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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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Increased channel activity in intact cells expressing homomeric P429L-, A475V-, or C1039Y-containing 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, P429L, A475V, or C1039Y SUR2 subunits in homomeric configuration, 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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fig2: Increased channel activity in intact cells expressing homomeric P429L-, A475V-, or C1039Y-containing 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, P429L, A475V, or C1039Y SUR2 subunits in homomeric configuration, 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: CS-associated mutations have been found throughout the coding sequence (Fig. 1). For the present study, we focused on two previously unexamined mutations (human A478V and C1043Y corresponding to rat A475V and C1039Y, respectively), located in the TMD1 and TMD2 segments, and P432L (corresponding to rat P429L), also located in the TMD1 region. To examine KATP channel activity in intact cells, we performed 86Rb+ efflux assays under four different conditions: basal, MI, in the presence of PIN, and MI and PIN combined (MI+PIN). As shown in Figs. 2 and 3, homomeric expression of SUR2A-P429L, A475V, or C1039Y channels, results in a significantly higher basal 86Rb+ efflux rate compared with WT (Figs. 2 A and 3 A). P429L- and A475V-based channels, but not those composed of C1039Y, also showed a significantly higher rate of efflux compared with WT channels under MI conditions and in the presence of PIN (Figs. 2, B and C, and 3, B and C), consistent with the GOF observed in the basal condition. Maximal efflux rates (estimated using simultaneous exposure to MI and PIN) of P429L and A475V were comparable to SUR2A-WT, implying similar channel densities at the cell surface (Figs. 2 D and 3 D). C1039Y showed significantly lower absolute fluxes in all stimulatory conditions (Figs. 2, B–D, and 3, B–D), implying a lower channel density at the membrane (Fig. 4).


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)

Increased channel activity in intact cells expressing homomeric P429L-, A475V-, or C1039Y-containing 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, P429L, A475V, or C1039Y SUR2 subunits in homomeric configuration, 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

fig2: Increased channel activity in intact cells expressing homomeric P429L-, A475V-, or C1039Y-containing 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, P429L, A475V, or C1039Y SUR2 subunits in homomeric configuration, 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: CS-associated mutations have been found throughout the coding sequence (Fig. 1). For the present study, we focused on two previously unexamined mutations (human A478V and C1043Y corresponding to rat A475V and C1039Y, respectively), located in the TMD1 and TMD2 segments, and P432L (corresponding to rat P429L), also located in the TMD1 region. To examine KATP channel activity in intact cells, we performed 86Rb+ efflux assays under four different conditions: basal, MI, in the presence of PIN, and MI and PIN combined (MI+PIN). As shown in Figs. 2 and 3, homomeric expression of SUR2A-P429L, A475V, or C1039Y channels, results in a significantly higher basal 86Rb+ efflux rate compared with WT (Figs. 2 A and 3 A). P429L- and A475V-based channels, but not those composed of C1039Y, also showed a significantly higher rate of efflux compared with WT channels under MI conditions and in the presence of PIN (Figs. 2, B and C, and 3, B and C), consistent with the GOF observed in the basal condition. Maximal efflux rates (estimated using simultaneous exposure to MI and PIN) of P429L and A475V were comparable to SUR2A-WT, implying similar channel densities at the cell surface (Figs. 2 D and 3 D). C1039Y showed significantly lower absolute fluxes in all stimulatory conditions (Figs. 2, B–D, and 3, B–D), implying a lower channel density at the membrane (Fig. 4).

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