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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.

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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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ATP sensitivity is decreased in C1039Y channels. Representative excised patch-current recordings from COSm6 cells coexpressing Kir6.2 and WT or CS mutant SUR2 subunits P429L, A475V, or C1039Y (A). Membrane potential was held at −50 mV, and negative currents (plotted as upward deflections) were recorded continuously on-cell and in inside-out excised patches exposed to KINT in the absence or presence of 0.01, 0.1, or 1 mM ATP; arrowheads mark the point of excision. (B) Dose–response data (mean ± SEM from 8–11 patches) was fit with Eq. 3 to estimate the ATP concentration for half-maximal inhibition Ki: WT (9 µM), P429L (9 µM), A475V (7 µM), and C1039Y (21 µM). (C) Maximum patch-current immediately after patch excision (mean ± SEM from 8–11 patches). *, P < 0.05 as compared with WT (unpaired t test). Error bars represent mean ± SEM.
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fig5: ATP sensitivity is decreased in C1039Y channels. Representative excised patch-current recordings from COSm6 cells coexpressing Kir6.2 and WT or CS mutant SUR2 subunits P429L, A475V, or C1039Y (A). Membrane potential was held at −50 mV, and negative currents (plotted as upward deflections) were recorded continuously on-cell and in inside-out excised patches exposed to KINT in the absence or presence of 0.01, 0.1, or 1 mM ATP; arrowheads mark the point of excision. (B) Dose–response data (mean ± SEM from 8–11 patches) was fit with Eq. 3 to estimate the ATP concentration for half-maximal inhibition Ki: WT (9 µM), P429L (9 µM), A475V (7 µM), and C1039Y (21 µM). (C) Maximum patch-current immediately after patch excision (mean ± SEM from 8–11 patches). *, P < 0.05 as compared with WT (unpaired t test). Error bars represent mean ± SEM.

Mentions: 86Rb+ flux assays provide evidence for overactivity in CS mutants, but do not provide any indication of underlying molecular mechanisms. We assessed the details of channel properties in inside-out excised patch-clamp electrophysiology experiments. Intrinsic sensitivity to ATP inhibition (in zero Mg2+) was similar for WT, P429L, and A475V channels (Ki = 7–9 µM). However, KATP channels expressing C1039Y exhibited a significant right shift in ATP sensitivity (Ki = 21.3 µM; Fig. 5 B). A diminished sensitivity to intracellular ATP may account, at least in part, for the increased activity of C1039Y-based KATP channels in basal conditions in the intact cell (Figs. 2 A and 3 A). Consistent with reduced channel density, the maximal current in zero ATP was significantly lower in C1039Y channels than in WT, P429L, or A475V channels (Fig. 5 C). The Po in the absence of inhibitory ATP (zero) was estimated by the application of PIP2 (Koster et al., 2005). After the application of PIP2, both WT and C1039Y channels lost ATP sensitivity; however, the current in zero ATP increased for WT channels but not for C1039Y channels (Fig. 6). The ratio of the initial maximum current in zero ATP to that in PIP2 (WT: 0.70 ± 0.11 vs. C1039Y: 1.26 ± 0.21) reflects marked increase in WT maximum Po but no increase for C1039Y.


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)

ATP sensitivity is decreased in C1039Y channels. Representative excised patch-current recordings from COSm6 cells coexpressing Kir6.2 and WT or CS mutant SUR2 subunits P429L, A475V, or C1039Y (A). Membrane potential was held at −50 mV, and negative currents (plotted as upward deflections) were recorded continuously on-cell and in inside-out excised patches exposed to KINT in the absence or presence of 0.01, 0.1, or 1 mM ATP; arrowheads mark the point of excision. (B) Dose–response data (mean ± SEM from 8–11 patches) was fit with Eq. 3 to estimate the ATP concentration for half-maximal inhibition Ki: WT (9 µM), P429L (9 µM), A475V (7 µM), and C1039Y (21 µM). (C) Maximum patch-current immediately after patch excision (mean ± SEM from 8–11 patches). *, P < 0.05 as compared with WT (unpaired t test). Error bars represent mean ± SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: ATP sensitivity is decreased in C1039Y channels. Representative excised patch-current recordings from COSm6 cells coexpressing Kir6.2 and WT or CS mutant SUR2 subunits P429L, A475V, or C1039Y (A). Membrane potential was held at −50 mV, and negative currents (plotted as upward deflections) were recorded continuously on-cell and in inside-out excised patches exposed to KINT in the absence or presence of 0.01, 0.1, or 1 mM ATP; arrowheads mark the point of excision. (B) Dose–response data (mean ± SEM from 8–11 patches) was fit with Eq. 3 to estimate the ATP concentration for half-maximal inhibition Ki: WT (9 µM), P429L (9 µM), A475V (7 µM), and C1039Y (21 µM). (C) Maximum patch-current immediately after patch excision (mean ± SEM from 8–11 patches). *, P < 0.05 as compared with WT (unpaired t test). Error bars represent mean ± SEM.
Mentions: 86Rb+ flux assays provide evidence for overactivity in CS mutants, but do not provide any indication of underlying molecular mechanisms. We assessed the details of channel properties in inside-out excised patch-clamp electrophysiology experiments. Intrinsic sensitivity to ATP inhibition (in zero Mg2+) was similar for WT, P429L, and A475V channels (Ki = 7–9 µM). However, KATP channels expressing C1039Y exhibited a significant right shift in ATP sensitivity (Ki = 21.3 µM; Fig. 5 B). A diminished sensitivity to intracellular ATP may account, at least in part, for the increased activity of C1039Y-based KATP channels in basal conditions in the intact cell (Figs. 2 A and 3 A). Consistent with reduced channel density, the maximal current in zero ATP was significantly lower in C1039Y channels than in WT, P429L, or A475V channels (Fig. 5 C). The Po in the absence of inhibitory ATP (zero) was estimated by the application of PIP2 (Koster et al., 2005). After the application of PIP2, both WT and C1039Y channels lost ATP sensitivity; however, the current in zero ATP increased for WT channels but not for C1039Y channels (Fig. 6). The ratio of the initial maximum current in zero ATP to that in PIP2 (WT: 0.70 ± 0.11 vs. C1039Y: 1.26 ± 0.21) reflects marked increase in WT maximum Po but no increase for C1039Y.

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