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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.

No MeSH data available.


Related in: MedlinePlus

Relative KATP conductance is markedly increased in basal and stimulated conditions in intact cells expressing homomeric C1039Y KATP channels. (A–C) The ratio of the rate constants for KATP-dependent 86Rb+ efflux (k2) in basal and PIN- or MI-stimulated conditions to the maximal activation (in MI+PIN) is plotted for WT and mutant channels. *, P < 0.05 as compared with WT (unpaired Student’s t test). Error bars represent mean ± SEM.
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fig4: Relative KATP conductance is markedly increased in basal and stimulated conditions in intact cells expressing homomeric C1039Y KATP channels. (A–C) The ratio of the rate constants for KATP-dependent 86Rb+ efflux (k2) in basal and PIN- or MI-stimulated conditions to the maximal activation (in MI+PIN) is plotted for WT and mutant channels. *, P < 0.05 as compared with WT (unpaired Student’s t test). Error bars represent mean ± SEM.

Mentions: After 24–48 h, transfected cells that fluoresced green under UV light were selected for analysis by excised patch-clamp experiments at room temperature in a perfusion chamber that allowed for the rapid switching of solutions. Kint solution (mM: 140 KCl, 10 HEPES, and 1 EGTA, pH 7.4) was used as the standard pipette (extracellular) and bath (cytoplasmic) solution in these experiments. ATP or ADP was added as indicated. The appropriate amounts of MgCl2 to be added in each Mg2+-nucleotide–containing solution to attain 0.5 mM of free Mg2+ were calculated by means of the CaBuf program (Katholieke Universiteit Leuven). Membrane patches were voltage clamped using an Axopatch 1D amplifier (Molecular Devices), and currents were recorded at −50 mV (pipette voltage, 50 mV) in on-cell and inside-out excised patch configuration. Data were typically filtered at 1 kHz and digitized at 5 kHz with a Digidata 1322A (Molecular Devices) A-D converter. pClamp and Axoscope software (Molecular Devices) were used for data acquisition. For ATP inhibition, [ATP]-response relationships (Fig. 4) were fitted by Eq. 3:(3)Irel={1+([ATP]Ki)H}−1,where Irel (relative current) is the current in the presence of a given concentration of ATP relative to current in zero ATP, Ki is the apparent ATP inhibition constant, and H is the Hill coefficient.


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)

Relative KATP conductance is markedly increased in basal and stimulated conditions in intact cells expressing homomeric C1039Y KATP channels. (A–C) The ratio of the rate constants for KATP-dependent 86Rb+ efflux (k2) in basal and PIN- or MI-stimulated conditions to the maximal activation (in MI+PIN) is plotted for WT and mutant channels. *, P < 0.05 as compared with WT (unpaired Student’s 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

fig4: Relative KATP conductance is markedly increased in basal and stimulated conditions in intact cells expressing homomeric C1039Y KATP channels. (A–C) The ratio of the rate constants for KATP-dependent 86Rb+ efflux (k2) in basal and PIN- or MI-stimulated conditions to the maximal activation (in MI+PIN) is plotted for WT and mutant channels. *, P < 0.05 as compared with WT (unpaired Student’s t test). Error bars represent mean ± SEM.
Mentions: After 24–48 h, transfected cells that fluoresced green under UV light were selected for analysis by excised patch-clamp experiments at room temperature in a perfusion chamber that allowed for the rapid switching of solutions. Kint solution (mM: 140 KCl, 10 HEPES, and 1 EGTA, pH 7.4) was used as the standard pipette (extracellular) and bath (cytoplasmic) solution in these experiments. ATP or ADP was added as indicated. The appropriate amounts of MgCl2 to be added in each Mg2+-nucleotide–containing solution to attain 0.5 mM of free Mg2+ were calculated by means of the CaBuf program (Katholieke Universiteit Leuven). Membrane patches were voltage clamped using an Axopatch 1D amplifier (Molecular Devices), and currents were recorded at −50 mV (pipette voltage, 50 mV) in on-cell and inside-out excised patch configuration. Data were typically filtered at 1 kHz and digitized at 5 kHz with a Digidata 1322A (Molecular Devices) A-D converter. pClamp and Axoscope software (Molecular Devices) were used for data acquisition. For ATP inhibition, [ATP]-response relationships (Fig. 4) were fitted by Eq. 3:(3)Irel={1+([ATP]Ki)H}−1,where Irel (relative current) is the current in the presence of a given concentration of ATP relative to current in zero ATP, Ki is the apparent ATP inhibition constant, and H is the Hill coefficient.

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