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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 KATP activity in both homDel and homT, del channels.Representative 86Rb+ efflux profile comparing untransfected COSm6 cells (Un) and cells transfected with WT, homS225T, homDel and homT, del channels in metabolic inhibition (A) and in basal conditions (B). Data points indicate means ± SEM of n = 4. NT  =  not transfected.
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pone-0063758-g001: Decreased KATP activity in both homDel and homT, del channels.Representative 86Rb+ efflux profile comparing untransfected COSm6 cells (Un) and cells transfected with WT, homS225T, homDel and homT, del channels in metabolic inhibition (A) and in basal conditions (B). Data points indicate means ± SEM of n = 4. NT  =  not transfected.

Mentions: We first assessed KATP channel activity in intact COS cells by 86Rb+ efflux assays. Surprisingly, these reveal much lower channel activity induced by metabolic inhibition (MI) in cells expressing homomeric deletion channels (referred to as homDel) or S225T plus deletion channels (referred to as homS225T, del) than in cells expressing WT channels (Fig. 1A). In addition, homDel and homS225T, del channels also display lower 86Rb+ efflux when compared to WT channels (Fig. 1B). On the other hand, homomeric S225T channels (referred to as homS225T) are slightly more active than WT channels in MI and basal conditions (Fig. 1A and 1B). Taken together, the data suggest that the homDel and homS225T, del channels result in loss of channel function. To further assess channel activity, we measured mutant/deletion channel activity from excised inside-out patch clamp recordings. Representative recordings of channel activities from WT, homS225T, homDel and homS225T, del are shown in Figure 2A. Both WT and homS225T channels display robust currents while only 20% of the patches from homDel and homS225T, del channels displayed even single channel levels of current, and 80% of the patches had no detectable currents (Fig. 2A) (n = 16–19), consistent with the Rb efflux data (Fig. 1). We therefore proceeded to assessment of protein expression level by Western blots. For this, cells were cotransfected with WT or mutant Kir6.2 cDNA and cDNA encoding N-terminally FLAG epitope-tagged SUR1 (referred to as fSUR1), which permits detection of fully assembled channel complexes expressed at the plasma membrane. When co-expressed with either homDel and homS225T, del, fSUR1 showed a significantly reduced complex-glycosylated band in Western blots (Fig. 2B). Reduced complex glycosylation reflects a reduced fraction of the protein that has exited the endoplasmic reticulum and moved past the medial Golgi wherein modification of N-linked glycosylation occurs. These results indicate that channels with deletion of these 7 amino acids (homDel and homS225T, del) likely have surface expression defects. On the other hand, the homS225T and WT channels exhibit a similar mature fSUR1 band suggesting that surface expression is unaffected by the point mutation. Interestingly, when the deletion channel was co-expressed with WT cDNA in 1∶1 ratio, the relative density of the mature fSUR1 band was markedly increased compared to the homozygous del expression, indicating rescue by the presence of WT Kir6.2 (see Discussion).


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 KATP activity in both homDel and homT, del channels.Representative 86Rb+ efflux profile comparing untransfected COSm6 cells (Un) and cells transfected with WT, homS225T, homDel and homT, del channels in metabolic inhibition (A) and in basal conditions (B). Data points indicate means ± SEM of n = 4. NT  =  not transfected.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063758-g001: Decreased KATP activity in both homDel and homT, del channels.Representative 86Rb+ efflux profile comparing untransfected COSm6 cells (Un) and cells transfected with WT, homS225T, homDel and homT, del channels in metabolic inhibition (A) and in basal conditions (B). Data points indicate means ± SEM of n = 4. NT  =  not transfected.
Mentions: We first assessed KATP channel activity in intact COS cells by 86Rb+ efflux assays. Surprisingly, these reveal much lower channel activity induced by metabolic inhibition (MI) in cells expressing homomeric deletion channels (referred to as homDel) or S225T plus deletion channels (referred to as homS225T, del) than in cells expressing WT channels (Fig. 1A). In addition, homDel and homS225T, del channels also display lower 86Rb+ efflux when compared to WT channels (Fig. 1B). On the other hand, homomeric S225T channels (referred to as homS225T) are slightly more active than WT channels in MI and basal conditions (Fig. 1A and 1B). Taken together, the data suggest that the homDel and homS225T, del channels result in loss of channel function. To further assess channel activity, we measured mutant/deletion channel activity from excised inside-out patch clamp recordings. Representative recordings of channel activities from WT, homS225T, homDel and homS225T, del are shown in Figure 2A. Both WT and homS225T channels display robust currents while only 20% of the patches from homDel and homS225T, del channels displayed even single channel levels of current, and 80% of the patches had no detectable currents (Fig. 2A) (n = 16–19), consistent with the Rb efflux data (Fig. 1). We therefore proceeded to assessment of protein expression level by Western blots. For this, cells were cotransfected with WT or mutant Kir6.2 cDNA and cDNA encoding N-terminally FLAG epitope-tagged SUR1 (referred to as fSUR1), which permits detection of fully assembled channel complexes expressed at the plasma membrane. When co-expressed with either homDel and homS225T, del, fSUR1 showed a significantly reduced complex-glycosylated band in Western blots (Fig. 2B). Reduced complex glycosylation reflects a reduced fraction of the protein that has exited the endoplasmic reticulum and moved past the medial Golgi wherein modification of N-linked glycosylation occurs. These results indicate that channels with deletion of these 7 amino acids (homDel and homS225T, del) likely have surface expression defects. On the other hand, the homS225T and WT channels exhibit a similar mature fSUR1 band suggesting that surface expression is unaffected by the point mutation. Interestingly, when the deletion channel was co-expressed with WT cDNA in 1∶1 ratio, the relative density of the mature fSUR1 band was markedly increased compared to the homozygous del expression, indicating rescue by the presence of WT Kir6.2 (see Discussion).

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