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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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Channel activity in cells expressing heteromeric P429L-, A475V-, or C1039Y-based KATP channels. 86Rb+ efflux as a function of time was measured in GFP-transfected control cells (dashed) and in cells transiently expressing reconstituted Kir6.2-based KATP channels with WT or 1:1 mixtures of WT and P429L, A475V, or C1039Y mutant SUR2 subunits in basal conditions (A), in the presence of MIs oligomycin and 2-deoxy-d-glucose (B), in the K+ channel opener PIN (C), or in the presence of both MI and PIN (D). The data represent means ± SEM of 6–10 experiments. Flux data were fit with Eq. 1 (GFP) to obtain the rate constant k1 or Eq. 2 to obtain the rate constants for KATP-dependent efflux, k2 (Fig. 3), where lines show mean fitted relationships.
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fig8: Channel activity in cells expressing heteromeric P429L-, A475V-, or C1039Y-based KATP channels. 86Rb+ efflux as a function of time was measured in GFP-transfected control cells (dashed) and in cells transiently expressing reconstituted Kir6.2-based KATP channels with WT or 1:1 mixtures of WT and P429L, A475V, or C1039Y mutant SUR2 subunits in basal conditions (A), in the presence of MIs oligomycin and 2-deoxy-d-glucose (B), in the K+ channel opener PIN (C), or in the presence of both MI and PIN (D). The data represent means ± SEM of 6–10 experiments. Flux data were fit with Eq. 1 (GFP) to obtain the rate constant k1 or Eq. 2 to obtain the rate constants for KATP-dependent efflux, k2 (Fig. 3), where lines show mean fitted relationships.

Mentions: All documented CS patients with mutations in ABCC9 are heterozygous. To mimic the predicted heteromeric composition of channels in such patients, we also expressed each mutation in a 1:1 ratio with SUR2A-WT plus Kir6.2, and assessed channel activity by 86Rb+ efflux assays (Figs. 8 and 9). In all conditions, P429L, A475V, and C1039Y heteromeric channels display no significant increases in the rate of 86Rb+ efflux compared with WT channels (Figs. 8 A and 9 A). When normalized to the maximal efflux rates, channels with heteromeric expression of C1039Y are still considerably less active than WT. In the heteromeric state, maximal C1039Y channel fluxes were markedly higher than in the homomeric state (Fig. 9 D), also implying a partial rescue of the surface expression of C1039Y subunits by WT subunits (Figs. 8, B–D, and 9, B–D).


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)

Channel activity in cells expressing heteromeric P429L-, A475V-, or C1039Y-based KATP channels. 86Rb+ efflux as a function of time was measured in GFP-transfected control cells (dashed) and in cells transiently expressing reconstituted Kir6.2-based KATP channels with WT or 1:1 mixtures of WT and P429L, A475V, or C1039Y mutant SUR2 subunits in basal conditions (A), in the presence of MIs oligomycin and 2-deoxy-d-glucose (B), in the K+ channel opener PIN (C), or in the presence of both MI and PIN (D). The data represent means ± SEM of 6–10 experiments. Flux data were fit with Eq. 1 (GFP) to obtain the rate constant k1 or Eq. 2 to obtain the rate constants for KATP-dependent efflux, k2 (Fig. 3), where lines show mean fitted relationships.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig8: Channel activity in cells expressing heteromeric P429L-, A475V-, or C1039Y-based KATP channels. 86Rb+ efflux as a function of time was measured in GFP-transfected control cells (dashed) and in cells transiently expressing reconstituted Kir6.2-based KATP channels with WT or 1:1 mixtures of WT and P429L, A475V, or C1039Y mutant SUR2 subunits in basal conditions (A), in the presence of MIs oligomycin and 2-deoxy-d-glucose (B), in the K+ channel opener PIN (C), or in the presence of both MI and PIN (D). The data represent means ± SEM of 6–10 experiments. Flux data were fit with Eq. 1 (GFP) to obtain the rate constant k1 or Eq. 2 to obtain the rate constants for KATP-dependent efflux, k2 (Fig. 3), where lines show mean fitted relationships.
Mentions: All documented CS patients with mutations in ABCC9 are heterozygous. To mimic the predicted heteromeric composition of channels in such patients, we also expressed each mutation in a 1:1 ratio with SUR2A-WT plus Kir6.2, and assessed channel activity by 86Rb+ efflux assays (Figs. 8 and 9). In all conditions, P429L, A475V, and C1039Y heteromeric channels display no significant increases in the rate of 86Rb+ efflux compared with WT channels (Figs. 8 A and 9 A). When normalized to the maximal efflux rates, channels with heteromeric expression of C1039Y are still considerably less active than WT. In the heteromeric state, maximal C1039Y channel fluxes were markedly higher than in the homomeric state (Fig. 9 D), also implying a partial rescue of the surface expression of C1039Y subunits by WT subunits (Figs. 8, B–D, and 9, B–D).

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