Limits...
Neto2- mice have impaired GABAergic inhibition and are susceptible to seizures.

Mahadevan V, Dargaei Z, Ivakine EA, Hartmann AM, Ng D, Chevrier J, Ormond J, Nothwang HG, McInnes RR, Woodin MA - Front Cell Neurosci (2015)

Bottom Line: Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized.We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2(-/-) neurons compared to wild-type controls.To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2(-/-) neurons had significant reductions in both their amplitude and frequency.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Systems Biology, University of Toronto Toronto, ON, Canada.

ABSTRACT
Neto2 is a transmembrane protein that interacts with the neuron-specific K(+)-Cl(-) cotransporter (KCC2) in the central nervous system (CNS). Efficient KCC2 transport is essential for setting the neuronal Cl(-) gradient, which is required for fast GABAergic inhibition. Neto2 is required to maintain the normal abundance of KCC2 in neurons, and increases KCC2 function by binding to the active oligomeric form of this cotransporter. In the present study, we characterized GABAergic inhibition and KCC2-mediated neuronal chloride homeostasis in pyramidal neurons from adult hippocampal slices. Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized. We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2(-/-) neurons compared to wild-type controls. To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2(-/-) neurons had significant reductions in both their amplitude and frequency. Based on the critical role of Neto2 in regulating GABAergic inhibition we rationalized that Neto2- mice would be prone to seizure activity. We found that Neto2- mice demonstrated a decrease in the latency to pentylenetetrazole (PTZ)-induced seizures and an increase in seizure severity.

No MeSH data available.


Related in: MedlinePlus

Native-KCC2 from adult Neto2- neurons exhibit decreased phosphorylated Ser940 status. (A) Two-dimensional BN-PAGE separation of native, total KCC2 and Ser940-phosphorylated KCC2 complexes from ~P30 hippocampus solubilized with C12E9, from WT or Neto2−/− mice; gel separations were immunoblotted with the antibodies indicated on right (total KCC2, phosphorylated Ser940KCC2). This blot is a representative example of three independent biological replicates. Majority of total KCC2 is phosphorylated at Ser940 residue in WT lysates while only a small proportion of total KCC2 is phosphorylated at Ser940 residue in Neto2- lysates. The dashed lines and arrows indicate the decrease in molecular weight of native-KCC2 in Neto2- lysates compared to WT. (B) Calibration of the 2D-BN-PAGE separation using native-ferritin marker composed of distinct molecular mass (220, 440 and 880 kDa). This allowed for determination of the approximate molecular mass of the native-KCC2 assemblies, during the absence or presence of Neto2. The existing Neto2-independent native Ser940 KCC2 migrates at a lower molecular weight in Neto2- lysates when compared to WT lysates. This indicates that Neto2 is an essential component of Ser940 phosphorylated native-KCC2.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4585209&req=5

Figure 3: Native-KCC2 from adult Neto2- neurons exhibit decreased phosphorylated Ser940 status. (A) Two-dimensional BN-PAGE separation of native, total KCC2 and Ser940-phosphorylated KCC2 complexes from ~P30 hippocampus solubilized with C12E9, from WT or Neto2−/− mice; gel separations were immunoblotted with the antibodies indicated on right (total KCC2, phosphorylated Ser940KCC2). This blot is a representative example of three independent biological replicates. Majority of total KCC2 is phosphorylated at Ser940 residue in WT lysates while only a small proportion of total KCC2 is phosphorylated at Ser940 residue in Neto2- lysates. The dashed lines and arrows indicate the decrease in molecular weight of native-KCC2 in Neto2- lysates compared to WT. (B) Calibration of the 2D-BN-PAGE separation using native-ferritin marker composed of distinct molecular mass (220, 440 and 880 kDa). This allowed for determination of the approximate molecular mass of the native-KCC2 assemblies, during the absence or presence of Neto2. The existing Neto2-independent native Ser940 KCC2 migrates at a lower molecular weight in Neto2- lysates when compared to WT lysates. This indicates that Neto2 is an essential component of Ser940 phosphorylated native-KCC2.

Mentions: 2D BN-PAGE analysis were performed as described (Schwenk et al., 2012; Mahadevan et al., 2014) with minor modifications. After the completion of the gel run, excised BN-PAGE lanes were equilibrated in Laemmli buffer containing SDS and DTT for 30 min at room temperature to denature the native proteins. After a brief rinse in SDS-PAGE running buffer, the excised BN-PAGE lanes were placed on a 6% SDS-PAGE gel for separation in the second dimension. After standard electroblotting of SDS-PAGE-resolved samples on PVDF membrane, the blots were subjected to western blotting analysis with Ms anti-KCC2 (developmental studies hybridoma bank, clone N1/12), Rb anti-Ser940 phosphorylated KCC2 (Phosphosolutions, p1551–940). The Ln (native-ferritin molecular weights 220 kDa, 440 kDa, 880 kDa) were plotted against the distance of migration of native-ferritin marker (in cms) in a 5% 1D BN-PAGE. Using this as a standard, the approximate molecular weights of Ser940 phosphorylated native-KCC2 were deduced after immune-blotting. Experiments in Figure 3 are representative results from three independent biological replicates.


Neto2- mice have impaired GABAergic inhibition and are susceptible to seizures.

Mahadevan V, Dargaei Z, Ivakine EA, Hartmann AM, Ng D, Chevrier J, Ormond J, Nothwang HG, McInnes RR, Woodin MA - Front Cell Neurosci (2015)

Native-KCC2 from adult Neto2- neurons exhibit decreased phosphorylated Ser940 status. (A) Two-dimensional BN-PAGE separation of native, total KCC2 and Ser940-phosphorylated KCC2 complexes from ~P30 hippocampus solubilized with C12E9, from WT or Neto2−/− mice; gel separations were immunoblotted with the antibodies indicated on right (total KCC2, phosphorylated Ser940KCC2). This blot is a representative example of three independent biological replicates. Majority of total KCC2 is phosphorylated at Ser940 residue in WT lysates while only a small proportion of total KCC2 is phosphorylated at Ser940 residue in Neto2- lysates. The dashed lines and arrows indicate the decrease in molecular weight of native-KCC2 in Neto2- lysates compared to WT. (B) Calibration of the 2D-BN-PAGE separation using native-ferritin marker composed of distinct molecular mass (220, 440 and 880 kDa). This allowed for determination of the approximate molecular mass of the native-KCC2 assemblies, during the absence or presence of Neto2. The existing Neto2-independent native Ser940 KCC2 migrates at a lower molecular weight in Neto2- lysates when compared to WT lysates. This indicates that Neto2 is an essential component of Ser940 phosphorylated native-KCC2.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585209&req=5

Figure 3: Native-KCC2 from adult Neto2- neurons exhibit decreased phosphorylated Ser940 status. (A) Two-dimensional BN-PAGE separation of native, total KCC2 and Ser940-phosphorylated KCC2 complexes from ~P30 hippocampus solubilized with C12E9, from WT or Neto2−/− mice; gel separations were immunoblotted with the antibodies indicated on right (total KCC2, phosphorylated Ser940KCC2). This blot is a representative example of three independent biological replicates. Majority of total KCC2 is phosphorylated at Ser940 residue in WT lysates while only a small proportion of total KCC2 is phosphorylated at Ser940 residue in Neto2- lysates. The dashed lines and arrows indicate the decrease in molecular weight of native-KCC2 in Neto2- lysates compared to WT. (B) Calibration of the 2D-BN-PAGE separation using native-ferritin marker composed of distinct molecular mass (220, 440 and 880 kDa). This allowed for determination of the approximate molecular mass of the native-KCC2 assemblies, during the absence or presence of Neto2. The existing Neto2-independent native Ser940 KCC2 migrates at a lower molecular weight in Neto2- lysates when compared to WT lysates. This indicates that Neto2 is an essential component of Ser940 phosphorylated native-KCC2.
Mentions: 2D BN-PAGE analysis were performed as described (Schwenk et al., 2012; Mahadevan et al., 2014) with minor modifications. After the completion of the gel run, excised BN-PAGE lanes were equilibrated in Laemmli buffer containing SDS and DTT for 30 min at room temperature to denature the native proteins. After a brief rinse in SDS-PAGE running buffer, the excised BN-PAGE lanes were placed on a 6% SDS-PAGE gel for separation in the second dimension. After standard electroblotting of SDS-PAGE-resolved samples on PVDF membrane, the blots were subjected to western blotting analysis with Ms anti-KCC2 (developmental studies hybridoma bank, clone N1/12), Rb anti-Ser940 phosphorylated KCC2 (Phosphosolutions, p1551–940). The Ln (native-ferritin molecular weights 220 kDa, 440 kDa, 880 kDa) were plotted against the distance of migration of native-ferritin marker (in cms) in a 5% 1D BN-PAGE. Using this as a standard, the approximate molecular weights of Ser940 phosphorylated native-KCC2 were deduced after immune-blotting. Experiments in Figure 3 are representative results from three independent biological replicates.

Bottom Line: Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized.We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2(-/-) neurons compared to wild-type controls.To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2(-/-) neurons had significant reductions in both their amplitude and frequency.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Systems Biology, University of Toronto Toronto, ON, Canada.

ABSTRACT
Neto2 is a transmembrane protein that interacts with the neuron-specific K(+)-Cl(-) cotransporter (KCC2) in the central nervous system (CNS). Efficient KCC2 transport is essential for setting the neuronal Cl(-) gradient, which is required for fast GABAergic inhibition. Neto2 is required to maintain the normal abundance of KCC2 in neurons, and increases KCC2 function by binding to the active oligomeric form of this cotransporter. In the present study, we characterized GABAergic inhibition and KCC2-mediated neuronal chloride homeostasis in pyramidal neurons from adult hippocampal slices. Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized. We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2(-/-) neurons compared to wild-type controls. To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2(-/-) neurons had significant reductions in both their amplitude and frequency. Based on the critical role of Neto2 in regulating GABAergic inhibition we rationalized that Neto2- mice would be prone to seizure activity. We found that Neto2- mice demonstrated a decrease in the latency to pentylenetetrazole (PTZ)-induced seizures and an increase in seizure severity.

No MeSH data available.


Related in: MedlinePlus