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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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Related in: MedlinePlus

Increased basal KATP activity in both hetDel and hetT, del channels.86Rb+ efflux of mutant Kir6.2 subunits coexpressed with WT subunits in 1∶1 DNA ratio, under metabolic inhibition (A) and in basal states (B). Data points indicate means ± SEM of n = 5. * indicates P<0.05 compared with WT by One-Way ANOVA analysis. NT  =  not transfected (no statistic given).
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pone-0063758-g003: Increased basal KATP activity in both hetDel and hetT, del channels.86Rb+ efflux of mutant Kir6.2 subunits coexpressed with WT subunits in 1∶1 DNA ratio, under metabolic inhibition (A) and in basal states (B). Data points indicate means ± SEM of n = 5. * indicates P<0.05 compared with WT by One-Way ANOVA analysis. NT  =  not transfected (no statistic given).

Mentions: The loss of surface expression of homDel or homS225T, del channels prohibited further detailed analysis of homomeric channels. We coexpressed the mutant/deletion subunits with WT Kir6.2 in equal ratio (plus SUR1) to simulate the heterozygous state that wil be present in vivo, and measured 86Rb+ efflux across the membrane: heteromeric S225T channels (S225T plus WT in 1∶1 DNA ratio and referred as hetS225T); heteromeric del226–232 channels (referred to as hetDel); heteromeric S225T plus del226–232 channels (referred as hetS225T, Del). In Figure 3A, all four channel types exhibit similar maximal Rb efflux in metabolic inhibition. HetS225T channels exhibit a slight, insignificant, increase in basal flux, but hetDel channels and hetS225T, Del channels both show significantly increased Rb efflux in the basal state, reflecting channel overactivity (Figure 3B).


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)

Increased basal KATP activity in both hetDel and hetT, del channels.86Rb+ efflux of mutant Kir6.2 subunits coexpressed with WT subunits in 1∶1 DNA ratio, under metabolic inhibition (A) and in basal states (B). Data points indicate means ± SEM of n = 5. * indicates P<0.05 compared with WT by One-Way ANOVA analysis. NT  =  not transfected (no statistic given).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063758-g003: Increased basal KATP activity in both hetDel and hetT, del channels.86Rb+ efflux of mutant Kir6.2 subunits coexpressed with WT subunits in 1∶1 DNA ratio, under metabolic inhibition (A) and in basal states (B). Data points indicate means ± SEM of n = 5. * indicates P<0.05 compared with WT by One-Way ANOVA analysis. NT  =  not transfected (no statistic given).
Mentions: The loss of surface expression of homDel or homS225T, del channels prohibited further detailed analysis of homomeric channels. We coexpressed the mutant/deletion subunits with WT Kir6.2 in equal ratio (plus SUR1) to simulate the heterozygous state that wil be present in vivo, and measured 86Rb+ efflux across the membrane: heteromeric S225T channels (S225T plus WT in 1∶1 DNA ratio and referred as hetS225T); heteromeric del226–232 channels (referred to as hetDel); heteromeric S225T plus del226–232 channels (referred as hetS225T, Del). In Figure 3A, all four channel types exhibit similar maximal Rb efflux in metabolic inhibition. HetS225T channels exhibit a slight, insignificant, increase in basal flux, but hetDel channels and hetS225T, Del channels both show significantly increased Rb efflux in the basal state, reflecting channel overactivity (Figure 3B).

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