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Functional characterization of a novel KCNJ11 in frame mutation-deletion associated with infancy-onset diabetes and a mild form of intermediate DEND: a battle between K(ATP) gain of channel activity and loss of channel expression.

Lin YW, Li A, Grasso V, Battaglia D, Crinò A, Colombo C, Barbetti F, Nichols CG - PLoS ONE (2013)

Bottom Line: The protein expression and gating effects of the resulting channels were assessed biochemically and electrophysiologically.Interestingly, homomeric channels for the combined deletion/mutation, or for the deletion alone, showed dramatically reduced channel expression at the cell membrane, which would underlie a reduced function in vivo.These results demonstrate that both the mis-sense mutation and the deleted region in the Kir6.2 subunit are important for control of the intrinsic channel gating and suggest that the clinical presentation could be affected by the competition between loss-of-function by reduced trafficking and enhanced channel gating.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, and Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT
ATP-sensitive potassium (K(ATP)) channels are widely distributed in various tissues and cell types where they couple cell metabolism to cell excitability. Gain of channel function (GOF) mutations in the genes encoding Kir6.2 (KCNJ11) or the associated regulatory ssulfonylurea receptor 1 subunit (ABCC8), cause developmental delay, epilepsy and neonatal diabetes (DEND) due to suppressed cell excitability in pancreatic β-cells and neurons. The objective of this study was to determine the molecular basis of infancy-onset diabetes and a mild form of intermediate DEND, resulting from a novel KCNJ11 in frame mutation plus deletion. The naturally occurring Kir6.2 mutation plus deletion (Ser225Thr, Pro226_Pro232del) as well as the isolated S225T mutation or isolated del226-232 deletion were coexpressed with SUR1 in COS cells in homozygous or heterozygous states. The protein expression and gating effects of the resulting channels were assessed biochemically and electrophysiologically. For both the deletion and point mutations, simulated heterozygous expression resulted in overall increased conductance in intact cells in basal conditions and rightward shifted ATP dose-response curves in excised patches, due to increased intrinsic open probability. Interestingly, homomeric channels for the combined deletion/mutation, or for the deletion alone, showed dramatically reduced channel expression at the cell membrane, which would underlie a reduced function in vivo. These results demonstrate that both the mis-sense mutation and the deleted region in the Kir6.2 subunit are important for control of the intrinsic channel gating and suggest that the clinical presentation could be affected by the competition between loss-of-function by reduced trafficking and enhanced channel gating.

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Decreased ATP sensitivity in both hetDel and hetT, del channels.(A) Representative currents recorded by inside-out excised patch-clamp technique from COSm6 cells expressing WT channels and hetT, del mutants. Patches were exposed to different concentrations of Mg-free ATP as indicated. (B) ATP dose-response relationships, fit by Hill equation as described in methods. Data points indicate means ± SEM of n = 5–7 patches. * indicates P<0.05 compared with WT by One-Way ANOVA analysis. The fitted K1/2 for WT, homS225T, hetS225T, hetDel, and hetT, del channels are 13.75, 25.06, 14.8, 22.87 and 43.94 (in μM) and the Hill coefficients are 1.6, 1.3, 1.1, 1.28 and 1.2, respectively.
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pone-0063758-g004: Decreased ATP sensitivity in both hetDel and hetT, del channels.(A) Representative currents recorded by inside-out excised patch-clamp technique from COSm6 cells expressing WT channels and hetT, del mutants. Patches were exposed to different concentrations of Mg-free ATP as indicated. (B) ATP dose-response relationships, fit by Hill equation as described in methods. Data points indicate means ± SEM of n = 5–7 patches. * indicates P<0.05 compared with WT by One-Way ANOVA analysis. The fitted K1/2 for WT, homS225T, hetS225T, hetDel, and hetT, del channels are 13.75, 25.06, 14.8, 22.87 and 43.94 (in μM) and the Hill coefficients are 1.6, 1.3, 1.1, 1.28 and 1.2, respectively.

Mentions: To characterize the mechanisms by which mutant/deletion subunits cause overall gain of channel function in the intact cell, we tested the ATP sensitivity of WT and mutant channels in either homozygous or heterozygous states. Representative recordings of WT and hetT, del channels in response to ATP (in the absence of Mg2+) are shown in Figure 4A. A summary of the [ATP]-response curves for WT and mutant channels is shown in Figure 4B. Both hetDel and homS225T channels exhibit slightly right-shifted dose-responses, with a further significantly right-shifted ATP sensitivity of hetS225T, del channels (Fig. 4B) suggesting that both the deletion and the mutation contribute to reduced ATP sensitivity.


Functional characterization of a novel KCNJ11 in frame mutation-deletion associated with infancy-onset diabetes and a mild form of intermediate DEND: a battle between K(ATP) gain of channel activity and loss of channel expression.

Lin YW, Li A, Grasso V, Battaglia D, Crinò A, Colombo C, Barbetti F, Nichols CG - PLoS ONE (2013)

Decreased ATP sensitivity in both hetDel and hetT, del channels.(A) Representative currents recorded by inside-out excised patch-clamp technique from COSm6 cells expressing WT channels and hetT, del mutants. Patches were exposed to different concentrations of Mg-free ATP as indicated. (B) ATP dose-response relationships, fit by Hill equation as described in methods. Data points indicate means ± SEM of n = 5–7 patches. * indicates P<0.05 compared with WT by One-Way ANOVA analysis. The fitted K1/2 for WT, homS225T, hetS225T, hetDel, and hetT, del channels are 13.75, 25.06, 14.8, 22.87 and 43.94 (in μM) and the Hill coefficients are 1.6, 1.3, 1.1, 1.28 and 1.2, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646792&req=5

pone-0063758-g004: Decreased ATP sensitivity in both hetDel and hetT, del channels.(A) Representative currents recorded by inside-out excised patch-clamp technique from COSm6 cells expressing WT channels and hetT, del mutants. Patches were exposed to different concentrations of Mg-free ATP as indicated. (B) ATP dose-response relationships, fit by Hill equation as described in methods. Data points indicate means ± SEM of n = 5–7 patches. * indicates P<0.05 compared with WT by One-Way ANOVA analysis. The fitted K1/2 for WT, homS225T, hetS225T, hetDel, and hetT, del channels are 13.75, 25.06, 14.8, 22.87 and 43.94 (in μM) and the Hill coefficients are 1.6, 1.3, 1.1, 1.28 and 1.2, respectively.
Mentions: To characterize the mechanisms by which mutant/deletion subunits cause overall gain of channel function in the intact cell, we tested the ATP sensitivity of WT and mutant channels in either homozygous or heterozygous states. Representative recordings of WT and hetT, del channels in response to ATP (in the absence of Mg2+) are shown in Figure 4A. A summary of the [ATP]-response curves for WT and mutant channels is shown in Figure 4B. Both hetDel and homS225T channels exhibit slightly right-shifted dose-responses, with a further significantly right-shifted ATP sensitivity of hetS225T, del channels (Fig. 4B) suggesting that both the deletion and the mutation contribute to reduced ATP sensitivity.

Bottom Line: The protein expression and gating effects of the resulting channels were assessed biochemically and electrophysiologically.Interestingly, homomeric channels for the combined deletion/mutation, or for the deletion alone, showed dramatically reduced channel expression at the cell membrane, which would underlie a reduced function in vivo.These results demonstrate that both the mis-sense mutation and the deleted region in the Kir6.2 subunit are important for control of the intrinsic channel gating and suggest that the clinical presentation could be affected by the competition between loss-of-function by reduced trafficking and enhanced channel gating.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, and Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT
ATP-sensitive potassium (K(ATP)) channels are widely distributed in various tissues and cell types where they couple cell metabolism to cell excitability. Gain of channel function (GOF) mutations in the genes encoding Kir6.2 (KCNJ11) or the associated regulatory ssulfonylurea receptor 1 subunit (ABCC8), cause developmental delay, epilepsy and neonatal diabetes (DEND) due to suppressed cell excitability in pancreatic β-cells and neurons. The objective of this study was to determine the molecular basis of infancy-onset diabetes and a mild form of intermediate DEND, resulting from a novel KCNJ11 in frame mutation plus deletion. The naturally occurring Kir6.2 mutation plus deletion (Ser225Thr, Pro226_Pro232del) as well as the isolated S225T mutation or isolated del226-232 deletion were coexpressed with SUR1 in COS cells in homozygous or heterozygous states. The protein expression and gating effects of the resulting channels were assessed biochemically and electrophysiologically. For both the deletion and point mutations, simulated heterozygous expression resulted in overall increased conductance in intact cells in basal conditions and rightward shifted ATP dose-response curves in excised patches, due to increased intrinsic open probability. Interestingly, homomeric channels for the combined deletion/mutation, or for the deletion alone, showed dramatically reduced channel expression at the cell membrane, which would underlie a reduced function in vivo. These results demonstrate that both the mis-sense mutation and the deleted region in the Kir6.2 subunit are important for control of the intrinsic channel gating and suggest that the clinical presentation could be affected by the competition between loss-of-function by reduced trafficking and enhanced channel gating.

Show MeSH
Related in: MedlinePlus