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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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Heterozygous S225T, deletion channels display higher channel open probability, assessed by the ‘PIP2’ method.(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 and PIP2 as indicated. (B) Mean estimated Po for various channels: WT (0.53±0.04); homS225T (0.62±0.04); hetS225T (0.59±0.03); hetDel (0.64±0.04); hetT, del (0.66+0.02). * indicates statistically significant difference compared with WT (Student's t-test, p-value <0.05). n = 6–12.
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pone-0063758-g006: Heterozygous S225T, deletion channels display higher channel open probability, assessed by the ‘PIP2’ method.(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 and PIP2 as indicated. (B) Mean estimated Po for various channels: WT (0.53±0.04); homS225T (0.62±0.04); hetS225T (0.59±0.03); hetDel (0.64±0.04); hetT, del (0.66+0.02). * indicates statistically significant difference compared with WT (Student's t-test, p-value <0.05). n = 6–12.

Mentions: To explore the potential structural basis of the mutagenic effects, we have examined the location of these residues by homology modeling of Kir6.2, based on the Kir2.2 structure [18]. (Figure 5A) This modeling makes clear that S225 and the S226–232 region are located far from the ATP binding pocket, and it is therefore unlikely that either the point mutation or the deletion directly affects ATP binding. Within the deleted region (Figure 5B), residues E227 and E229 have been reported to form inter-subunit ion pairs and thereby affect the intrinsic open probability of the channel [14], [19], such that the open probability of mutations E227K and E229K is greater than that of WT channels [19]. Deletion of these residues might also increase intrinsic open probability and we therefore estimated the open probably for these channels, using the ‘PIP2 method’ [17], assessing the increase of channel activity achieved by exposure of excised patches to saturating exogenous PIP2. Figure 6A displays representative recordings of WT and hetS225T, del channel activity before and after PIP2 application. Open probability (Po) is then calculated as:


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)

Heterozygous S225T, deletion channels display higher channel open probability, assessed by the ‘PIP2’ method.(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 and PIP2 as indicated. (B) Mean estimated Po for various channels: WT (0.53±0.04); homS225T (0.62±0.04); hetS225T (0.59±0.03); hetDel (0.64±0.04); hetT, del (0.66+0.02). * indicates statistically significant difference compared with WT (Student's t-test, p-value <0.05). n = 6–12.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063758-g006: Heterozygous S225T, deletion channels display higher channel open probability, assessed by the ‘PIP2’ method.(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 and PIP2 as indicated. (B) Mean estimated Po for various channels: WT (0.53±0.04); homS225T (0.62±0.04); hetS225T (0.59±0.03); hetDel (0.64±0.04); hetT, del (0.66+0.02). * indicates statistically significant difference compared with WT (Student's t-test, p-value <0.05). n = 6–12.
Mentions: To explore the potential structural basis of the mutagenic effects, we have examined the location of these residues by homology modeling of Kir6.2, based on the Kir2.2 structure [18]. (Figure 5A) This modeling makes clear that S225 and the S226–232 region are located far from the ATP binding pocket, and it is therefore unlikely that either the point mutation or the deletion directly affects ATP binding. Within the deleted region (Figure 5B), residues E227 and E229 have been reported to form inter-subunit ion pairs and thereby affect the intrinsic open probability of the channel [14], [19], such that the open probability of mutations E227K and E229K is greater than that of WT channels [19]. Deletion of these residues might also increase intrinsic open probability and we therefore estimated the open probably for these channels, using the ‘PIP2 method’ [17], assessing the increase of channel activity achieved by exposure of excised patches to saturating exogenous PIP2. Figure 6A displays representative recordings of WT and hetS225T, del channel activity before and after PIP2 application. Open probability (Po) is then calculated as:

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