obtained with channels expressed in heteromeric mixture with WT subunits (Fig. 10, B–D). As discussed below, this glibenclamide insensitivity is consistent with similar findings for SUR1 mutations that produce increased activity of resultant KATP channels.

Bottom Line: For P429L and A475V mutants, sensitivity to ATP inhibition was comparable to WT channels, but activation by MgADP was significantly greater.C1039Y-dependent channels were significantly less sensitive to inhibition by ATP or by glibenclamide, but MgADP activation was comparable to WT.The results indicate that these three CS mutations all lead to overactive K(ATP) channels, but at least two mechanisms underlie the observed gain of function: decreased ATP inhibition and enhanced MgADP activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology and Physiology, and Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, Saint Louis, MO 63110 Department of Cell Biology and Physiology, and Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, Saint Louis, MO 63110.

No MeSH data available.


Related in: MedlinePlus